-
[show abstract]
[hide abstract]
ABSTRACT: DNA microarrays have become a mainstream tool in experimental plant biology. The constant improvements in the technological platforms have enabled the development of the tiling DNA microarrays that cover the whole genome, which in turn catalyzed the wide variety of creative applications of such microarrays in the areas as diverse as global studies of genetic variation, DNA-binding proteins, DNA methylation, and chromatin and transcriptome dynamics. This chapter attempts to summarize such applications as well as discusses some technical and strategic issues that are particular to the use of tiling microarrays.
Methods in molecular biology (Clifton, N.J.) 02/2009; 553:39-56.
-
[show abstract]
[hide abstract]
ABSTRACT: A pervasive theme in development is that dynamic changes in gene expression drive developmental progression; yet in studies of gene expression, the general RNA decay pathways have historically played second fiddle to transcription. However, recent advances in this field have revealed a surprising degree of mRNA specificity for particular branches of these RNA decay pathways. General cytoplasmic mRNA decay typically initiates with deadenylation, following which the deadenylated mRNA can continue decay from the 3'-end through the action of the exosome, or it can undergo 5'-to-3' decay. Functional characterization of exosome subunits using inducible knock-outs uncovered a surprising complexity of molecular phenotypes and RNA substrates. Decay in the 5'-to-3' direction requires decapping, which is carried out by the decapping complex in Processing bodies (PBs). Recent analyses of decapping mutants have also revealed substrate specificity and roles in translational regulation. In addition, recent studies of specialized pathways such as nonsense-mediated decay and silencing reveal interactions with the general RNA decay pathways.
Current opinion in plant biology 12/2008; 12(1):96-102. · 10.33 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Errors in the mRNP biogenesis pathway can lead to retention of mRNA in discrete, transcription-site-proximal foci. This RNA remains tethered adjacent to the transcription site long after transcriptional shutoff. Here we identify Sus1, Thp1, and Sac3 as factors required for the persistent tethering of such foci (dots) to their cognate genes. We also show that the prolonged association of previously activated GAL genes with the nuclear periphery after transcriptional shutoff is similarly dependent on the Sac3-Thp1-Sus1-Cdc31 complex. We suggest that the complex associates with nuclear mRNP and that mRNP properties influence the association of dot-confined mRNA with its gene of origin as well as the post-transcriptional retention of the cognate gene at the nuclear periphery. These findings indicate a coupling between the mRNA-to-gene and gene-to-nuclear periphery tethering. Taken together with other recent findings, these observations also highlight the importance of nuclear mRNP to the mobilization of active genes to the nuclear rim.
RNA 02/2008; 14(1):66-77. · 5.09 Impact Factor
-
Julia A Chekanova,
Brian D Gregory,
Sergei V Reverdatto,
Huaming Chen,
Ravi Kumar,
Tanya Hooker,
Junshi Yazaki,
Pinghua Li,
Nikolai Skiba,
Qian Peng,
Jose Alonso,
Vladimir Brukhin,
Ueli Grossniklaus,
Joseph R Ecker, Dmitry A Belostotsky
[show abstract]
[hide abstract]
ABSTRACT: The exosome complex plays a central and essential role in RNA metabolism. However, comprehensive studies of exosome substrates and functional analyses of its subunits are lacking. Here, we demonstrate that as opposed to yeast and metazoans the plant exosome core possesses an unanticipated functional plasticity and present a genome-wide atlas of Arabidopsis exosome targets. Additionally, our study provides evidence for widespread polyadenylation- and exosome-mediated RNA quality control in plants, reveals unexpected aspects of stable structural RNA metabolism, and uncovers numerous novel exosome substrates. These include a select subset of mRNAs, miRNA processing intermediates, and hundreds of noncoding RNAs, the vast majority of which have not been previously described and belong to a layer of the transcriptome that can only be visualized upon inhibition of exosome activity. These first genome-wide maps of exosome substrates will aid in illuminating new fundamental components and regulatory mechanisms of eukaryotic transcriptomes.
Cell 01/2008; 131(7):1340-53. · 32.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Multiple studies indicate that mRNA processing defects cause mRNAs to accumulate in discrete nuclear foci or dots, in mammalian cells as well as yeast. To investigate this phenomenon, we have studied a series of GAL reporter constructs integrated into the yeast genome adjacent to an array of TetR-GFP-bound TetO sites. mRNA within dots is predominantly post-transcriptional, and dots are adjacent to but distinct from their transcription site. These reporter genes also localize to the nuclear periphery upon gene induction, like their endogenous GAL counterparts. Surprisingly, this peripheral localization persists long after transcriptional shutoff, and there is a comparable persistence of the RNA in the dots. Moreover, dot disappearance and gene delocalization from the nuclear periphery occur with similar kinetics after transcriptional shutoff. Both kinetics depend in turn on reporter gene 3'-end formation signals. Our experiments indicate that gene association with the nuclear periphery does not require ongoing transcription and suggest that the mRNPs within dots may make a major contribution to the gene-nuclear periphery tether.
The EMBO Journal 10/2006; 25(18):4253-62. · 9.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Although initially believed to act exclusively as translational repressors, microRNAs (miRNAs) are now known to target complementary messenger RNA (mRNA) transcripts for either translational repression or cleavage via the RNA-induced silencing complex (RISC) ([1], reviewed in ref. 2). The current model postulates that mature miRNAs are incorporated into the RISC, bind target mRNAs based on complementarity, and guide cleavage of mRNA targets with perfect or nearly perfect complementarity and translational repression of targets with lower complementarity (2). The translational repression mechanism of miRNA-mediated gene regulation, which is common in animals but also exists in plants, is not well understood mechanistically. Conversely, miRNA-directed mRNA cleavage by RISC is common in plants, but also occurs in animals (3). This chapter focuses on the mRNA cleavage by miRNA-programmed RISC, and, specifically, on characterizing the products of such cleavage.
Methods in molecular biology (Clifton, N.J.) 02/2006; 342:73-85.
-
[show abstract]
[hide abstract]
ABSTRACT: The extensive mechanistic and regulatory interconnections between the various events of mRNA biogenesis are now recognized as a fundamental principle of eukaryotic gene expression, yet the specific details of the coupling between the various steps of mRNA biogenesis do differ, and sometimes dramatically, between the different kingdoms. In this review, we emphasize examples where plants must differ in this respect from other eukaryotes, and highlight a recurring trend of recruiting the conserved, versatile functional modules, which have evolved to support the general mRNA biogenesis reactions, for plant-specific functions. We also argue that elucidating the inner workings of the plant 'mRNA factory' is essential for accomplishing the ambitious goal of building the 'virtual plant'.
Trends in Plant Science 08/2005; 10(7):347-53. · 11.05 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Deadenylation of mRNA is often the first and rate-limiting step in mRNA decay. PARN, a poly(A)-specific 3' --> 5' ribonuclease which is conserved in many eukaryotes, has been proposed to be primarily responsible for such a reaction, yet the importance of the PARN function at the whole-organism level has not been demonstrated in any species. Here, we show that mRNA deadenylation by PARN is essential for viability in higher plants (Arabidopsis thaliana). Yet, this essential requirement for the PARN function is not universal across the phylogenetic spectrum, because PARN is dispensable in Fungi (Schizosaccharomyces pombe), and can be at least severely downregulated without any obvious consequences in Metazoa (Caenorhabditis elegans). Development of the Arabidopsis embryos lacking PARN (AtPARN), as well as of those expressing an enzymatically inactive protein, was markedly retarded, and ultimately culminated in an arrest at the bent-cotyledon stage. Importantly, only some, rather than all, embryo-specific transcripts were hyperadenylated in the mutant embryos, suggesting that preferential deadenylation of a specific select subset of mRNAs, rather than a general deadenylation of the whole mRNA population, by AtPARN is indispensable for embryogenesis in Arabidopsis. These findings indicate a unique, nonredundant role of AtPARN among the multiple plant deadenylases.
RNA 08/2004; 10(8):1200-14. · 5.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Eukaryotic poly(A) binding protein (PABP) is a ubiquitous, essential cellular factor with well-characterized roles in translational initiation and mRNA turnover. In addition, there exists genetic and biochemical evidence that PABP has an important nuclear function. Expression of PABP from Arabidopsis thaliana, PAB3, rescues an otherwise lethal phenotype of the yeast pab1Delta mutant, but it neither restores the poly(A) dependent stimulation of translation, nor protects the mRNA 5' cap from premature removal. In contrast, the plant PABP partially corrects the temporal lag that occurs prior to the entry of mRNA into the decay pathway in the yeast strains lacking Pab1p. Here, we examine the nature of this lag-correction function. We show that PABP (both PAB3 and the endogenous yeast Pab1p) act on the target mRNA via physically binding to it, to effect the lag correction. Furthermore, substituting PAB3 for the yeast Pab1p caused synthetic lethality with rna15-2 and gle2-1, alleles of the genes that encode a component of the nuclear pre-mRNA cleavage factor I, and a factor associated with the nuclear pore complex, respectively. PAB3 was present physically in the nucleus in the complemented yeast strain and was able to partially restore the poly(A) tail length control during polyadenylation in vitro, in a poly(A) nuclease (PAN)-dependent manner. Importantly, PAB3 in yeast also promoted the rate of entry of mRNA into the translated pool, rescued the conditional lethality, and alleviated the mRNA export defect of the nab2-1 mutant when overexpressed. We propose that eukaryotic PABPs have an evolutionarily conserved function in facilitating mRNA biogenesis and export.
RNA 01/2004; 9(12):1476-90. · 5.09 Impact Factor
-
Dmitry A Belostotsky
[show abstract]
[hide abstract]
ABSTRACT: Eukaryotic poly(A)-binding protein (PABP) is a ubiquitous, essential factor involved in mRNA biogenesis, translation, and turnover. Most eukaryotes examined have only one or a few PABPs. In contrast, eight expressed PABP genes are present in Arabidopsis thaliana. These genes fall into three distinct classes, based on highly concordant results of (i) phylogenetic analysis of the amino acid sequences of the encoded proteins, (ii) analysis of the intron number and placement, and (iii) surveys of gene expression patterns. Representatives of each of the three classes also exist in the rice genome, suggesting that the diversification of the plant PABP genes has occurred prior to the split of monocots and dicots >or=200 MYA. Experiments with the recombinant PAB3 protein suggest the possibility of a negative feedback regulation, as well as of cross-regulation between the Arabidopsis PABPs that belong to different classes but are simultaneously expressed in the same cell type. Such a high complexity of the plant PABPs might enable a very fine regulation of organismal growth and development at the post-transcriptional level, compared with PABPs of other eukaryotes.
Genetics 01/2003; 163(1):311-9. · 4.01 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The exosome, an evolutionarily conserved complex of multiple 3'-->5' exoribonucleases, is responsible for a variety of RNA processing and degradation events in eukaryotes. In this report Arabidopsis thaliana AtRrp4p is shown to be an active 3'-->5' exonuclease that requires a free 3'-hydroxyl and degrades RNA hydrolytically and distributively, releasing nucleoside 5'-monophosphate products. AtRrp4p behaves as an approximately 500 kDa species during sedimentation through a 10-30% glycerol gradient, co-migrating with AtRrp41p, another exosome subunit, and it interacts in vitro with AtRrp41p, suggesting that it is also present in the plant cell as a subunit of the exosome. We found that, in addition to a previously reported S1-type RNA-binding domain, members of the Rrp4p family of proteins contain a KH-type RNA-binding domain in the C-terminal half and show that either domain alone can bind RNA. However, only the full-length protein is capable of degrading RNA and interacting with AtRrp41p.
Nucleic Acids Research 03/2002; 30(3):695-700. · 8.03 Impact Factor
