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Interference probing of rRNA with snoRNPs: A novel approach for functional mapping of RNA in vivo
Abstract
Synthesis of eukaryotic ribosomal RNAs (rRNAs) includes methylation of scores of nucleotides at the 2'-O-ribose position (Nm) by small nucleolar RNP complexes (snoRNPs). Sequence specificity is provided by the snoRNA component through base-pairing of a guide sequence with rRNA. Here, we report that methylation snoRNPs can be targeted to many new sites in yeast rRNA, by providing the snoRNA with a novel guide sequence, and that in some cases growth and translation activity are strongly impaired. Novel snoRNAs can be expressed individually or by a unique library strategy that yields guide sequences specific for a large target region. Interference effects were observed for sites in both the small and large subunits, including the reaction center region. Targeting guide RNAs to nucleotides flanking the sensitive sites caused little or no defect, indicating that methylation is responsible for the interference rather than a simple antisense effect or misguided chaperone function. To our knowledge, this is the only approach that has been used to mutagenize the backbone of rRNA in vivo.
... We next investigated the effect of PFAR inhibition on [PSI + ] propagation by altering PFAR rRNA nucleotides using site-directed methylation 32,33 . Site-directed methylation is based on the hijacking of naturally encoded small nucleolar RNAs (snoRNA) to specifically methylate nucleotides at rRNA level and can be achieved by replacing the cellular snoRNA guide element by a custom guide sequence 32 . ...
... The nucleotide to be modified is targeted through specific base pairing of the snoRNA guide sequence with its rRNA substrate (Fig. 3A). Adding an unnatural methyl moiety to a rRNA specific nucleotide using the cellular machinery is equivalent to a gene-product point mutation 33 . We focused on U2862 and G2863 nucleotides which are collectively conserved in bacterial, yeast and human domain V (Fig. 3B), involved in ribosome protein folding in vitro 14 , able to bind 6AP and GA 14 and not naturally methylated 34 . ...
... Expression of PFAR-snoRNA targeting nucleotide U2862 led to a larger proportion of [psi − ] red cells than cells transformed by PFAR-snoRNA targeting nucleotide G2863 and negative controls (Fig. 3C), suggesting nucleotide U2862 methylation markedly impairs [PSI + ] propagation. As expected, forced methylation of U1757 known to induce growth defect in yeast 33 and of catalytic adenine A2820, caused major growth interference and no transformants were obtained. [PSI + ] instability in PFAR-snoRNA-U2862 expressing cells was not due to modification of the 25S:18S ratio (Fig. 3D) 35 or to onset of cellular stress (induction of Hsp104p or Hsp70p expression, Fig. 3E). ...
6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI⁺] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI⁺]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI⁺] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases.
... On induction, the novel snoRNA is produced, assembled into a snoRNP, and directs modification to a nucleotide specified by the new (13nt) guide element. Because this method has been described in a ''Methods'' chapter (Liu et al., 2001), we refer the reader to that source and to a study in which this strategy was used to mutagenize the backbone of rRNA and analyze the resulting effects (Liu and Fournier, 2004). Engineered H/ACA box snoRNAs have also been used in yeast to target pseudouridylation to novel rRNA sites (Bortolin et al., 1999;King, McCully, and Fournier, unpublished results). ...
... Alternately, if deletion of an entire snoRNA leads to elimination of an additional modification, the desired modification can be maintained by providing an artificial guide snoRNA ( Fig. 13.4A). The technology is well established for the C/D guide snoRNAs and involves insertion of a customized guide sequence into a designed host snoRNA (Liu and Fournier, 2004;Liu et al., 2001). In principle, such manipulations can be done for the box H/ACA snoRNAs; however, as noted previously, we have not found engineering guides for C to be straightforward as of yet (unpublished results and see Bortolin et al., 1999). ...
... Ten mCi of [ 35 S]methionine (1175 Ci/mmol; Perkin-Elmer) is added to 10 ml of culture. At this point (zero time point), 0.5 ml of labeled cells is taken and mixed immediately with 0.2 ml ice-cold stop solution (70 ml of unlabeled 60 mM D,L-methionine, 1 ml of 10 mg/ml cycloheximide, and 129 ml of 50% TCA) (Liu and Fournier, 2004). Next, four or five samples of labeled cells are taken at intervals of one to a few minutes, depending on the severity of the effects with modification loss. ...
The small nucleolar RNAs (snoRNAs) are associated with proteins in ribonucleoprotein complexes called snoRNPs ("snorps"). These complexes create modified nucleotides in preribosomal RNA and other RNAs and participate in nucleolytic cleavages of pre-rRNA. The various reactions occur in site-specific fashion, and the mature rRNAs are ultimately incorporated into cytoplasmic ribosomes. Most snoRNAs exist in two structural classes, and most members in each class are involved in nucleotide modification reactions. Guide snoRNAs in the "box C/D" class target methylation of the 2'-hydroxyl moiety, to form 2'-O-methylated nucleotides (Nm), whereas guide snoRNAs in the "box H/ACA" class target specific uridines for conversion to pseudouridine (Psi). The rRNA nucleotides modified in this manner are numerous, totaling approximately 100 in yeast and twice that number in humans. Although the chemistry of the modifications and the factors involved in their formation are largely explained, very little is known about the influence of the copious snoRNA-guided nucleotide modifications on rRNA activity and ribosome function. Among eukaryotic organisms the sites of rRNA modification and the corresponding guide snoRNAs have been best characterized in S. cerevisiae, making this a model organism for analyzing the consequences of modification. This chapter presents approaches to characterizing rRNA modification effects in yeast and includes strategies for evaluating a variety of specific rRNA functions. To aid in planning, a package of bioinformatics tools is described that enables investigators to correlate guide function with targeted ribosomal sites in several contexts. Genetic procedures are presented for depleting modifications at one or more rRNA sites, including ablation of all Nm or Psi modifications made by snoRNPs, and for introducing modifications at novel sites. Methods are also included for characterizing modification effects on cell growth, antibiotic sensitivity, rRNA processing, formation of various rRNP complexes, translation activity, and rRNA structure within the ribosome.
... Previous studies have exploited designer snoRNAs to direct modifications to novel RNA targets (Cavaille et al. 1996b). This has been used as an alternative to mutagenesis to explore ribosome function (Liu and Fournier 2004;Liu et al. 2008), and to influence modification-sensitive processes such as nonsense suppression (Karijolich and Yu 2011). As a simple proof-of-concept, we tested whether a mutant snoRNA could increase acetylation of an h45-ITS1 substrate that is poorly recognized by endogenous SNORD13 (Fig. 6A). ...
... An additional motivation for studying snoRNAs lies in their potential to be engineered to direct RNA modifications to novel targets (Cavaille et al. 1996b;Liu and Fournier 2004). As an initial step in this direction, we demonstrated that a SNORD13 mutant can be used to drive acetylation of an ectopic pre-rRNA substrate that is not efficiently modified in endogenous cells. ...
N4-acetylcytidine (ac4C) is an RNA nucleobase found in all domains of life. Establishment of ac4C in helix 45 (h45) of human 18S ribosomal RNA (rRNA) requires the combined activity of the acetyltransferase NAT10 and the box C/D snoRNA SNORD13. However, the molecular mechanisms governing RNA-guided nucleobase acetylation in humans remain unexplored. After applying comparative sequence analysis and site-directed mutagenesis to provide evidence that SNORD13 folds into three main RNA helices, we report two assays that enable the study of SNORD13-dependent RNA acetylation in human cells. First, we demonstrate that ectopic expression of SNORD13 rescues h45 in a SNORD13 knockout cell line. Next, we show mutant snoRNAs can be used in combination with nucleotide resolution ac4C sequencing to define structure and sequence elements critical for SNORD13 function. Finally, we develop a second method that reports on the substrate specificity of endogenous NAT10-SNORD13 via mutational analysis of an ectopically-expressed pre-rRNA substrate. By combining mutational analysis of these reconstituted systems with nucleotide resolution ac4C sequencing, our studies reveal plasticity in the molecular determinants underlying RNA-guided cytidine acetylation that is distinct from deposition of other well-studied rRNA modifications (e.g. pseudouridine). Overall, our studies provide a new approach to reconstitute RNA-guided cytidine acetylation in human cells as well as nucleotide resolution insights into the mechanisms governing this process.
... Previous studies have exploited designer snoRNAs to direct modifications to novel RNA targets. 16 This has been used as an alternative to mutagenesis to explore ribosome function, 37,38 and to influence modification-sensitive processes such as nonsense suppression. 39 As a simple proof- of-concept, we tested whether a mutant snoRNA could increase acetylation of an h45-ITS1 substrate that is poorly recognized by endogenous SNORD13 (Figure 6a). ...
... ; https://doi.org/10.1101/2022.05.12.491732 doi: bioRxiv preprint An additional motivation for studying snoRNAs lies in their potential to be engineered to direct RNA modifications to novel targets. 16,37 As an initial step in this direction, we demonstrated that a SNORD13 mutant can be used to drive acetylation of an ectopic pre-rRNA substrate that is not efficiently modified in endogenous cells. It is important to specify that this represents an extremely simple model system, and the extent of SNORD13's reprogrammability, as well as whether analogues may direct NAT10 to novel substrates, remains unknown. ...
N4-acetylcytidine (ac ⁴ C) is an RNA nucleobase found in all domains of life. Establishment of ac ⁴ C in helix 45 (h45) of human 18S ribosomal RNA (rRNA) requires the combined activity of the acetyltransferase NAT10 and the box C/D snoRNA SNORD13. However, the molecular mechanisms governing RNA-guided nucleobase acetylation in humans remain unexplored. Here we report two assays that enable the study of SNORD13-dependent RNA acetylation in human cells. First, we demonstrate that ectopic expression of SNORD13 rescues h45 in a SNORD13 knockout cell line. Next, we show mutant snoRNAs can be used in combination with nucleotide resolution ac ⁴ C sequencing to define structure and sequence elements critical for SNORD13 function. Finally, we develop a second method that reports on the substrate specificity of endogenous NAT10-SNORD13 via mutational analysis of an ectopically-expressed pre-rRNA substrate. By combining mutational analysis of these reconstituted systems with nucleotide resolution ac ⁴ C sequencing, our studies reveal plasticity in the molecular determinants underlying RNA-guided cytidine acetylation that is distinct from deposition of other well-studied rRNA modifications (e.g. pseudouridine). Overall, our studies provide a new approach to reconstitute RNA-guided cytidine acetylation in human cells as well as nucleotide resolution insights into the mechanisms governing this process.
Abstract Figure
... Bu ajanlar, ribonükleazların nükleofilik N ve O atomları ve fosfat omurgasının O atomu üzerinden nükleofilik yer değiştirme reaksiyonlarına girerler. 9,10 Tek iplikli nükleik asitler metilasyona karşı daha savunmasız olduğundan, birçok RNA bu hasara karşı DNA'dan daha hassas olabilir. rRNA moleküllerinin sentezi öncelikle bir prekürsör RNA (pre-rRNA) molekülünün oluşumuyla başlar. ...
... snoRNP'lerin maya rRNA'sında normal şartlarda metillenmemesi gereken alanların metilasyonuna yönlendirilmek üzere deneysel olarak yeniden yapılandırılması, hücre büyümesinde bozukluğa ve protein sentezinin inhibisyonuna neden olabilir. 10 Alkilasyon rastgele meydana geldiğinde, zararlı etki kaynaklarından birisi olarak baz eşleşme etkileşimlerinin engellenmesi söylenebilir. Örneğin metilasyon translasyondaki tRNA-rRNA ve mRNA-tRNA etkileşimlerini etkileyebilir ve teorik olarak siRNA ve miRNA fonksiyonu gibi baz eşleşmesine dayanan diğer RNA işlevlerine müdahale edebilir. ...
... Most artificial modifications at positions unmodified in nature have no physiological consequences. Those leading to growth defects are also associated with slow protein synthesis, consistent with the interference effect observed for nucleotides already identified as involved in tRNA binding, subunit association or decoding [19]. Interestingly, the expression of a few engineered snoRNAs did not alter modification status. ...
Ribosomal RNA is a major component of the ribosome. This RNA plays a crucial role in ribosome functioning by ensuring the formation of the peptide bond between amino acids and the accurate decoding of the genetic code. The rRNA carries many chemical modifications that participate in its maturation, the formation of the ribosome and its functioning. In this review, we present the different modifications and how they are deposited on the rRNA. We also describe the most recent results showing that the modified positions are not 100% modified, which creates a heterogeneous population of ribosomes. This gave rise to the concept of specialized ribosomes that we discuss. The knowledge accumulated in the yeast Saccharomyces cerevisiae is very helpful to better understand the role of rRNA modifications in humans, especially in ribosomopathies.
... Interestingly, a high-throughput screen based on expression of an artificial snoRNA guiding an rRNA 2 -O-methylation that impairs cellular growth (46), identified Dbp3 as a potential regulator of rRNA 2 -O-methylation (Nicholas Watkins and Robert van Nues, unpublished). This suggested that the pre-rRNA processing defects observed in the absence of Dbp3 and its function in ribosome assembly may be linked to rRNA 2 -O-methylation-guiding box C/D snoRNPs. ...
RNA helicases play important roles in diverse aspects of RNA metabolism through their functions in remodelling ribonucleoprotein complexes (RNPs), such as pre-ribosomes. Here, we show that the DEAD box helicase Dbp3 is required for efficient processing of the U18 and U24 intron-encoded snoRNAs and 2′-O-methylation of various sites within the 25S ribosomal RNA (rRNA) sequence. Furthermore, numerous box C/D snoRNPs accumulate on pre-ribosomes in the absence of Dbp3. Many snoRNAs guiding Dbp3-dependent rRNA modifications have overlapping pre-rRNA basepairing sites and therefore form mutually exclusive interactions with pre-ribosomes. Analysis of the distribution of these snoRNAs between pre-ribosome-associated and ‘free’ pools demonstrated that many are almost exclusively associated with pre-ribosomal complexes. Our data suggest that retention of such snoRNPs on pre-ribosomes when Dbp3 is lacking may impede rRNA 2′-O-methylation by reducing the recycling efficiency of snoRNPs and by inhibiting snoRNP access to proximal target sites. The observation of substoichiometric rRNA modification at adjacent sites suggests that the snoRNPs guiding such modifications likely interact stochastically rather than hierarchically with their pre-rRNA target sites. Together, our data provide new insights into the dynamics of snoRNPs on pre-ribosomal complexes and the remodelling events occurring during the early stages of ribosome assembly.
... 104,121,123,124 Because there is ∼4−6 times more RNA than DNA in the cell, RNA damage can have a deleterious effect on gene expression. Undesired RNA modifications in yeast rRNAs 125 and mammalian tRNAs 107 have been shown to inhibit protein synthesis due to inhibition of rRNA−tRNA base pairing interactions. 43 In addition, unwanted RNA modifications can cause RNAs to misfold and can result in mispairing of ncRNA−mRNA interactions leading to altered mRNA decay rates and production of aberrant proteins. ...
An emerging molecular understanding of RNA alkylation and its removal are transforming our knowledge of RNA biology and its interplay with cancer chemotherapy responses. DNA modifications are known to perform critical functions depending on the genome template, including gene expression, DNA replication timing, and DNA damage protection. Yet, current results suggest that the chemical diversity of DNA modifications pales in comparison to those on RNA. Over 150 RNA modifications have been identified to date, and their complete functional implications are still being unveiled. These include intrinsic roles such as proper processing and RNA maturation; emerging evidence has furthermore uncovered RNA modification ‘readers’, seemingly analogous to those identified for histone modifications. These modification recognition factors may regulate mRNA stability, localization, and interaction with translation machinery, affecting gene expression. Not surprisingly, tumors differentially modulate factors involved in expressing these marks, contributing both to tumorigenesis and responses to alkylating chemotherapy. Here we describe current understanding of RNA modifications and their removal, with a focus primarily on methylation and alkylation as functionally relevant changes to the transcriptome. Intriguingly, some of the same RNA modifications elicited by physiological processes are also produced by alkylating agents, thus blurring the lines between what is a physiological mark versus a damage-induced modification. Furthermore, we find that high gene expression of enzymes with RNA dealkylation activity is a sensitive readout for poor survival in four different cancer types, underscoring the likely importance of examining RNA dealkylation mechanisms to cancer biology and for cancer treatment and prognosis.
... The rRNA modification clusters are essential for cell growth (5)(6)(7)(8)(9)(10) while loss of some individual modifications increases cellular sensitivity to stress and ribosome-specific antibiotics (11). The specificity of modification is important as 2 -Omethylation of the wrong site can affect ribosome formation and/or function (12)(13)(14). Furthermore, with the recent observations that some sites in the rRNA are not 2 -Omethylated to a 100% (15,16) and the interest in 'specialised' ribosomes (17), there is renewed interest in the mechanism controlling the complexity of rRNA modification. ...
Eukaryotic box C/D small nucleolar (sno)RNPs catalyse the site-specific 2'-O-methylation of ribosomal RNA. The RNA component (snoRNA) contains guide regions that base-pair with the target site to select the single nucleotide to be modified. The terminal C/D and internal C'/D' motifs in the snoRNA, adjacent to the guide region, function as binding sites for the snoRNP proteins including the enzymatic subunit fibrillarin/Nop1. Four yeast snoRNAs are unusual in that they are predicted to methylate two nucleotides in a single target region. In each case, the internal C'/D' motifs from these snoRNAs differ from the consensus. Our data indicate that the C'/D' motifs in snR13, snR48 and U18 form two alternative structures that lead to differences in the position of the proteins bound to this motif. We propose that each snoRNA forms two different snoRNPs, subtly different in how the proteins are bound to the C'/D' motif, leading to 2'-O-methylation of different nucleotides in the target region. For snR48 and U18, the unusual C'/D' alone is enough for the modification of two nucleotides. However, for the snR13 snoRNA the unusual C'/D' motif and extra base-pairing, which stimulates rRNA 2'-O-methylation, are both critical for multiple modifications in the target region.
... In contrast to DNA, for which the modification, beyond its accelerated rate of depurination, is not toxic [41], in RNA, N7 methylation interferes with Hoogsteen base pairing and how certain proteins recognize RNA. In addition to N7 methylation, modifications to the Watson-Crick face of the nucleobase are expected to be detrimental to RNA function [42,43] similar to analogous studies on DNA replication. It is worth remembering that the list of possible modifications is extensive due to the diverse nature of the damaging agents (many groups can be added, from methyl to bulky aromatic groups) as well as the atom of the nucleobase on which these groups can be added. ...
The "central dogma" of molecular biology describes how information contained in DNA is transformed into RNA and finally into proteins. In order for proteins to maintain their functionality in both the parent cell and subsequent generations, it is essential that the information encoded in DNA and RNA remains unaltered. DNA and RNA are constantly exposed to damaging agents, which can modify nucleic acids and change the information they encode. While much is known about how cells respond to damaged DNA, the importance of protecting RNA has only become appreciated over the past decade. Modification of the nucleobase through oxidation and alkylation has long been known to affect its base-pairing properties during DNA replication. Similarly, recent studies have begun to highlight some of the unwanted consequences of chemical damage on mRNA decoding during translation. Oxidation and alkylation of mRNA appear to have drastic effects on the speed and fidelity of protein synthesis. As some mRNAs can persist for days in certain tissues, it is not surprising that it has recently emerged that mRNA-surveillance and RNA-repair pathways have evolved to clear or correct damaged mRNA.
... In order to demonstrate the applicability of the method for the detection of changes in methylation patterns, we analyzed whole-cell (WC) RNA from wild-type (wt) yeast, a knock-out strain in which one of the guide RNAs (snR67) and its corresponding methyl group at position LSU-G2619 has been deleted, [11] and a knock-in strain that carries an artificial snoRNA which guides methylation at LSU-U2954 [12] (Figure 3). LSU-G2619 is a universally conserved modification in the P-site and LSU-U2954 is a critical residue for P-site tRNA binding (Figure 3 C). ...
Ribose methylations are the most abundant chemical modifications of ribosomal RNA and are critical for ribosome assembly and fidelity of translation. Many aspects of ribose methylations have been difficult to study due to lack of efficient mapping methods. Here, we present a sequencing-based method (RiboMeth-seq) and its application to yeast ribosomes, presently the best-studied eukaryotic model system. We demonstrate detection of the known as well as new modifications, reveal partial modifications and unexpected communication between modification events, and determine the order of modification at several sites during ribosome biogenesis. Surprisingly, the method also provides information on a subset of other modifications. Hence, RiboMeth-seq enables a detailed evaluation of the importance of RNA modifications in the cells most sophisticated molecular machine. RiboMeth-seq can be adapted to other RNA classes, for example, mRNA, to reveal new biology involving RNA modifications.
... A series of snoRNA-analogues were constructed to guide 2 -O-methylation of rRNA nucleotides critical for ribosome functioning. Targeted nucleotides were selected by adaptation of published data on the sensitivity of S.cerevisiae to de novo 2 -O-methylation of rRNA (Liu and Fournier 2004). Transfection of MCF-7 with rRNA-directed snoRNA analogues induced 2 -O-methylation of targeted rRNA nucleotides. ...
In order to determine the biological functions of extracellular RNA, we have designed and synthesized single- and double-stranded
analogues of human extracellular RNAs and analyzed the influences of these analogues on both the proliferation and the viability
of cultured human adenocarcinoma cells MCF-7. Transfection of MCF-7 cells by snoRNA analogues, which were targeted to de novo
2′-O-methylation of nucleotides of pre-mRNA, resulted in a partial impairment of pre-mRNA splicing. SnoRNA analogues directed
to rRNA induced 2′-O-methylation of targeted nucleotides of rRNA. Transfection by Alu RNA reduced the viability of MCF-7 cells. These data emphasize
the role of circulating RNA as efficient inter-cellular messengers and highlight the structures of circulating RNA as a promising
basis for the development of new biologically active substances.
KeywordsCirculating RNA-Alu RNA-C/D box RNA
... Methylation activity of various C 0 /D 0 motifs was analysed in vivo in W303 (MATa/MATá; leu2-3112 trp1-1 can1-100 ura3-1 ade2-1 his3- 11,15; [phi þ ]) using an artificial snoRNA construct inserted in the intron of the actin gene. The actin/snoRNA cassette (a PCR amplified 1-kb Bam HI–Xba I fragment derived from pFL45/ACT/ XK; Laszlo et al, 1996) was placed under the control of the GAL1 promoter (amplified as a 1-kb Eco R1–Bam HI fragment from pBL143; Liu and Fournier, 2004) and cloned into pRS416 in which the Acc65 I and Xho I sites of the multiple cloning site had been deleted. Artificial snoRNAs were assembled from oligonucleotides and cloned in between the unique Xho I and Acc65 I sites present within the actin intron (Figure 3A; SupplementaryFigure S10; KissLaszlo et al, 1996). ...
2'-O-methylation of eukaryotic ribosomal RNA (r)RNA, essential for ribosome function, is catalysed by box C/D small nucleolar (sno)RNPs. The RNA components of these complexes (snoRNAs) contain one or two guide sequences, which, through base-pairing, select the rRNA modification site. Adjacent to the guide sequences are protein-binding sites (the C/D or C'/D' motifs). Analysis of >2000 yeast box C/D snoRNAs identified additional conserved sequences in many snoRNAs that are complementary to regions adjacent to the rRNA methylation site. This 'extra base-pairing' was also found in many human box C/D snoRNAs and can stimulate methylation by up to five-fold. Sequence analysis, combined with RNA-protein crosslinking in Saccharomyces cerevisiae, identified highly divergent box C'/D' motifs that are bound by snoRNP proteins. In vivo rRNA methylation assays showed these to be active. Our data suggest roles for non-catalytic subunits (Nop56 and Nop58) in rRNA binding and support an asymmetric model for box C/D snoRNP organization. The study provides novel insights into the extent of the snoRNA-rRNA interactions required for efficient methylation and the structural organization of the snoRNPs.
... Yeast strains LMA439 (MATa ade2 arg4 leu2-3,112 trp1-289 ura3-52 NOP1-CBP-TEV-protA::TRP1) (37) and YGALSNU13 (MATa his3⌬1 leu2⌬0 met15⌬0 LYS2 ura3⌬0 kanMX6: PGAL1-SNU13) (8) were provided by A. Jacquier and R. T. O'Keefe, respectively. Plasmids pBL150 (25) and pRS415 were provided by M. J. Fournier. Plasmid pRS413-snu13 (8) was provided by R. T. O'Keefe, and plasmid pFL45/ snR5Nco (19) was provided by T. Kiss. ...
Box C/D ribonucleoprotein particles guide the 2′-O-ribose methylation of target nucleotides in both archaeal and eukaryotic RNAs. These complexes contain two functional centers,
assembled around the C/D and C′/D′ motifs in the box C/D RNA. The C/D and C′/D′ RNPs of the archaeal snoRNA-like RNP (sRNP)
are spatially and functionally coupled. Here, we show that similar coupling also occurs in eukaryotic box C/D snoRNPs. The
C/D RNP guided 2′-O-methylation when the C′/D′ motif was either mutated or ablated. In contrast, the C′/D′ RNP was inactive
as an independent complex. Additional experiments demonstrated that the internal C′/D′ RNP is spatially coupled to the terminal
box C/D complex. Pulldown experiments also indicated that all four core proteins are independently recruited to the box C/D
and C′/D′ motifs. Therefore, the spatial-functional coupling of box C/D and C′/D′ RNPs is an evolutionarily conserved feature
of both archaeal and eukaryotic box C/D RNP complexes.
... Box C/D snoRNAs have been localized to the nucleoli and Cajal bodies, where they naturally direct 29-O-methylation of rRNAs and snRNAs (Darzacq et al. 2002;Deryusheva and Gall 2009;Tycowski et al. 2009). Interestingly, when expressed in the cell, artificial snoRNAs can also efficiently guide rRNA modifications at target sites (Cavaille et al. 1996;Ni et al. 1997;Liu et al. 2001Liu et al. , 2008Liu and Fournier 2004). ...
The 2'-OH group of the branch point adenosine is a key moiety to initiate pre-mRNA splicing. We use RNA-guided RNA modification to target the pre-mRNA branch point adenosine for 2'-O-methylation, with the aim of blocking pre-mRNA splicing in vertebrate cells. We show that, under certain conditions, injection of a branch point-specific artificial box C/D RNA into Xenopus oocytes effectively 2'-O-methylates adenovirus pre-mRNA at the target nucleotide. However, 2'-O-methylation at the authentic branch point activates a host of cryptic branch points, thus allowing splicing to continue. These cryptic sites are mapped, and mutated. Upon injection, pre-mRNA free of cryptic branch points fails to splice when the branch point-specific box C/D RNA is present. However, 2'-O-methylation at the branch point does not prevent pre-mRNA from being assembled into pre-catalytic spliceosome-like complexes prior to the first chemical step of splicing. Our results demonstrate that RNA-guided pre-mRNA modification can occur in the nucleoplasm of vertebrate cells, thus offering a powerful tool for molecular biology research.
... While methylation is a naturally occurring rRNA modification, only specific, largely conserved positions are methylated in a process requiring a large class of small nucleolar ribonucleoproteins (snoRNPs) to guide methylation. Indeed, experimental re-engineering of snoRNPs to direct methylation of normally unmethylated sites in yeast rRNA can cause growth defects and inhibition of protein synthesis (Liu and Fournier, 2004). When alkylation occurs at random, one source of deleterious effects is the inhibition of base-pairing interactions. ...
Damage to RNA from ultraviolet light, oxidation, chlorination, nitration, and akylation can include chemical modifications to nucleobases as well as RNA-RNA and RNA-protein crosslinking. In vitro studies have described a range of possible damage products, some of which are supported as physiologically relevant by in vivo observations in normal growth, stress conditions, or disease states. Damage to both messenger RNA and noncoding RNA may have functional consequences, and work has begun to elucidate the role of RNA turnover pathways and specific damage recognition pathways in clearing cells of these damaged RNAs.
... Our findings reveal a possible functional or structural importance of the 2 0-hydroxyl group of A2451 for peptide bond formation. This interpretation is compatible with recent in vivo data showing that snoRNA-guided methylation of the 2 0-hydroxyl group at A2451 resulted in a lethal growth phenotype in yeast (27). In the crystallographic structure of the PTC, this hydroxyl group was found to be in hydrogen bonding distance to the reactive amino group of the A-site substrate (11). ...
The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystallography showed that
A2451 of 23S rRNA makes the closest approach to the attacking amino group of aminoacyl-tRNA. Mutations of A2451 had relatively
small effects on transpeptidation and failed to unequivocally identify the crucial functional group(s). Here, we employed
an in vitro reconstitution system for chemical engineering the peptidyl transferase center by introducing non-natural nucleosides at
position A2451. This allowed us to investigate the peptidyl transfer reaction performed by a ribosome that contained a modified
nucleoside at the active site. The main finding is that ribosomes carrying a 2′-deoxyribose at A2451 showed a compromised
peptidyl transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A2451
had only little impact on peptide bond formation, as long as the 2′-hydroxyl was present. This implicates a functional or
structural role of the 2′-hydroxyl group at A2451 for transpeptidation.
Ribosomal RNA (rRNA) maturation is a complex process that involves chemical modifications of the bases or sugar residues of specific nucleotides. One of the most abundant types of rRNA modifications, ribose 2'-O-methylation, is guided by ribonucleoprotein complexes containing small nucleolar box C/D RNAs. Since the majority of 2'-O-methylated nucleotides are located in the most conserved regions of rRNA that comprise functionally important centers of the ribosome, an alteration in a 2'-O-methylation profile can affect ribosome assembly and function. One of the key approaches for localization of 2'-O-methylated nucleotides in long RNAs is a method based on the termination of reverse transcription. The current study presents an adaptation of this method for the use of fluorescently labeled primers and analysis of termination products by capillary gel electrophoresis on an automated genetic analyzer. The developed approach allowed us to analyze the influence of the synthetic analogues of box C/D RNAs on post-transcriptional modifications of human 28S rRNA in MCF-7 cells. It has been established that the transfection of MCF-7 cells with a box C/D RNA analogue leads to an enhanced modification level of certain native sites of 2'-O-methylation in the target rRNA. The observed effect of synthetic RNAs on the 2'-O-methylation of rRNA in human cells demonstrates a path towards targeted regulation of rRNA post-transcriptional maturation. The described approach can be applied in the development of novel diagnostic methods for detecting diseases in humans.
Parkfield, California, which experienced M 6.0 earthquakes in 1934, 1966, and 2004, is one of the few locales for which geodetic observations span multiple earthquake cycles. We undertake a comprehensive study of deformation over the most recent earthquake cycle and explore the results in the context of ge-odetic data collected prior to the 1966 event. Through joint inversion of the variety of Parkfield geodetic measurements (trilateration, two-color laser, and Global Posi-tioning System), including previously unpublished two-color data, we estimate the spatial distribution of slip and slip rate along the San Andreas using a fault geometry based on precisely relocated seismicity. Although the three most recent Parkfield earthquakes appear complementary in their along-strike distributions of slip, they do not produce uniform strain release along strike over multiple seismic cycles. Since the 1934 earthquake, more than 1 m of slip deficit has accumulated on portions of the fault that slipped in the 1966 and 2004 earthquakes, and an average of 2 m of slip deficit exists on the 33 km of the fault southeast of Gold Hill to be released in a future, perhaps larger, earthquake. It appears that the fault is capable of partially releasing stored strain in moderate earth-quakes, maintaining a disequilibrium through multiple earthquake cycles. This com-plicates the application of simple earthquake recurrence models that assume only the strain accumulated since the most recent event is relevant to the size or timing of an upcoming earthquake. Our findings further emphasize that accumulated slip deficit is not sufficient for earthquake nucleation. Online material: Model fault geometry, fit to the data for the inversions, and model resolution.
Small nucleolar RNAs (snoRNAs) play a key role in ribosomal RNA (rRNA) biogenesis. Box C/D snoRNAs guide the site-specific 2'-O-ribose methylation of nucleotides in rRNAs and small nuclear RNAs (snRNAs). A number of box C/D snoRNAs and their fragments have recently been reported to regulate post-transcriptional modifications and the alternative splicing of pre-mRNA. Artificial analogues of U24 snoRNAs directed to nucleotides in 28S and 18S rRNAs, as well as pre-mRNAs and mature mRNAs of human heat shock cognate protein (hsc70), were designed and synthesized in this study. It was found that after the transfection of MCF-7 human cells with artificial box C/D RNAs in complex with lipofectamine, snoRNA analogues penetrated into cells and accumulated in the cytoplasm and nucleus. It was demonstrated that the transfection of cultured human cells with artificial box C/D snoRNA targeted to pre-mRNAs induce partial splicing impairments. It was found that transfection with artificial snoRNAs directed to 18S and 28S rRNA nucleotides, significant for ribosome functioning, induce a decrease in MCF-7 cell viability.
Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants.
Eukaryotic rRNAs contain scores of two major types of nucleotide modifications, 2'-O-methylation (Nm) and pseudouridylation (Psi). Both types are known to alter the stability and dynamics of RNA folding. In Eukaryotes, these modifications are created by small nucleolar RNPs (snoRNPs) with site-specificity provided by the snoRNA component. Little is yet known about the influence of such modifications on ribosome synthesis or activity, although in a few cases depletions of natural modifications have impaired ribosome function. Our previous work showed that targeting Nm modifications to non-natural sites in yeast rRNA can severely impair cell growth, however, the underlying basis of the interference effects were not described. Here, we show that targeting Nm formation to several individual sensitive sites in the peptidyl transferase center (PTC) strongly impairs ribosome accumulation and activity. Methylation was detected for all sites targeted, suggesting that the non-natural modification is the basis of the interference effects. For certain sensitive sites, the translation rate was reduced by 70-100%, due to: (1) a marked decrease (28-50%) in ribosomal subunits caused by slower pre-rRNA processing and mainly faster rRNA turn over and, (2) impaired activity of the surviving ribosomes. This last finding infers that the mis-targeted methylations compromise PTC function. The discovery that a new methylation can trigger robust rRNA degradation indicates that modification effects are monitored for quality control. These findings imply that nucleotide modifications can serve as evolutionary constraints and that snoRNP mutations expected to occur in nature can cause human disease.
Posttranscriptional, site-specific adenosine to inosine (A-to-I) base conversions, designated as RNA editing, play significant roles in generating diversity of gene expression. However, little is known about how and in which cellular compartments RNA editing is controlled. Interestingly, the two enzymes that catalyze RNA editing, adenosine deaminases that act on RNA (ADAR) 1 and 2, have recently been demonstrated to dynamically associate with the nucleolus. Moreover, we have identified a brain-specific small RNA, termed MBII-52, which was predicted to function as a nucleolar C/D RNA, thereby targeting an A-to-I editing site (C-site) within the 5-HT2C serotonin receptor pre-mRNA for 2'-O-methylation. Through the subcellular targeting of minigenes that contain natural editing sites, we show that ADAR2- but not ADAR1-mediated RNA editing occurs in the nucleolus. We also demonstrate that MBII-52 forms a bona fide small nucleolar ribonucleoprotein particle that specifically decreases the efficiency of RNA editing by ADAR2 at the targeted C-site. Our data are consistent with a model in which C/D small nucleolar RNA might play a role in the regulation of RNA editing.
The ribosomal peptidyl transferase center is a ribozyme catalyzing peptide bond synthesis in all organisms. We applied a novel modified nucleoside interference approach to identify functional groups at 9 universally conserved active site residues. Owing to their immediate proximity to the chemical center, the 23S rRNA nucleosides A2451, U2506 and U2585 were of particular interest. Our study ruled out U2506 and U2585 as contributors of vital chemical groups for transpeptidation. In contrast the ribose 2'-OH of A2451 was identified as the prime ribosomal group with potential functional importance. This 2'-OH renders almost full catalytic power to the ribosome even when embedded into an active site of six neighboring 2'-deoxyribose nucleosides. These data highlight the unique functional role of the A2451 2'-OH for peptide bond synthesis among all other functional groups at the ribosomal peptidyl transferase active site.
The non-protein-coding transcriptional output of the cell is far greater than previously thought. Although the functions, if any, of the vast majority of these RNA transcripts remain elusive, out of those for which functions have already been established, most act as RNA guides for protein enzymes. Common features of these RNAs provide clues about the evolutionary constraints that led to the development of RNA-guided proteins and the specific biological environments in which target specificity and diversity are most crucial to the cell.
When isolating ribonucleoprotein (RNP) complexes by an affinity selection approach, tagging the RNA component can prove to be strategically important. This is especially true for purifying single types of snoRNPs, because in most cases the snoRNA is thought to be the only unique component. Here, we present a general strategy for selecting specific snoRNPs that features a high-affinity tag in the snoRNA and another in a snoRNP core protein. The RNA tag (called U1hpII) is a small (26 nt) stem-loop domain from human U1 snRNA. This structure binds with high affinity (K(D)=10(-11)M) to the RRM domain of the snRNP protein U1A. In our approach, the U1A protein contains a unique affinity tag and is coexpressed in vivo with the tagged snoRNA to yield snoRNP-U1A complexes with two unique protein tags-one in the bound U1A protein and the other in the snoRNP core protein. This scheme has been used effectively to select C/D and H/ACA snoRNPs, including both processing and modifying snoRNPs, and the snoRNA and core proteins are highly enriched. Depending on selection stringency other proteins are isolated as well, including an RNA helicase involved in snoRNP release from pre-rRNA and additional proteins that function in ribosome biogenesis. Tagging the snoRNA component alone is also effective when U1A is expressed with a myc-Tev-protein A fusion sequence. Combined with reduced stringency, enrichment of the U14 snoRNP with this latter system revealed potential interactions with two other snoRNPs, including one processing snoRNP involved in the same cleavages of pre-rRNA.
Development of transformable vectors for thermophilic archaea requires the characterization of appropriate selectable marker genes. Many antibiotic inhibitors of protein biosynthesis are known to bind to rRNA; therefore, we screened 14 for their capacity to inhibit growth of the thermophilic archaeon Sulfolobus acidocaldarius. Carbomycin, celesticetin, chloramphenicol, puromycin, sparsomycin, tetracycline, and thiostrepton all inhibited growth by different degrees. Spontaneous drug-resistant mutants were isolated from plates containing celesticetin or chloramphenicol. Six mutants from each plate exhibited a C-2585-to-U transition in the peptidyl transferase loop of 23S rRNA (corresponding to C-2452 in Escherichia coli 23S rRNA). The single-site mutation also conferred resistance to carbomycin. The mutated 23S rRNA gene provides a potentially useful and dominant marker for a thermophilic archael vector.
Previous experiments have shown that the top of helix 90 of 23S rRNA is highly important for the ribosomal peptidyltransferase activity and might be part of the donor (P) site. Developing on these studies, mutations in the 23S rRNA at the highly conserved positions G2505, G2582, and G2583 were investigated. None of the mutations affected assembly, subunit association, or the capacity of tRNA binding to A and P sites. A "selective transpeptidation assay" revealed that the mutations specifically impaired peptide bond formation. Results with a modified "fragment" assay using the minimal donor substrate pA-fMet are consistent with a model where the nucleotides psiGG2582 form a binding pocket for C75 of the tRNA.
rRNA plays an important role in function of peptidyl transferase, the catalytic center of the ribosome responsible for the peptide bond formation. Proper placement of the peptidyl transferase substrates, peptidyl-tRNA and aminoacyl-tRNA, is essential for catalysis of the transpeptidation reaction and protein synthesis. In this report, we define a small set of rRNA nucleotides that are most likely directly involved in binding of tRNA in the functional sites of the large ribosomal subunit. By binding biotinylated tRNA substrates to randomly modified large ribosomal subunits from Escherichia coli and capturing resulting complexes on the avidin resin, we identified four nucleotides in the large ribosomal subunit rRNA (positions G2252, A2451, U2506, and U2585) whose modifications prevent binding of a peptidyl-tRNA analog in the P site and one residue (U2555) whose modification interferes with transfer of peptidyl moiety to puromycin. These nucleotides represent a subset of positions protected by tRNA analogs from chemical modification and significantly narrow the number of 23S rRNA nucleotides that may be directly involved in tRNA binding in the ribosomal functional sites.
Development of transformable vectors for thermophilic archaea requires the characterization of appropriate selectable marker genes. Many antibiotic inhibitors of protein biosynthesis are known to bind to rRNA; therefore, we screened 14 for their capacity to inhibit growth of the thermophilic archaeon Sulfolobus acidocaldarius. Carbomycin, celesticetin, chloramphenicol, puromycin, sparsomycin, tetracycline, and thiostrepton all inhibited growth by different degrees. Spontaneous drug-resistant mutants were isolated from plates containing celesticetin or chloramphenicol. Six mutants from each plate exhibited a C-2585-to-U transition in the peptidyl transferase loop of 23S rRNA (corresponding to C-2452 in Escherichia coli 23S rRNA). The single-site mutation also conferred resistance to carbomycin. The mutated 23S rRNA gene provides a potentially useful and dominant marker for a thermophilic archael vector.
Site-specific 2'-O-ribose methylation of eukaryotic rRNAs is guided by small nucleolar RNAs (snoRNAs). The methylation guide snoRNAs carry long perfect complementaries to rRNAs. These antisense elements are located either in the 5' half or in the 3' end region of the snoRNA, and are followed by the conserved D' or D box motifs, respectively. An uninterrupted helix formed between the rRNA and the antisense element of the snoRNA, in conjunction with the adjacent D' or D box, constitute the recognition signal for the putative methyltransferase. Here, we have identified an additional essential box element common to methylation guide snoRNAs, termed the C' box. We show that the C' box functions in concert with the D' box and plays a crucial role in the methyltransfer reaction directed by the upstream antisense element and the D' box. We also show that an internal fragment of U24 methylation guide snoRNA, encompassing the upstream antisense element and the D' and C' box motifs, can support the site-specific methylation of rRNA. This strongly suggests that the C box of methylation guide snoRNAs plays an essential role in the methyltransfer reaction guided by the 3'-terminal antisense element and the D box of the snoRNA.
Small nucleolar RNAs (snoRNAs) are intron encoded or expressed from monocistronic independent transcription units, or, in the case of plants, from polycistronic clusters. We show that the snR190 and U14 snoRNAs from the yeast Saccharomyces cerevisiae are co-transcribed as a dicistronic precursor which is processed by the RNA endonuclease Rnt1, the yeast ortholog of bacterial RNase III. RNT1 disruption results in a dramatic decrease in the levels of mature U14 and snR190 and in accumulation of dicistronic snR190-U14 RNAs. Addition of recombinant Rnt1 to yeast extracts made from RNT1 disruptants induces the chase of dicistronic RNAs into mature snoRNAs, showing that dicistronic RNAs correspond to functional precursors stalled in the processing pathway. Rnt1 cleaves a dicistronic transcript in vitro in the absence of other factors, separating snR190 from U14. Thus, one of the functions of eukaryotic RNase III is, as for the bacterial enzyme, to liberate monocistronic RNAs from polycistronic transcripts.
Small nucleolar RNAs (snoRNAs) are involved in cleavage of rRNA, modification of rRNA nucleotides and, perhaps, other aspects
of ribosome biogenesis in eukaryotic cells. Scores of snoRNAs have been discovered in recent years from various eukaryotes,
and the total number is predicted to be up to 200 different snoRNA species per individual organism. We have created a comprehensive
database for snoRNAs from the yeast Saccharomyces cerevisiae which allows easy access to detailed information about each species known (almost 70 snoRNAs are featured). The database
consists of three major parts: (i) a utilities section; (ii) a master table; and (iii) a collection of tables for the individual
snoRNAs. The utilities section provides an introduction to the database. The master table lists all known s.cerevisiae snoRNAs and their major properties. Information in the individual tables includes: alternate names, size, family classification,
genomic organization, sequences (with major features identified), GenBank accession numbers, occurrence of homologues, gene
disruption phenotypes, functional properties and associated RNAs and proteins. All information is accompanied with appropriate
literature references. The database is available on the World Wide Web (http://www.bio.umass.edu/biochem/rna-sequence/Yeast_snoRNA_Database/snoRNA_DataBase.html), and should be useful for a wide range of snoRNA studies.
Small nucleolar RNAs (snoRNAs) are required for ribose 2′-O-methylation of eukaryotic ribosomal RNA. Many of the genes for
this snoRNA family have remained unidentified in Saccharomyces cerevisiae, despite the availability of a complete genome sequence. Probabilistic modeling methods akin to those used in speech recognition
and computational linguistics were used to computationally screen the yeast genome and identify 22 methylation guide snoRNAs,
snR50 to snR71. Gene disruptions and other experimental characterization confirmed their methylation guide function. In total,
51 of the 55 ribose methylated sites in yeast ribosomal RNA were assigned to 41 different guide snoRNAs.
The nucleolus has long been known as a functionally highly specialized subnuclear compartment where synthesis, posttranscriptional modification, and processing of cytoplasmic rRNAs take place. In this study, we demonstrate that the nucleolus contains all the
trans
-acting factors that are responsible for the accurate and efficient synthesis of the eight 2′-O-methylated nucleotides and three pseudouridine residues carried by the mammalian U6 spliceosomal small nuclear RNA. Factors mediating the formation of pseudouridine residues in the U3 small nucleolar RNA are also present and functionally active in the nucleolus. For selection of the correct target nucleotides in the U6 and U3 RNAs, the nucleolar 2′-O-methylation and pseudouridylation factors rely on short sequences located around the target nucleotide to be modified. This observation further underscores a recently proposed role for small nucleolar guide RNAs in the 2′-O-methylation of the U6 spliceosomal RNA (K. T. Tycowski, Z.-H. You, P. J. Graham, and J. A. Steitz, Mol. Cell 2:629–638, 1998). We demonstrate that a novel 2′-O-methylated nucleotide can be generated in the yeast U6 RNA by use of an artificial 2′-O-methylation small nucleolar guide RNA. We also show that a short fragment of the 5.8S rRNA, when expressed as part of the human U6 RNA, is faithfully 2′-O-methylated and pseudouridylated. These results are most consistent with a trafficking pathway in which the U6 spliceosomal RNA cycles through the nucleolus to undergo nucleolar RNA-directed modifications.
Using the atomic structures of the large ribosomal subunit fromHaloarcula marismortui and its complexes with two substrate analogs, we establish that the ribosome is a ribozyme and address the catalytic properties
of its all-RNA active site. Both substrate analogs are contacted exclusively by conserved ribosomal RNA (rRNA) residues from
domain V of 23S rRNA; there are no protein side-chain atoms closer than about 18 angstroms to the peptide bond being synthesized. The mechanism
of peptide bond synthesis appears to resemble the reverse of the acylation step in serine proteases, with the base of A2486
(A2451 in Escherichia coli) playing the same general base role as histidine-57 in chymotrypsin. The unusual pK
a (where K
a is the acid dissociation constant) required for A2486 to perform this function may derive in part from its hydrogen bonding
to G2482 (G2447 in E. coli), which also interacts with a buried phosphate that could stabilize unusual tautomers of these two bases. The polypeptide
exit tunnel is largely formed by RNA but has significant contributions from proteins L4, L22, and L39e, and its exit is encircled
by proteins L19, L22, L23, L24, L29, and L31e.
The large ribosomal subunit catalyzes peptide bond formation and binds initiation, termination, and elongation factors. We
have determined the crystal structure of the large ribosomal subunit fromHaloarcula marismortui at 2.4 angstrom resolution, and it includes 2833 of the subunit's 3045 nucleotides and 27 of its 31 proteins. The domains
of its RNAs all have irregular shapes and fit together in the ribosome like the pieces of a three-dimensional jigsaw puzzle
to form a large, monolithic structure. Proteins are abundant everywhere on its surface except in the active site where peptide
bond formation occurs and where it contacts the small subunit. Most of the proteins stabilize the structure by interacting
with several RNA domains, often using idiosyncratically folded extensions that reach into the subunit's interior.
We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron
density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent
with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major
elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the
intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation
with intersubunit movement.
Posttranscriptional modifications were mapped in helices 90-92 of 23S rRNA from the following phylogenetically diverse organisms: Haloarcula marismortui, Sulfolobus acidocaldarius, Bacillus subtilis, and Bacillus stearothermophilus. Helix 92 is a component of the ribosomal A-site, which contacts the aminoacyl-tRNA during protein synthesis, implying that posttranscriptional modifications in helices 90-92 may be important for ribosome function. RNA fragments were isolated from 23S rRNA by site-directed RNase H digestion. A novel method of mapping modifications by analysis of short, nucleotide-specific, RNase digestion fragments with Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was utilized. The MALDI-MS data were complemented by two primer extension techniques using reverse transcriptase. One technique utilizes decreasing concentrations of deoxynucleotide triphosphates to map 2'-O-ribose methylations. In the other, the rRNA is chemically modified, followed by mild alkaline hydrolysis to map pseudouridines (psis). A total of 10 posttranscriptionally methylated nucleotides and 6 psis were detected in the five organisms. Eight of the methylated nucleotides and one psi have not been reported previously. The distribution of modified nucleotides and their locations on the surface of the ribosomal peptidyl transferase cleft suggests functional importance.
Functional Escherichia coli 50S ribosomal subunits can be reconstituted from their natural rRNA and protein components. However, when the assembly is performed with in vitro-transcribed 23S rRNA, the reconstitution efficiency is diminished by four orders of magnitude. We tested a variety of chemical chaperones (compounds that are typically used for protein folding), putative RNA chaperones (proteins) and ribosome-targeted antibiotics (small-molecule ligands) that might be reasoned to aid in folding and assembly. Addition of the osmolyte trimethylamine-oxide (TMAO) and the ketolide antibiotic telithromycin (HMR3647) to the reconstitution stimulates its efficiency up to 100-fold yielding a substantially improved system for the in vitro analysis of mutant ribosomes.
Translation elongation factor 1β (EF-1β) is a member of the family of guanine nucleotide exchange factors, proteins whose activities are important for the regulation of G proteins critical to many cellular processes. EF-1β is a highly conserved protein that catalyzes the exchange of bound GDP for GTP on EF-1α, a required step to ensure continued protein synthesis. In this work, we demonstrate that the highly conserved C-terminal region of
Saccharomyces cerevisiae
EF-1β is sufficient for normal cell growth. This region of yeast and metazoan EF-1β and the metazoan EF-1β-like protein EF-1δ is highly conserved. Human EF-1β, but not human EF-1δ, is functional in place of yeast EF-1β, even though both EF-1β and EF-1δ have previously been shown to have guanine nucleotide exchange activity in vitro. Based on the sequence and functional homology, mutagenesis of two C-terminal residues identical in all EF-1β protein sequences was performed, resulting in mutants with growth defects and sensitivity to translation inhibitors. These mutants also enhance translational fidelity at nonsense codons, which correlates with a reduction in total protein synthesis. These results indicate the critical function of EF-1β in regulating EF-1α activity, cell growth, translation rates, and translational fidelity.
Die ultraschallkontrollierte Radiofrequenzablation (RFA) wird seit geraumer Zeit zur Behandlung maligner Lebertumoren eingesetzt, jedoch ist das Verfahren nicht auf dieses Organ begrenzt. Eine Patientin mit symptomatischer postmenopausaler Osteoporose, zusätzlich seit 1995 bestehendem primären Hyperparathyreoidismus und stärksten Wirbelsäulenschmerzen lehnte eine Operation der Nebenschilddrüse ab. Sonographisch zeigte sich eine 16 mm große, echoarme Raumforderung dorsal des rechten oberen Schilddrüsenpols. Osteodensitometrie der LWS: ausgeprägte Osteoporose (88 % einer altersentsprechenden Gruppe). Erhöhung des Serum-Kalzium-Spiegels auf 3,1mmol/l und des Serum-Parathormon-Spiegels auf 274 pg/dl (N: 10-50). Bei Wunsch nach einem alternativen Therapieverfahren seitens der Patientin wurde eine perkutane ultraschallkontrollierte RFA des solitären Nebenschilddrüsenadenoms durchgeführt. Postinterventionell kam es zu einer Normalisierung des Parathormonspiegels und des Serum-Kalzium-Spiegels. Der Nachbeobachtungszeitraum beträgt ein Jahr. Erstmals konnte bei der Patientin postinterventionell eine suffiziente Osteoporosetherapie durchgeführt werden. Die durchgeführte Osteodensitometrie zeigte eine deutliche Verbesserung des Befundes der LWS im Vergleich zum Vorjahr (98 % einer altersentsprechenden Gruppe). Bei symptomatischem primären Hyperparathyreoidismus kann die RFA in besonderen Situationen eine therapeutische Alternative darstellen.
A gene encoding a yeast homologue of translation elongation factor 1λ (EF-1λ), TEF3, was isolated as a gene dosage extragenic suppressor of the coldsensitive phenotype of the Saccharomyces cerevisiae drs2 mutant. The drs2 mutant is deficient in the assembly of 40S ribosomal subunits. We have identified a second gene, TEF4, that encodes a protein highly related to both the Tef3p protein (Tef3p), and EF-1λ isolated from other organisms. In contrast
to TEF3, the TEF4 gene contains an intron. Gene disruptions showed that neither gene is required for mitotic growth. Haploid spores containing
disruptions of both genes are viable and have no defects in ribosomal subunit composition or polyribosomes. Unlike TEF3, extra copies of TEF4 do not suppress the cold-sensitive 40S ribosomal subunit deficiency of a drs2 strain. Lowstringency genomic Southern hybridization analysis indicates there may be additional yeast genes related to TEF3 and TEF4.
Typescript. Thesis (Ph. D.)--University of Massachusetts at Amherst, 1998. Includes bibliographical references (leaves 188-199).
An alignment of 75 phylogenetically diverse large subunit ribosomal RNA sequences was created and searched for secondary and tertiary structure elements by computer. The search revealed four unknown secondary structural pairings, two internal loop closings, and five short-range tertiary interactions--two of which were pairings of an unusual type. One brings a loop together with two other loops previously known to be paired, and one involves a nucleotide within a presumed tetraloop. The latter interaction constrains the RNA structure near the ribosomal E-site, where two base pairs previously suggested to be in parallel orientation are now proven. No clear phylogenetic evidence for direct base pairing between the large and small subunit rRNA was found.
This chapter discusses the currently available information on the modified nucleotides, especially in man, the vertebrates and in yeast. Information on the modified nucleotides cannot be obtained simply by sequencing rDNA. Instead, a number of structural and kinetic techniques have been applied to the direct analysis of the modified nucleotides in rRNAs and ribosomal precursor RNAs. The most detailed knowledge has come from the studies on vertebrates including man, and from the yeast, Saccharomyces. Almost all of the modified nucleotides that have been located are in the conserved structural core of rRNA. This brings the modified nucleotides into the central arena of rRNA structure and function. There is no simple theme of primary or secondary structure among the modification sites. The diversity of modification sites is discussed in the chapter, and this diversity raises challenging questions concerning the molecular recognition processes that bring about the modifications. Further insight into these processes may come from the long established finding that most modifications occur very rapidly upon ribosomal precursor RNA. A related finding, also long established, is that inhibition of methylation of precursors of rRNA is associated with failure of ribosome maturation. However, the precise roles of the modified nucleotides in the maturation and working life of the ribosome remain unknown.
A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.
The Howard Hughes Medical Institute, Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine,
New Haven, CT 06536-0812, USA. A set of small nuclear RNAs of 50 to 200 nucleotides resides in the nucleolus of the cell,
the region of the nucleus in which ribosomes are made. J. A. Steitz and K. T. Tycowski discuss what is known about how these
RNAs participate in the conversion of the long precursor RNAs to the mature 18S, 5.8S, and 28S rRNAs of the ribosome.
U14 is a conserved small nucleolar RNA (snoRNA) required for processing of yeast 18S rRNA. The presence of two long sequences (13 and 14 nucleotides) with strong complementarity to 18S rRNA suggests that U14 base-pairs with pre-rRNA. Evidence of direct binding was developed by showing that mutations in these U14 elements mimic U14 depletion and that function can be rescued by a compensatory sequence change in 18S RNA. The U14 elements are functionally interdependent, indicating that both participate in binding. Folding models predict that binding might occur through both rRNA elements simultaneously. Potential roles of U14 in rRNA folding, maturation, and ribosome assembly are discussed. U14 is one of several snoRNAs with long complementarities to rRNA and the first snoRNA in this class shown to interact directly with rRNA.
The present review summarizes key progress made in characterizing the small nucleolar RNAs (snoRNAs) of eukaryotic cells. Recent studies have shown snoRNA populations to be substantially more complex than anticipated initially. Many newly discovered snoRNAs are synthesized by an intron-processing pathway, which provides a potential mechanism for coordinating nuclear RNA synthesis. Several snoRNAs and snoRNP proteins are known to be needed for processing of ribosomal RNA, but precise functions remain to be defined. In principle, snoRNAs could have several roles in ribosome synthesis including: folding of pre-rRNA, formation of rRNP substrates, catalyzing RNA cleavages, base modification, assembly of pre-ribosomal subunits, and export of product rRNP particles.
Human ribosomes contain more than 200 modified nucleotides. These are made up as follows: more than 100 2'-O-methyl groups, 10 methylated bases, about 95 pseudouridines and at least one other modification. Other mammalian sources that have been examined, as well as the lower vertebrate Xenopus laevis, show very similar patterns of nucleotide modifications, especially as revealed by oligonucleotide fingerprinting for methyl groups. Most of the methyl groups have been located along the rRNA primary structure by matching oligonucleotide sequence data to the complete sequences derived from rDNA. Nearly all of the methyls are in conserved core regions. Saccharomyces carlsbergensis ribosomes contain about 55% as many methyls as vertebrate ribosomes. The locations of most of the S carlsbergensis methyls are also known. However, of the numerous other eukaryotes whose rRNA sequences have been determined indirectly from rDNA, few have yielded detailed data on modified nucleotides. This is in part because the methods applied to vertebrate and yeast ribosomes are highly laborious and are not universally applicable. Therefore in the final part of this paper we briefly review other methods that have been applied to the detection and localization of modified nucleotides in rRNA. In particular, we outline progress towards developing a method whereby reverse transcription shows characteristic pausing at most of the 2'-O-methylation sites in human and Xenopus 18S rRNA. 2'-O-Methylation pauses are distinguishable from most other interruptions; the 2'-O-methyl pauses occur more strongly at low than at high dNTP concentration, whereas most other interruptions are independent of dNTP concentration.
A growing subset of small nucleolar RNAs (snoRNAs) contains long stretches of sequence complementarity to conserved sequences in mature ribosomal RNA (rRNA). This article reviews current knowledge about these complementarities and proposes that these antisense snoRNAs might function in pre-rRNA folding, base modification and ribosomal ribonucleoprotein assembly, in some cases acting as RNA chaperones.
Ribosomal binding sites were investigated for the diverse group of antibiotics: anisomycin, anthelmycin, blasticidin S, bruceantin, carbomycin, chloramphenicol, griseoviridin, narciclasine, T2 toxin, tylosin and virginiamycin M1 all of which are considered to inhibit the peptidyl transferase reaction by different mechanisms. The drugs also exhibit differing degrees of specificity for bacterial, archaeal and eukaryotic ribosomes despite a high level of conservation of sequence and secondary structure at the peptidyl transferase centre of the 23 S-like rRNAs. The drug binding sites were characterized by incubating each antibiotic with ribosomes from a bacterium, an archaeon and a eukaryote and chemically probing the 23 S-like rRNA. The complexity of the changes in reactivity ranged from one or two nucleotides (anthelmycin, narciclasine) to eight or nine (virginiamycin M1) and it was inferred, at least for those drugs producing complex changes, that they induce, and stabilize, a particular functional conformer in the peptidyl transferase centre. The results were correlated with literature data on both ribosomal ligand binding and the putative inhibitory mechanisms of the drugs, and the following inferences are made concerning the fine structure of the peptidyl transferase centre. (1) An irregular secondary structural motif, which includes unpaired A2439 (Escherichia coli numbering), lies close to the catalytic centre; (2) nucleotides A2451 and C2452 contribute to a site for the binding of the side chains of aromatic amino acids; (3) the P-substrate site encompasses U2585, U2506 and, possibly, a site in domain IV (A1787), and (4) the sequence A2058 to A2062 and nucleotide U2609 contribute to, or modulate, the start of the peptide channel. No drug effects were found that could be directly attributed to an A-site and the possibility is raised that, if it exists, it consists mainly of ribosomal proteins. However, two drugs T2 toxin and virginiamycin M1 protected the only nucleotide in the peptidyl transferase loop region (C2394) associated with the E-site. Finally, it is proposed that the putative sub-sites are physically separated, that some drugs bind to more than one of them, and that they are conformationally interdependent.
Genes for three novel yeast snRNAs have been identified and tested for essentiality. Partial sequence information was developed
for RNA extracted from isolated nuclei and the respective gene sequences were discovered by screening a DNA sequence database.
The three RNAs contain 222, 188 and 183 nucleotides and are designated snR31, snR32 and snR33, respectively. Each RNA is derived
from a single copy gene. The SNR31 gene is adjacent to a gene for an unnamed protein associated with the cap-binding protein
elF-4E. The SNR32 gene is next to a gene for ribosomal protein L41 and the gene for SNR33 is on chromosome III, between two
open reading frames with no known function. Genetic disruption analyses showed that none of the three snRNAs is required for
growth. The new RNAs bring the number of nonspliceosomal snRNAs characterized thus far in S.cerevisiae to 14, of which only
three are essential.
We have generated temperature-sensitive lethal point mutations in the small nucleolar RNA-associated protein fibrillarin (encoded by the NOP1 gene in yeast) and analyzed their effects on ribosome synthesis. The five alleles tested all prevent synthesis of normal ribosomes, but in dramatically different ways. At the non-permissive temperature, the nop1.2 and nop1.5 alleles prevent synthesis of both 18S and 25S rRNA and all pre-rRNA species except the 35S primary transcript. In contrast, the nop1.3, nop1.4, and nop1.7 alleles do not strongly impair processing. In nop1.3 strains, nucleolar methylation of pre-rRNA is strongly inhibited; late, cytoplasmic methylation of 18S rRNA and tRNA methylation continue. The nop1.4 and nop1.7 alleles result in the synthesis of cytoplasmic 60S ribosomal subunits with strongly aberrant mobilities on sucrose gradients even at the permissive temperature, owing to the impairment of a late step in ribosome assembly. Thus, all major posttranscriptional activities in ribosome synthesis, pre-rRNA processing, pre-rRNA modification, and ribosome assembly are dependent on fibrillarin.
Eukaryotic cells contain many fibrillarin-associated small nucleolar RNAs (snoRNAs) that possess long complementarities to mature rRNAs. Characterization of 21 novel antisense snoRNAs from human cells followed by genetic depletion and reconstitution studies on yeast U24 snoRNA provides evidence that this class of snoRNAs is required for site-specific 2'-O-methylation of preribosomal RNA (pre-rRNA). Antisense sno-RNAs function through direct base-pairing interactions with pre-rRNA. The antisense element, together with the D or D' box of the snoRNA, provide the information necessary to select the target nucleotide for the methyltransfer reaction. The conclusion that sno-RNAs function in covalent modification of the sugar moieties of ribonucleotides demonstrates that eukaryotic small nuclear RNAs have a more versatile cellular function than earlier anticipated.
Sparsomycin is a universal and powerful inhibitor of peptide bond formation which, in contrast to many other ribosome-targeted antibiotics, does not produce footprints on rRNA. A mutant of an archaeon Halobacterium halobium has been isolated that exhibits resistance to sparsomycin. Resistant cells possessed a mutation in the 23 S rRNA, where C2518 (C2499 in Escherichia coli) was substituted by U. Introduction of the C2518U mutation into the chromosomal 23 S rRNA gene of wild-type H. halobium rendered cells resistant to sparsomycin, demonstrating that a single nucleotide alteration in the rRNA is sufficient to confer resistance. Accordingly, ribosomes containing mutant 23 S rRNA exhibited increased tolerance to sparsomycin in vitro. Mutations of two other nucleotide positions in the peptidyl transferase center, C2471 and U2519 (C2452 and U2500 in E. coli), conferred resistance to low concentrations of sparsomycin. The location of the sparsomycin resistance mutations reveals the possible site of drug binding and/or action. Our findings provide further support for the idea that rRNA may be directly involved in interaction with antibiotics and the catalysis of the peptide bond formation.
A growing number of small nucleolar RNAs (snoRNAs) are intron-encoded, contain the characteristic box C (UGAUGA) and box D (CUGA) motifs and exhibit long complementarities to conserved sequences in mature rRNAs. We have identified nine additional members of this family, U32 to U40. All but one are encoded in introns of ribosomal protein genes in vertebrates: U32 to U35 in rpL13a, U36 in rpL7a and U38 to U40 in rpS8. By contrast, U37 is encoded in elongation factor 2 gene. Interestingly, U32 and U36 each contain two complementarities (one to 18 S and the other to 28 S rRNA). U32 to U40 are fibrillarin-associated, devoid of a 5'-trimethyl-cap and display an exclusively nucleolar localization. They are all metabolically stable and roughly as abundant as previously reported members of this family. Characterization of their homologs in distant species shows that their 10 to 14 nt long rRNA complementarities are conserved. A clue on the function of this snoRNA family is provided by the comparative analysis of the largely expanded collection of their conserved duplexes with rRNA. Not only does each duplex span at least one site of 2'-O-ribose methylation in the rRNA but the modification site is always at the same position in the duplex, paired to the fifth nucleotide upstream from a box D motif in the snoRNA. Consistent with the notion that each snoRNA of this family guides one particular methylation along the rRNA sequence, we have detected several pairs of snoRNAs with overlapping complementarities to rRNA tracts with vicinal sites of ribose methylations. In each case, the two overlapping complementarities are shifted from each other by a distance equal to the spacing between the methylated sites which are thus found at the same position within each of the mutually exclusive duplexes. Finally, we have also identified, within three previously known snoRNAs, novel antisense elements able to form a canonical duplex around ribose-methylated sites in rRNA, which further supports the conclusion that the duplex structure provides the 2'-O-methyltransferase with the appropriate site-specificity on the substrate.
Eukaryotic ribosomal RNAs are post-transcriptionally modified by methylation at the ribose sugar of specific nucleotides. This takes place in the nucleolus and involves a family of small nucleolar RNAs (snoRNAs) with long regions (10-21 nucleotides) complementary to rRNA sequences spanning the methylation site--a complementary snoRNA is required for methylation at a specific site. Here we show that altering the sequence of the snoRNA is sufficient to change the specificity of methylation. Mammalian cells transfected with a snoRNA engineered to be complementary to an arbitrary rRNA sequence direct the methylation of the predicted nucleotide in that sequence. We have further identified structural features, both of the guide and substrate RNA, required for methylation and have used these to design an exogenous transcript, devoid of rRNA sequence, that is site-specifically methylated when coexpressed with an appropriate guide snoRNA. Endogenous non-ribosomal RNA can thus be targeted, possibly providing a highly selective tool for the alteration of gene expression at the post-transcriptional level.
An RNA region associated with the donor substrate site, located at the base of the peptidyl transferase loop of 23 S rRNA, was subjected to a comprehensive single-site mutational study. Growth phenotypes of Escherichia coli cells were characterized on induction of synthesis of the mutated rRNAs and the mutated ribosomes were tested, selectively, for their capacity to generate peptide bonds under the conditions of the "fragment" assay. Most of the mutants exhibited dominant or recessive lethal growth phenotypes and, in general, defective growth correlated with low activities in peptide bond formation, although exceptions were observed with normal growth and low activities, and vice versa. All these phenotypes are consistent with defects occurring in the structure of the ribosomal donor site and/or the capacity of the donor substrate to enter or leave this site. A compensating base change approach was employed to test for Watson-Crick base-pairing interactions between the -CCA end of the P-site bound tRNA(Phe) and this region of the peptidyl-transferase loop. Single nucleotide substitutions were introduced into the -CCA end of tRNA(Phe) and the ability of the 3'-terminal pentanucleotide fragments to act as donor substrates was examined for ribosomes carrying the different mutated 23 S rRNAs. No evidence was found for the occurrence of Watson-Crick base-pairing interactions. However, the data are consistent with the formation of a Hoogsteen pair between the 3'-terminal adenosine base of the donor substrate and U2585 of the 23 S rRNA.
Previous experiments have shown that the phylogenetically conserved G2252 of 23 S rRNA forms a Watson-Crick base-pair with C74 of peptidyl-tRNA. In the studies presented here, site-directed mutations were introduced at two other conserved positions in 23 S rRNA, G2251 and U2585, that were previously implicated in interaction of the CCA acceptor end of tRNA with the 50 S subunit P site. The mutant 23 S rRNAs were characterized by determining (1) the in vivo phenotypes, (2) the ability of mutant ribosomes to bind tRNA oligonucleotide fragments in vitro, using footprinting with allele-specific primer extension and (3) the ability of mutant ribosomes to catalyze peptide bond formation using a chimeric reconstitution approach. Mutations at either position confer a dominant lethal phenotype when the mutant 23 S rRNA is coexpressed with the endogenous wild-type 23 S rRNA. Mutations at 2585 disrupt binding of the wild-type (CCA) tRNA oligonucleotide fragment and cause a modest decrease in the peptidyl transferase activity of reconstituted ribosomes. By contrast, mutations at 2251 abolish both binding of the wild-type (CCA) tRNA fragment and peptidyl transferase activity using the wild-type tRNA fragment. In neither case was the loss of binding or peptidyl transferase activity suppressed by mutations in the tRNA oligonucleotide fragment. Chemical modification analysis revealed that mutations at 2251 perturb the reactivity of bases 2584 to 2586, providing further evidence that the 2250 loop of 23 S rRNA interacts, either directly or indirectly, with the 2585 region in the central loop of domain V of 23 S rRNA.
The ribosome is a large multifunctional complex composed of both RNA and proteins. Biophysical methods are yielding low-resolution structures of the overall architecture of ribosomes, and high-resolution structures of individual proteins and segments of rRNA. Accumulating evidence suggests that the ribosomal RNAs play central roles in the critical ribosomal functions of tRNA selection and binding, translocation, and peptidyl transferase. Biochemical and genetic approaches have identified specific functional interactions involving conserved nucleotides in 16S and 23S rRNA. The results obtained by these quite different approaches have begun to converge and promise to yield an unprecedented view of the mechanism of translation in the coming years.
We have studied the effect of subunit association on the accessibility of nucleotides in 23S and 5S rRNA. Escherichia coli 50S subunits and 70S ribosomes were subjected to a combination of chemical probes and the sites of attack identified by primer extension. Since the ribose groups and all of the bases were probed, the present study provides a comprehensive map of the nucleotides that are likely to be involved in subunit-subunit interactions. Upon subunit association, the bases of 22 nucleotides and the ribose groups of more than 60 nucleotides are protected in 23S rRNA; no changes are seen in 5S rRNA. Interestingly, the bases of nucleotides A1866, A1891 and A1896, and G2505 become more reactive to chemical probes, indicating localized rearrangement of the structure of the 50S subunit upon association with the 30S subunit. Most of the protected nucleotides are located in four stem-loop structures around positions 715, 890, 1700, and 1920. In free 50S subunits, virtually all of the ribose groups in these four regions are strongly cleaved by hydroxyl radicals, suggesting that these stems protrude from the 50S subunit. When the 30S subunit is bound, most of the ribose groups in the 715, 890, 1700 and 1920 stem-loops are protected, as are many bases in and around the corresponding apical loops. Intriguingly, three of the protected regions of 23S rRNA are known to be linked via tertiary interactions to features of the peptidyl transferase center. Together with the juxtaposition of the subunit-protected regions of 16S rRNA with the small subunit tRNA binding sites, our findings suggest the existence of a communication pathway between the codon-anticodon binding sites of the 30S subunit with the peptidyl transferase center of the 50S subunit via rRNA-rRNA interactions.
In vitro transcripts of Bacillus stearothermophilus 23S rRNA can be reconstituted into catalytically active 50S ribosomal subunits with an efficiency only 3-4-fold lower than that of natural 23S rRNA. Thus, post-transcriptional modifications in 23S rRNA are not essential for the assembly or function of the 50S subunit of the ribosome. This reconstitution sytem has been used to characterize the peptidyl transferase activity of site-directed mutations in 23S rRNA at positions G2252, U2506, U2584, and A2602 (Escherichia coli numbering), demonstrating its potential for the analysis of the role played by 23S rRNA in the function of the 50S subunit of the ribosome.
In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA. Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (Psi) synthase. Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known Psi synthase, and shares the "KP" and "XLD" conserved sequence motifs found in the catalytic domains of three distinct families of known and putative Psi synthases. To gain additional evidence on the role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative Psi synthase domain of Cbf5p. Yeast strains expressing these mutated cbf5 genes in a cbf5Delta null background are viable at 25 degrees C but display pronounced cold- and heat-sensitive growth phenotypes. Most of the mutants contain reduced levels of Psi in rRNA at extreme temperatures. Substitution of alanine for an aspartic acid residue in the conserved XLD motif of Cbf5p (mutant cbf5D95A) abolishes in vivo pseudouridylation of rRNA. Some of the mutants are temperature sensitive both for growth and for formation of Psi in the rRNA. In most cases, the impaired growth phenotypes are not relieved by transcription of the rRNA from a polymerase II-driven promoter, indicating the absence of polymerase I-related transcriptional defects. There is little or no abnormal accumulation of pre-rRNAs in these mutants, although preferential inhibition of 18S rRNA synthesis is seen in mutant cbf5D95A, which lacks Psi in rRNA. A subset of mutations in the Psi synthase domain impairs association of the altered Cbf5p proteins with selected box H/ACA snoRNAs, suggesting that the functional catalytic domain is essential for that interaction. Our results provide additional evidence that Cbf5p is the Psi synthase component of box H/ACA snoRNPs and suggest that the pseudouridylation of rRNA, although not absolutely required for cell survival, is essential for the formation of fully functional ribosomes.
Most box C/D small nucleolar RNAs (snoRNAs) direct the formation of 2'-O-methylated nucleotides in ribosomal RNA and, apparently, other RNAs present in the nucleolar complex. Sites to be modified are selected by a long (>10-nt) antisense guide sequence in the snoRNA and a distance measurement from a box D or D' element that follows the snoRNA guide sequence. Modification of the substrate occurs in the region of complementarity, at a position five nucleotides upstream from box D/D'. Methylation can be targeted to novel sites by expressing a snoRNA with a new guide sequence. In some cases methylation impairs the growth rate of the cell, indicating that a functionally important nucleotide has been altered. With a view to harnessing snoRNA-directed methylation for functional mapping, we have developed a method for constructing libraries of snoRNA genes that, in principle, can introduce methylation point mutations into any rRNA segment of interest. The strategy and procedures are described here, and preliminary results are presented that show the feasibility of using this technology to probe a region of the yeast large subunit rRNA that includes the core of the peptidyltransferase center.
A universally conserved adenosine, A2451, within the ribosomal peptidyl transferase center has been proposed to act as a general acid-base catalyst during peptide bond formation. Evidence in support of this proposal came from pH-dependent dimethylsulfate (DMS) modification within Escherichia coli ribosomes. A2451 displayed reactivity consistent with an apparent acidity constant (pKa) near neutrality, though pH-dependent structural flexibility could not be rigorously excluded as an explanation for the enhanced reactivity at high pH. Here we present three independent lines of evidence in support of the alternative interpretation. First, A2451 in ribosomes from the archaebacteria Haloarcula marismortui displays an inverted pH profile that is inconsistent with proton-mediated base protection. Second, in ribosomes from the yeast Saccharomyces cerevisiae, C2452 rather than A2451 is modified in a pH-dependent manner. Third, within E. coli ribosomes, the position of A2451 modification (N1 or N3 imino group) was analyzed by testing for a Dimroth rearrangement of the N1-methylated base. The data are more consistent with DMS modification of the A2451 N1, a functional group that, according to the 50S ribosomal crystal structure, is solvent inaccessible without structural rearrangement. It therefore appears that pH-dependent DMS modification of A2451 does not provide evidence either for or against a general acid-base mechanism of protein synthesis. Instead the data suggest that there is pH-dependent conformational flexibility within the peptidyl transferase center, the exact nature and physiological relevance of which is not known.
Ribosomal RNAs (rRNAs) from all sources contain modified nucleosides, whose numbers range from a few in mitochondrial rRNA to more than 200 in the complete rRNAs of some higher eukaryotes. In eukaryotic rRNA the great majority of modified nucleosides are 2'-O-methylated nucleosides or pseudouridines. The locations of most of the 2'-O-methylated nucleosides in rRNA from some representative eukaryotes are known from studies whose aim was full characterization of rRNA methylation. More recently, and particularly in connection with the discovery of methylation guide RNAs, it is often required to check for the presence or absence of 2'-O-methyl nucleosides at specified locations within rRNA. Three methods that can be applied for such "local" objectives are reviewed. Two of the methods are based on primer extension by reverse transcriptase. They exploit, respectively, a tendency of 2'-O-methyl groups to impede reverse transcriptase at low dNTP concentrations, or the resistance of phosphodiester bonds adjacent to 2'-O-methyl groups to alkaline hydrolysis. Examples of these methods are summarized. Although the two methods are relatively straightforward, they suffer from various experimental limitations, as discussed. The third method is technically more sophisticated but is capable of overcoming the limitations of the first two methods. It is based on the resistance of a target 2'-O-methylated site to cleavage by RNase H when the site is hybridized to an appropriate chimeric oligonucleotide. An overview of the approaches and methods now available for the complete mapping of 2'-O-methyl groups in rRNA is presented.
Pseudouridines are found in virtually all ribosomal RNAs but their function is unknown. There are four to eight times more pseudouridines in eukaryotes than in eubacteria. Mapping 19 Haloarcula marismortui pseudouridines on the three-dimensional 50S subunit does not show clustering. In bacteria, specific enzymes choose the site of pseudouridine formation. In eukaryotes, and probably also in archaea, selection and modification is done by a guide RNA-protein complex. No unique specific role for ribosomal pseudouridines has been identified. We propose that pseudouridine's function is as a molecular glue to stabilize required RNA conformations that would otherwise be too flexible.
The development of three-dimensional maps of the modified nucleotides in the ribosomes of Escherichia coli and yeast has revealed that most (approximately 95% in E. coli and 60% in yeast) occur in functionally important regions. These include the peptidyl transferase centre, the A, P and E sites of tRNA- and mRNA binding, the polypeptide exit tunnel, and sites of subunit-subunit interaction. The correlations suggest that many ribosome functions benefit from nucleotide modification.
The ribosomal peptidyl transferase center is responsible for two fundamental reactions, peptide bond formation and nascent peptide release, during the elongation and termination phases of protein synthesis, respectively. We used in vitro genetics to investigate the functional importance of conserved 23S rRNA nucleotides located in the peptidyl transferase active site for transpeptidation and peptidyl-tRNA hydrolysis. While mutations at A2451, U2585, and C2063 (E. coli numbering) did not significantly affect either of the reactions, substitution of A2602 with C or its deletion abolished the ribosome ability to promote peptide release but had little effect on transpeptidation. This indicates that the mechanism of peptide release is distinct from that of peptide bond formation, with A2602 playing a critical role in peptide release during translation termination.
Crystal structures of tRNA mimics complexed with the large ribosomal subunit of Deinococcus radiodurans indicate that remote interactions determine the precise orientation of tRNA in the peptidyl-transferase center (PTC). The PTC tolerates various orientations of puromycin derivatives and its flexibility allows the conformational rearrangements required for peptide-bond formation. Sparsomycin binds to A2602 and alters the PTC conformation. H69, the intersubunit-bridge connecting the PTC and decoding site, may also participate in tRNA placement and translocation. A spiral rotation of the 3' end of the A-site tRNA around a 2-fold axis of symmetry identified within the PTC suggests a unified ribosomal machinery for peptide-bond formation, A-to-P-site translocation, and entrance of nascent proteins into the exit tunnel. Similar 2-fold related regions, detected in all known structures of large ribosomal subunits, indicate the universality of this mechanism.