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ABSTRACT: Eukaryotic gene expression is orchestrated on a genome-wide scale through several post-transcriptional mechanisms. Of these, alternative pre-mRNA splicing expands the proteome diversity and modulates mRNA stability through downstream RNA quality control (QC) pathways including nonsense-mediated decay (NMD) of mRNAs containing premature termination codons and nuclear retention and elimination (NRE) of intron-containing transcripts. Although originally identified as mechanisms for eliminating aberrant transcripts, a growing body of evidence suggests that NMD and NRE coupled with deliberate changes in pre-mRNA splicing patterns are also used in a number of biological contexts for deterministic control of gene expression. Here we review recent studies elucidating molecular mechanisms and biological significance of these gene regulation strategies with a specific focus on their roles in nervous system development and physiology.
Molecular and Cellular Neuroscience 01/2013; · 3.66 Impact Factor
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ABSTRACT: Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3'-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context.
Genes & development 06/2012; 26(11):1209-23. · 12.08 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are 19- to 25-nt-long non-coding RNAs that regulate gene expression by base-pairing with target mRNAs and reducing their stability or translational efficiency. Mammalian miRNAs function in association with four closely related Argonaute proteins, AGO1-4. All four proteins contain the PAZ and the MID domains interacting with the miRNA 3' and 5' termini, respectively, as well as the PIWI domain comprising an mRNA 'slicing' activity in the case of AGO2 but not AGO1, AGO3 and AGO4. However, the slicing mode of the miRNA-programmed AGO2 is rarely realized in vivo and the four Argonautes are thought to play largely overlapping roles in the mammalian miRNA pathway. Here, we show that the average length of many miRNAs is diminished during nervous system development as a result of progressive shortening of the miRNA 3' ends. We link this modification with an increase in the fractional abundance of Ago2 in the adult brain and identify a specific structural motif within the PAZ domain that enables efficient trimming of miRNAs associated with this but not the other three Argonautes. Taken together, our data suggest that mammalian Argonautes may define the length and possibly biological activity of mature mammalian miRNAs in a developmentally controlled manner.
Nucleic Acids Research 04/2012; 40(14):6808-20. · 8.03 Impact Factor
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ABSTRACT: RNA-binding protein HuR modulates the stability and translational efficiency of messenger RNAs (mRNAs) encoding essential components of the cellular proliferation, growth and survival pathways. Consistent with these functions, HuR levels are often elevated in cancer cells and reduced in senescent and quiescent cells. However, the molecular mechanisms that control HuR expression are poorly understood. Here we show that HuR protein autoregulates its abundance through a negative feedback loop that involves interaction of the nuclear HuR protein with a GU-rich element (GRE) overlapping with the HuR major polyadenylation signal (PAS2). An increase in the cellular HuR protein levels stimulates the expression of long HuR mRNA species containing an AU-rich element (ARE) that destabilizes the mRNAs and thus reduces the protein production output. The PAS2 read-through occurs due to a reduced recruitment of the CstF-64 subunit of the pre-mRNA cleavage stimulation factor in the presence of the GRE-bound HuR. We propose that this mechanism maintains HuR homeostasis in proliferating cells. Since only the nuclear HuR is expected to contribute to the auto-regulation, our model may explain the longstanding observation that the increase in the total HuR expression in cancer cells often correlates with the accumulation of its substantial fraction in the cytoplasm.
Nucleic Acids Research 09/2011; 40(2):787-800. · 8.03 Impact Factor
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ABSTRACT: Sequence-specific gene silencing by short hairpin (sh) RNAs has recently emerged as an indispensable tool for understanding gene function and a promising avenue for drug discovery. However, a wider biomedical use of this approach is hindered by the lack of straightforward methods for achieving uniform expression of shRNAs in mammalian cell cultures. Here we report a high-efficiency and low-background (HILO) recombination-mediated cassette exchange (RMCE) technology that yields virtually homogeneous cell pools containing doxycycline-inducible shRNA elements in a matter of days and with minimal efforts. To ensure immediate utility of this approach for a wider research community, we modified 11 commonly used human (A549, HT1080, HEK293T, HeLa, HeLa-S3, and U2OS) and mouse (CAD, L929, N2a, NIH 3T3, and P19) cell lines to be compatible with the HILO-RMCE process. Because of its technical simplicity and cost efficiency, the technology will be advantageous for both low- and high-throughput shRNA experiments. We also provide evidence that HILO-RMCE will facilitate a wider range of molecular and cell biology applications by allowing one to rapidly engineer cell populations expressing essentially any transgene of interest.
Proceedings of the National Academy of Sciences 08/2011; 108(31):12799-804. · 9.68 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are approximately 22-nucleotide-long noncoding RNAs that normally function by suppressing translation and destabilizing messenger RNAs bearing complementary target sequences. Some miRNAs are expressed in a cell- or tissue-specific manner and may contribute to the establishment and/or maintenance of cellular identity. Recent studies indicate that tissue-specific miRNAs may function at multiple hierarchical levels of gene regulatory networks, from targeting hundreds of effector genes incompatible with the differentiated state to controlling the levels of global regulators of transcription and alternative pre-mRNA splicing. This multilevel regulation may allow individual miRNAs to profoundly affect the gene expression program of differentiated cells.
Science 04/2008; 319(5871):1789-90. · 31.20 Impact Factor
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ABSTRACT: Both microRNAs and alternative pre-mRNA splicing have been implicated in the development of the nervous system (NS), but functional interactions between these two pathways are poorly understood. We demonstrate that the neuron-specific microRNA miR-124 directly targets PTBP1 (PTB/hnRNP I) mRNA, which encodes a global repressor of alternative pre-mRNA splicing in nonneuronal cells. Among the targets of PTBP1 is a critical cassette exon in the pre-mRNA of PTBP2 (nPTB/brPTB/PTBLP), an NS-enriched PTBP1 homolog. When this exon is skipped, PTBP2 mRNA is subject to nonsense-mediated decay (NMD). During neuronal differentiation, miR-124 reduces PTBP1 levels, leading to the accumulation of correctly spliced PTBP2 mRNA and a dramatic increase in PTBP2 protein. These events culminate in the transition from non-NS to NS-specific alternative splicing patterns. We also present evidence that miR-124 plays a key role in the differentiation of progenitor cells to mature neurons. Thus, miR-124 promotes NS development, at least in part by regulating an intricate network of NS-specific alternative splicing.
Molecular Cell 09/2007; 27(3):435-48. · 14.18 Impact Factor
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ABSTRACT: RNA silencing refers to a group of RNA-induced gene-silencing mechanisms that developed early in the eukaryotic lineage, probably for defence against pathogens and regulation of gene expression. In plants, protozoa, fungi, and nematodes, but apparently not insects and vertebrates, it involves a cell-encoded RNA-dependent RNA polymerase (cRdRP) that produces double-stranded RNA triggers from aberrant single-stranded RNA. We report the 2.3-A resolution crystal structure of QDE-1, a cRdRP from Neurospora crassa, and find that it forms a relatively compact dimeric molecule, each subunit of which comprises several domains with, at its core, a catalytic apparatus and protein fold strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription. This evolutionary link between the two enzyme types suggests that aspects of RNA silencing in some organisms may recapitulate transcription/replication pathways functioning in the ancient RNA-based world.
PLoS Biology 01/2007; 4(12):e434. · 11.45 Impact Factor
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ABSTRACT: The RNA-dependent RNA polymerase, QDE-1, is a component of the RNA silencing pathway in Neurospora crassa. The enzymatically active carboxy-terminal fragment QDE-1 DeltaN has been expressed in Saccharomyces cerevisiae in the presence and absence of selenomethionine (SeMet). The level of SeMet incorporation was estimated by mass spectrometry to be approximately 98%. Both native and SeMet proteins were crystallized in space group P2(1) with unit cell parameters a=101.2, b=122.5, c=114.4A, beta=108.9 degrees , and 2 molecules per asymmetric unit. The native and SeMet crystals diffract to 2.3 and 3.2A, respectively, the latter are suitable for MAD structure determination.
Journal of Structural Biology 02/2005; 149(1):111-5. · 3.41 Impact Factor
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ABSTRACT: This review summarizes the combined insights from recent structural and functional studies of viral RNA-dependent RNA polymerases (RdRPs) with the primary focus on the mechanisms of initiation of RNA synthesis. Replication of RNA viruses has traditionally been approached using a combination of biochemical and genetic methods. Recently, high-resolution structures of six viral RdRPs have been determined. For three RdRPs, enzyme complexes with metal ions, single-stranded RNA and/or nucleoside triphosphates have also been solved. These advances have expanded our understanding of the molecular mechanisms of viral RNA synthesis and facilitated further RdRP studies by informed site-directed mutagenesis. What transpires is that the basic polymerase right hand shape provides the correct geometrical arrangement of substrate molecules and metal ions at the active site for the nucleotidyl transfer catalysis, while distinct structural elements have evolved in the different systems to ensure efficient initiation of RNA synthesis. These elements feed the template, NTPs and ions into the catalytic cavity, correctly position the template 3' terminus, transfer the products out of the catalytic site and orchestrate the transition from initiation to elongation.
Journal of General Virology 06/2004; 85(Pt 5):1077-93. · 3.36 Impact Factor
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ABSTRACT: Genome replication and transcription of riboviruses are catalyzed by an RNA-dependent RNA polymerase (RdRP). RdRPs are normally associated with other virus- or/and host-encoded proteins that modulate RNA polymerization activity and template specificity. The polymerase complex of double-stranded dsRNA viruses is a large icosahedral particle (inner core) containing RdRP as a minor constituent. In phi6 and other dsRNA bacteriophages from the Cystoviridae family, the inner core is composed of four virus-specific proteins. Of these, protein P2, or Pol subunit, has been tentatively identified as RdRP by sequence comparisons, but the role of this protein in viral RNA synthesis has not been studied until recently. Here, we overview the work on the Pol subunits of phi6 and related viruses from the standpoints of function, structure and evolution.
Virus Research 05/2004; 101(1):45-55. · 2.94 Impact Factor
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ABSTRACT: The RNA-dependent RNA polymerase of bacteriophage phi6 transcribes mRNA from the three segments of the dsRNA viral genome. We have cocrystallized RNA oligonucleotides with the polymerase, revealing the mode of binding of RNA templates. This binding is somewhat different from that previously seen for DNA oligomers, leading to additional RNA-protein hydrogen bonds, consistent with a preference for RNA. Activation of the RNA/polymerase complex by the addition of substrate and Mg2+ initiates a single round of reaction within the crystal to form a dead-end complex that partially collapses within the enzyme active site. By replacing Mg2+ with Ca2+, we have been able to capture the inhibited complex which shows distortion that explains the structural basis for the inhibition of such polymerases by Ca2+.
Structure 03/2004; 12(2):307-16. · 6.35 Impact Factor
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ABSTRACT: RNA viruses are remarkably adaptable to changing environments. This is medically important because it enables pathogenic viruses to escape the immune response and chemotherapy and is of considerable theoretical interest since it allows the investigation of evolutionary processes within convenient time scales. A number of earlier studies have addressed the dynamics of adapting RNA virus populations. However, it has been difficult to monitor the trajectory of molecular changes in RNA genomes in response to selective pressures. To address the problem, we developed a novel in vitro evolution system based on a recombinant double-stranded RNA bacteriophage, phi 6, containing a beta-lactamase (bla) gene marker. Carrier-state bacterial cells are resistant to ampicillin, and after several passages, they become resistant to high concentrations of another beta-lactam antibiotic, cefotaxime, due to mutations in the virus-borne bla gene. We monitored the changes in bla cDNAs induced by cefotaxime selection and observed an initial explosion in sequence variants with multiple mutations throughout the gene. After four passages, a stable, homogeneous population of bla sequences containing three specific nonsynonymous mutations was established. Of these, two mutations (E104K and G238S) have been previously reported for beta-lactamases from cefotaxime-resistant bacterial isolates. These results extend our understanding of the molecular mechanisms of viral adaptation and also demonstrate the possibility of using an RNA virus as a vehicle for directed evolution of heterologous proteins.
Journal of Virology 03/2004; 78(4):2114-20. · 5.40 Impact Factor
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ABSTRACT: The packaging of genomic RNA in members of the Cystoviridae is performed by P4, a hexameric protein with NTPase activity. Across family members such as Phi6, Phi8 and Phi13, the P4 proteins show low levels of sequence identity, but presumably have similar atomic structures. Initial structure-determination efforts for P4 from Phi6 and Phi8 were hampered by difficulties in obtaining crystals that gave ordered diffraction. Diffraction from crystals of full-length P4 showed a variety of disorder and anisotropy. Subsequently, crystals of Phi13 P4 were obtained which yielded well ordered diffraction to 1.7 A. Comparison of the packing arrangements of P4 hexamers in different crystal forms and analysis of the disorder provides insights into the flexibility of this family of proteins, which might be an integral part of their biological function.
Acta Crystallographica Section D Biological Crystallography 01/2004; 59(Pt 12):2337-41. · 12.62 Impact Factor
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ABSTRACT: Genomes of complex viruses have been demonstrated, in many cases, to be packaged into preformed empty capsids (procapsids). This reaction is performed by molecular motors translocating nucleic acid against the concentration gradient at the expense of NTP hydrolysis. At present, the molecular mechanisms of packaging remain elusive due to the complex nature of packaging motors. In the case of the double-stranded RNA bacteriophage phi 6 from the Cystoviridae family, packaging of single-stranded genomic precursors requires a hexameric NTPase, P4. In the present study, the purified P4 proteins from two other cystoviruses, phi 8 and phi 13, were characterized and compared with phi 6 P4. All three proteins are hexameric, single-stranded RNA-stimulated NTPases with alpha/beta folds. Using a direct motor assay, we found that phi 8 and phi 13 P4 hexamers translocate 5' to 3' along ssRNA, whereas the analogous activity of phi 6 P4 requires association with the procapsid. This difference is explained by the intrinsically high affinity of phi 8 and phi 13 P4s for nucleic acids. The unidirectional translocation results in RNA helicase activity. Thus, P4 proteins of Cystoviridae exhibit extensive similarity to hexameric helicases and are simple models for studying viral packaging motor mechanisms.
Journal of Biological Chemistry 12/2003; 278(48):48084-91. · 4.77 Impact Factor
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ABSTRACT: To continue the molecular characterization of RNA-dependent RNA polymerases of dsRNA bacteriophages (Cystoviridae), we purified and biochemically characterized the wild-type (wt) and a temperature-sensitive (ts) point mutant of the polymerase subunit (Pol) from bacteriophage phi12. Interestingly, initiation by both wt and the ts phi12 Pol was notably more sensitive to increased temperatures than the elongation step, the absolute value of the nonpermissive temperature being lower for the ts enzyme. Experiments with the Pol subunit of related cystovirus phi6 revealed a similar differential sensitivity of the initiation and elongation steps. This is consistent with the previous result showing that de novo initiation by RdRp from dengue virus is inhibited at elevated temperatures, whereas the elongation phase is relatively thermostable. Overall, these data suggest that de novo RNA-dependent RNA synthesis in many viral systems includes a specialized thermolabile state of the RdRp initiation complex.
Virology 10/2003; 314(2):706-15. · 3.35 Impact Factor
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ABSTRACT: In most double-stranded RNA (dsRNA) viruses, RNA transcription occurs inside a polymerase (Pol) complex particle, which contains an RNA-dependent RNA Pol subunit as a minor component. Only plus- but not minus-sense copies of genomic segments are produced during this reaction. In the case of phi6, a dsRNA bacteriophage from the Cystoviridae family, isolated Pol synthesizes predominantly plus strands using virus-specific dsRNAs in vitro, thus suggesting that Pol template preferences determine the transcriptional polarity. Here, we dissect transcription reactions catalyzed by Pol complexes and Pol subunits of two other cystoviruses, phi8 and phi13. While both Pol complexes synthesize exclusively plus strands over a wide range of conditions, isolated Pol subunits can be stimulated by Mn(2+) to produce minus-sense copies on phi13 dsRNA templates. Importantly, all three Pol subunits become more prone to the native-like plus-strand synthesis when the dsRNA templates (including phi13 dsRNA) are activated by denaturation before the reaction. Based on these and earlier observations, we propose a model of transcriptional polarity in Cystoviridae controlled on two independent levels: Pol affinity to plus-strand initiation sites and accessibility of these sites to the Pol in a single-stranded form.
Journal of Virology 02/2003; 77(2):1195-203. · 5.40 Impact Factor
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ABSTRACT: Recent genetic data suggest that proteins homologous to a plant RNA-dependent RNA polymerase (RdRP) play a central role in posttranscriptional gene silencing (PTGS) in many organisms. We show here that purified recombinant protein QDE-1, a genetic component of PTGS ("quelling") in the fungus Neurospora crassa, possesses RNA polymerase activity in vitro. The full-length enzyme and its enzymatically active C-terminal fragment perform two different reactions on single-stranded RNA templates, synthesizing either extensive RNA chains that form template-length duplexes or approximately 9-21-mer complementary RNA oligonucleotides scattered along the entire template. QDE-1 supports both de novo and primer-dependent initiation mechanisms. These results suggest that several distinct activities of cell-encoded RdRPs can be employed for efficient PTGS in vivo.
Molecular Cell 01/2003; 10(6):1417-27. · 14.18 Impact Factor
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ABSTRACT: Like most RNA polymerases, the polymerase of double-strand RNA bacteriophage phi6 (phi6pol) is capable of primer-independent initiation. Based on the recently solved phi6pol initiation complex structure, a four-amino acid-long loop (amino acids 630-633) has been suggested to stabilize the first two incoming NTPs through stacking interactions with tyrosine, Tyr(630). A similar loop is also present in the hepatitis C virus polymerase, another enzyme capable of de novo initiation. Here, we use a series of phi6pol mutants to address the role of this element. As predicted, mutants at the Tyr(630) position are inefficient in initiation de novo. Unexpectedly, when the loop is disordered by changing Tyr(630)-Lys(631)-Trp(632) to GSG, phi6pol becomes a primer-dependent enzyme, either extending complementary oligonucleotide or, when the template 3' terminus can adopt a hairpin-like conformation, utilizing a "copy-back" initiation mechanism. In contrast to the wild-type phi6pol, the GSG mutant does not require high GTP concentration for its optimal activity. These findings suggest a general model for the initiation of de novo RNA synthesis.
Journal of Biological Chemistry 06/2002; 277(19):17117-24. · 4.77 Impact Factor
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ABSTRACT: Like most RNA polymerases, the polymerase of double-strand RNA bacteriophage φ6 (φ6pol) is capable of primer-independent initiation.
Based on the recently solved φ6pol initiation complex structure, a four-amino acid-long loop (amino acids 630–633) has been
suggested to stabilize the first two incoming NTPs through stacking interactions with tyrosine, Tyr630. A similar loop is also present in the hepatitis C virus polymerase, another enzyme capable of de novo initiation. Here, we use a series of φ6pol mutants to address the role of this element. As predicted, mutants at the Tyr630 position are inefficient in initiation de novo. Unexpectedly, when the loop is disordered by changing Tyr630-Lys631-Trp632 to GSG, φ6pol becomes a primer-dependent enzyme, either extending complementary oligonucleotide or, when the template 3′
terminus can adopt a hairpin-like conformation, utilizing a “copy-back” initiation mechanism. In contrast to the wild-type
φ6pol, the GSG mutant does not require high GTP concentration for its optimal activity. These findings suggest a general model
for the initiation ofde novo RNA synthesis.
Journal of Biological Chemistry 05/2002; 277(19):17117-17124. · 4.77 Impact Factor
