15 research outputs found
Structure of the Rna15 RRM–RNA complex reveals the molecular basis of GU specificity in transcriptional 3′-end processing factors
Rna15 is a core subunit of cleavage factor IA (CFIA), an essential transcriptional 3′-end processing factor from Saccharomyces cerevisiae. CFIA is required for polyA site selection/cleavage targeting RNA sequences that surround polyadenylation sites in the 3′-UTR of RNA polymerase-II transcripts. RNA recognition by CFIA is mediated by an RNA recognition motif (RRM) contained in the Rna15 subunit of the complex. We show here that Rna15 has a strong and unexpected preference for GU containing RNAs and reveal the molecular basis for a base selectivity mechanism that accommodates G or U but discriminates against C and A bases. This mode of base selectivity is rather different to that observed in other RRM-RNA structures and is structurally conserved in CstF64, the mammalian counterpart of Rna15. Our observations provide evidence for a highly conserved mechanism of base recognition amongst the 3′-end processing complexes that interact with the U-rich or U/G-rich elements at 3′-end cleavage/polyadenylation sites
Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to Xylella fastidiosa infection
<p>Abstract</p> <p>Background</p> <p>Plant cytochrome P450 monooxygenases (CYP) mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to <it>Xylella fastidiosa </it>(<it>Xf</it>) infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase <it>CYP736B </it>gene in both disease resistant and susceptible grapevines.</p> <p>Results</p> <p>Cloning of genomic DNA and cDNA revealed that the <it>CYP736B </it>gene was composed of two exons and one intron with GT as a donor site and AG as an acceptor site. <it>CYP736B </it>transcript was up-regulated in PD-resistant plants and down-regulated in PD-susceptible plants 6 weeks after <it>Xf </it>inoculation. However, <it>CYP736B </it>expression was very low in stem tissues at all evaluated time points. 5'RACE and 3'RACE sequence analyses revealed that there were three candidate transcription start sites (TSS) in the upstream region and three candidate polyadenylation (PolyA) sites in the downstream region of <it>CYP736B</it>. Usage frequencies of each transcription initiation site and each polyadenylation site varied depending on plant genotype, developmental stage, tissue, and treatment. These results demonstrate that expression of <it>CYP736B </it>is regulated developmentally and in response to <it>Xf </it>infection at both transcriptional and post-transcriptional levels. Multiple transcription start and polyadenylation sites contribute to regulation of <it>CYP736B </it>expression.</p> <p>Conclusions</p> <p>This report provides evidence that the cytochrome P450 monooxygenase <it>CYP736B </it>gene is involved in defense response at a specific stage of <it>Xf </it>infection in grapevines; multiple transcription initiation and polyadenylation sites exist for <it>CYP736B </it>in grapevine; and coordinative and selective use of transcription initiation and polyadenylation sites play an important role in regulation of <it>CYP736B </it>expression during growth, development and response to <it>Xf </it>infection.</p
Sequence and position requirements for uridylate-rich downstream elements of polyadenylation signals.
We have defined the positional and sequence requirements of U-rich downstream elements using a simian virus 40 late polyadenylation signal containing a substituted downstream region. A UUUUU element will significantly increase the efficiency of 3' end processing when placed between 6 and 25 bases downstream from the cleavage site. Positions in this interval closer than 15 bases from the cleavage site, however, were noticeably less efficient. Placement of the UUUUU element between +20 and +25 caused a partial shift in cleavage site usage to a CA motif at +4. Mutational analysis indicated that the sequence requirements at individual positions of the UUUUU element were somewhat flexible. Changing more than one base of the UUUUU sequence, however, severely diminished the ability of the element to mediate efficient 3' end processing. Finally, although hnRNP C proteins specifically interact with U-rich sequences, this protein--RNA interaction is not required for efficient in vitro polyadenylation
Regulation and Function of the Dscam1 Extended 3’ UTR mRNA
More than 50% of genes in species from Drosophila to human undergo alternative polyadenylation (APA). It has been shown that neuronal tissues are highly enriched for usage of transcripts with extended alternative 3’ untranslated regions, which are products of APA. Specific function of these longer 3’ UTR transcripts are largely unknown; however, because the 3’ UTR can regulate transcript stability, translational efficiency, and localization, it is plausible that 3’ UTR switching in nervous tissues may serve important biological functions. We chose to study the extended 3’ UTR of Dscam1 as it is one of several axon guidance genes that express an extended 3’ UTR mRNA isoform. We created a Dscam1 extended 3’ UTR mutant using the relatively new CRISPR/CAS9 system. We show that the Dscam1 extended 3’ UTR loss of function mutants have severe deficiencies in locomotor activity and display an increased mortality rate. We have identified that in S2 cells the RNA binding protein ELAV promotes use of the extended 3’ UTR of Dscam1 and also show that ELAV is capable of binding at least two putative binding sites located on the 3’ UTR of Dscam1. Elucidating the important function of these extended 3’ UTRs will benefit not only our knowledge of axon guidance, but may reveal the function of a pervasive neural phenomenon
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Structural and Biochemical Characterizations of the Symplekin-Ssu72-CTD Complex in Pre-mRNA 3' end Processing
RNA polymerase II (RNAP II) transcribes essentially all messenger RNAs (mRNAs) in eukaryotes. The C-terminal domain (CTD) of its largest subunit contains consensus heptad repeats Y₁S₂P₃T₄S₅P₆S₇. Dynamic post-translational modifications of the CTD regulate RNAP II transcriptional activity and also facilitate transcription-coupled RNA processing events. One important mark is phosphorylation at Ser5 position, whose level peaks during transcription initiation but gradually diminishes toward the 3' end of genes. Ssu72 is a known CTD pSer5 phosphatase. Recent studies identified a binding partner of Ssu72, symplekin, which is an essential scaffold protein in pre-mRNA 3' end processing. Little is known about the molecular function of symplekin and neither do we understand how the symplekin-Ssu72 interaction couples pre-mRNA 3' processing to transcription. We first determined the crystal structure of the symplekin-Ssu72-CTD phosphopeptide complex. The N-terminal domain of symplekin embraces Ssu72 with its HEAT-repeat motif, serving as a typical molecular scaffold. Strikingly, the CTD phosphopeptide bound to the active site of Ssu72 has the peptide bond between pSer5 and Pro6 in the cis configuration, distinct from all known CTD conformations, which were exclusively in trans. While it was generally believed that only the trans peptide bond is recognized by proline-directed serine/threonine phosphatases or kinases, our discovery demonstrates for the first time that Ssu72 targets the energetically less-favorable cis peptide bond. In addition, we found that the binding of symplekin and also the presence of a proline cis-trans isomerase can stimulate the phosphatase activity of Ssu72 in vitro. The symplekin-Ssu72 interaction as well as the catalytic activity of Ssu72 is required in our transcription-coupled polyadenylation assay. Overall, our study has important implications for the regulation of RNAP II transcription by cis-trans isomerization of the CTD and will help us understand how CTD modifications influence the recruitment of pre-mRNA 3' end processing factors in a transcription-coupled manner. Recent studies showed that Ssu72 is also a phosphatase of CTD pSer7, which is involved in small nuclear RNA transcription and 3' end processing. However, a pSer7 phosphatase activity appears to be inconsistent with our structure because pSer7 is followed by Tyr1' of the next repeat rather than a proline, and it is unlikely for the pSer7-Tyr1' peptide bond to be in cis configuration. To solve this conundrum, we determined the crystal structure of the pSer7 CTD peptide bound to Ssu72. Surprisingly, the backbone of the pSer7 CTD runs in an opposite direction compared with the pSer5 CTD, allowing a trans pSer7-Pro6 peptide bond to be accommodated in the active site. However, Ssu72 has a much lower affinity for pSer7 than pSer5 and several structural features are detrimental for the catalytic activity towards pSer7. Consistent with these observations, our in vitro assays showed that the dephosphorylation of pSer7 by Ssu72 is ~4000-fold lower than that of pSer5. This further characterization of Ssu72 not only presents the first phosphatase in the literature that recognizes peptide substrates in both directions but also provides a more comprehensive understanding on CTD regulation by phosphatases from a structural perspective. Another protein, Rtr1, was recently suggested to function as a pSer5 phosphatase in a zinc-dependent fashion, separately or redundantly with Ssu72. We solved the crystal structure of Rtr1 and discovered a new type of zinc finger with no close structural homologs. Unexpectedly, Rtr1 does not present any evidence of an active site and it lacks detectable phosphatase activity in all our assays. We believe that, based on our results, Rtr1 does not have catalytic ability but instead indirectly regulate the phosphorylation state of the CTD. In summary, our studies on the symplein-Ssu72-CTD complex as well as Rtr1 have revealed several novel structural features that are essential for the CTD regulation at the atomic level. These results will also shed light on understanding the mechanism by which RNAP II transcription and RNA processing are coupled
ISOLATION AND CHARACTERIZATION OF THE FOUR ARABIDOPSIS THALIANA POLY(A) POLYMERASE GENES
Poly(A) tail addition to pre-mRNAs is a highly coordinated and essential step in mRNA maturation involving multiple cis- and trans-acting factors. The trans-acting factor, poly(A) polymerase (PAP) plays an essential role in the polyadenylation of mRNA precursors. The Arabidopsis thaliana genome contains four putative PAP genes. We have found, using in silico analysis and transgenic plants expressing GUS under the control of the four PAP promoters, that each of these genes is expressed in overlapping, yet unique patterns. This gives rise to the possibility that these genes are not redundant and may be essential for plant survival. To further test this, inducible RNAi and T-DNA mutagenized plants were obtained and analyzed. Plants lacking all, or most, of each PAP gene product, due to RNAi induction, were not viable at any of the stages of plant growth tested. Furthermore, T-DNA PCR analysis determined that no plants containing a homozygous mutation, were viable. This data reveals that lack of any of the four PAP gene products has a significant effect on the plants ability of survive, thus indicating that each PAP gene is essential. Finally, transient expression experiments with each of the full length PAP cDNAs fused to GFP showed that the PAP I, PAP II and PAP IV gene products are localized throughout the nucleus and within nuclear speckles. The cellular localization of PAP III could not be determined
Symplekin and Transforming Acidic Coiled-Coil Containing Protein 3 Support the Cancer Cell Mitotic Spindle
An increased rate of proliferation in cancer cells, combined with abnormalities in spindle architecture, places tumors under increased mitotic stress. Previously, our laboratory performed a genome-wide paclitaxel chemosensitizer screen to identify genes whose depletion sensitizes non-small cell lung cancer (NSCLC) cells to mitotic stress induced by paclitaxel treatment. This screen uncovered a cohort of genes that are required for viability only in the presence of paclitaxel. Two genes uncovered in this screen were the polyadenylation scaffold symplekin and the gametogenic protein transforming acidic coiled-coil containing protein 3 (TACC3). Herein, we examine the impact of polyadenylation and gametogenesis on the tumor cell mitotic spindle. First, we demonstrate that depletion of SYMPK and other polyadenylation components sensitizes many NSCLC cells, but not normal immortalized lines, to paclitaxel by inducing mitotic errors and leading to abnormal mitotic progression. Second, we demonstrate that multiple gametogenic genes are required for normal microtubule dynamics and mitotic spindle formation in the presence of paclitaxel. Additionally, we show that the gametogenic protein TACC3 is uniquely required for mitosis only in transformed cell lines but not normal immortalized cell lines and that this unique dependency can be targeted in vitro with a small molecule. These studies reveal an unanticipated dependence of the cancer cell mitotic spindle on polyadenylation and gametogenic genes. We propose that, faced with mitotic stress, cancer cells develop conditional dependencies on processes such as polyadenylation that occur in all cells and emergent dependencies on gametogenic genes that are overexpressed in tumor cells.Doctor of Philosoph
Role of Cnot3 in gene regulation and cell cycle progression
Gene expression is a process that is tightly regulated by many factors. Different genes are transcribed not only in a cell specific manner but are also differentially expressed at different stages of the cell cycle. Cnot3 is part of the CCR4-NOT deadenylation complex, which is involved in the turnover of mRNAs in the cytoplasm and has also been shown to have roles in regulating transcription and cell proliferation and in maintaining ES cell pluripotency. Previous work demonstrated that Cnot3 interacts directly with Aurora B kinase and is phosphorylated by Aurora B in an in vitro assay. Aurora B and Cnot3 co-localise at active gene promoters in resting B cells. Since Aurora B is a cell cycle kinase, I have developed a cell synchronization system to analyse the role of the Cnot3-Aurora B interaction at different stages of the cell cycle in primary B cells. Using this system, I have demonstrated that the interaction between Cnot3 and Aurora B varies during cell cycle progression. In vitro analysis showed that the interaction occurs through the NOT box domain of Cnot3. Mass spectrometry analysis of Cnot3 interactors, performed on nuclear extracts from B cells in the early G1 and G2 phases of the cell cycle, identified interactions with many factors that are known to have roles in transcription regulation and RNA processing. Interaction of Cnot3 with Histone H1 was confirmed using a peptide binding assay, suggesting a potential role in chromatin organization. Cnot3 was also shown to interact with Xrn2, a 5’-3’ exoribonuclease that is involved in RNA turnover and termination of transcription. ChIP analysis demonstrated promoter binding of Cnot3 at a number of cell cycle stages. Cnot3 shows cell cycle dependent binding to promoters of a wide range of active genes, including promoters that are not directly involved in cell cycle regulation. Genome wide analysis using ChIP sequencing revealed changes in the binding profiles of Cnot3 at promoters and enhancers during cell cycle progression. A Cnot3 conditional knock out mouse has been generated, which will be used to test the functional importance of these observations.Open Acces