Browsing by Subject "RNA Splicing"
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Item Characterization of U2AF26, a Paralog of the Splicing Factor U2AF35(2004-08-19) Shepard, Jeremiah Brian; Lynch, Kristen W.; McKnight, Steven L.The essential splicing factor U2 auxiliary factor (U2AF) mediates 3' splice site recognition during spliceosome assembly. The mammalian U2AF is composed of a large subunit, U2AF65, and a small subunit, U2AF35. U2AF65 recognizes the pyrimidine tract and U2AF35 binds to the AG dinucleotide, both of which are specific 3' splice site sequence motifs. In the present work U2AF26, a paralog of the conventional U2AF35, has been studied. U2AF26 shares 84% primary amino acid identity with U2AF35, suggesting functional homology. However, U2AF26 has two amino acid substitutions in ribonulceoprotein consensus sequence-2 (RNP-2) and significant differences within the RS domain, two regions thought to be important for the function of U2AF35. The goal of this study was to characterize the functional differences between the two small subunits. Western blot analysis revealed that U2AF26 protein expression varies relative to U2AF35 in different mouse tissues. Site-specific crosslinking analysis of sixteen permutations of the nucleotide composition upstream and downstream of the AG indicates that U2AF26 and U2AF35 bind to the UAGG motif with the highest affinity. Interestingly, U2AF26 binds the UAGU motif better than U2AF35. This observation suggests that U2AF26 and U2AF35 have overlapping binding affinities, but that U2AF26 might be capable of recognizing a specific 3' splice site motif better than U2AF35. Initial evidence suggested that U2AF26 is regulated by circadian rhythm. Analysis of U2AF26 over a 24-hour period in the mouse forebrain indicates that expression of the full length transcript does not change significantly, but the alternative splicing of the U2AF26 transcript fluctuates during the day:night cycle. Examination of U2AF26 alternative splicing in other tissues revealed that this splicing event is temporally regulated in the liver, but with a two-peaked pattern of splicing. Further analysis of other alternative splicing events in the liver indicates that the polypyrimidine tract binding (PTB) transcript is regulated in a similar manner. The two-peaked pattern of splicing in the liver suggests that the alternative splicing of U2AF26 and PTB is not regulated by circadian rhythm. However, this is the first time it has been observed that pre-mRNA splicing changes as a function of the day:night cycle.Item Characterization of Ubiquitin Ligase Targeting by Anticancer Sulfonamides(2020-08-01T05:00:00.000Z) Ting, Tabitha Chung-Yan; De Martino, George; Nijhawan, Deepak; Yu, Hongtao; DeBose-Boyd, Russell A.Aryl sulfonamides are small molecules that are selectively toxic to a subset of human cancer cell lines. Clinical trials of the aryl sulfonamide indisulam have resulted in modest clinical activity against a subset of solid tumors. Recent work revealed that indisulam recruits the RNA binding protein RBM39 to DCAF15, a component of the CRL4-DCAF15 E3 ubiquitin ligase. This recruitment results in RBM39 ubiquitination and degradation, leading to splicing defects and cancer cell death (Han et al., 2017; Uehara et al., 2017). The mechanism of action of sulfonamides is similar to that of immunomodulatory drugs (IMiDs), which recruit substrates to the closely related CRL4-CRBN E3 ubiquitin ligase for ubiquitination. Known for their roles in inhibiting embryonic development and cancer cell growth, IMiDs exert their pleiotropic effects by targeting a variety of substrate proteins to the CRL4-CRBN E3. Despite major advances in our understanding of aryl sulfonamides, it is unclear whether sulfonamides also target multiple substrates or modulate the endogenous function of the CRL4-DCAF15 E3 ligase. This dissertation describes our efforts to define the requirements for RBM39 ubiquitination, identify other substrates that are recruited to the CRL4-DCAF15 E3 ligase, and further our understanding of the cellular consequences of indisulam treatment. In Chapters 2 and 3, we define the components required for RBM39 ubiquitination using a combination of in vitro and in vivo techniques. In Chapters 4 and 5, we identify putative endogenous substrates and a previously undescribed neo-substrate recruited to the CRL4-DCAF15 for ubiquitination. In Chapter 6, we characterize the cellular consequences of indisulam treatment and neo-substrate degradation. In aggregate, this work aims to contribute to our understanding of the sulfonamide mechanism of action and the field of targeted protein degradation.Item Chemical Intervention of Influenza Virus mRNA Nuclear Export(2020-07-15) Esparza, Matthew Aaron; Shay, Jerry W.; Fontoura, Beatriz; Grinnell, Frederick; Minna, John D.Influenza A viruses are human pathogens with limited therapeutic options, making it crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. We show that influenza virus utilizes nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies, and exports the spliced M2 mRNA from the nucleus. In addition, gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway, inhibits M mRNA nuclear export without significantly altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that inhibits nuclear export of a subset of viral and cellular mRNAs via the mRNA export factor UAP56 without altering bulk cellular mRNA nuclear export. These findings underscore specific nuclear export requirements for viral mRNA nuclear export. This RNA export inhibitor also impaired replication of diverse influenza virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.Item Combinatorial Regulation of Signal-Induced CD45 Exon Repression by hnRNP L and PSF(2007-08-08) Melton, Alexis Allyson; Lynch, Kristen W.CD45 is a hematopoetic-specific tyrosine phoshatase. In resting T cells three variable exons are partially repressed, and following antigen challenge, these exons are more highly repressed. Previous work identified the ESS1 silencer element that functions to mediate exon 4 silencing under resting conditions by binding to hnRNP L. ESS1 is also sufficient to confer the activation-induced increase in exon repression, and this document describes two mechanisms responsible for mediating this effect. First, hnRNP L silencing function is slightly increased in activated cells as compared to resting cells. Additionally, PSF binds to the ESS1 complex in a signal-dependent manner and provides a significant increase in repressive activity. Further investigation shows these two mechanisms are largely independent but show some functional crosstalk, and while neither of these mechanisms is sufficient in isolation, the combination of these two effects accounts for an increase in exon silencing that is of similar magnitude to the total observed change in splicing in response to cellular activation.Item Context-Dependent Function of the Splicing Factor hnRNP L(2010-11-02) Motta-Mena, Laura Beatriz; Lynch, Kristen W.Based on the number of genes impacted (~95% of humans genes), alternative splicing is one of the most extensively used mechanisms for generating proteomic diversity and cellular complexity. Splicing of pre-mRNAs is carried out by a highly specialized, RNA-based macromolecular enzyme known as the spliceosome. The spliceosome is made up of 5 small nuclear RNP (snRNP) complexes (U1, U2, U4/U6, and U5), all of which consist of a uridine-rich snRNA and multiple proteins. Importantly, the spliceosome is not a pre-formed enzyme but instead forms through the step-wise assembly of the snRNP complexes on the pre-mRNA. Mechanistically, the selection of exons or splice sites during alternative splicing occurs by modulating the assembly of the spliceosome on a pre-mRNA. Ultimately, the decision to include or exclude an exon into the final mRNA is based on the integration of both the synergistic and antagonistic forces between groups of protein regulators and between protein regulators and the snRNP complexes. An excellent model system to illustrate the mechanisms of alternative splicing, as well as the physiologic significance of this mode of regulation, is the human CD45 gene. HnRNP L binds to a motif present in both CD45 variable exons 4 and 5 to affect their coordinate repression. Previously, it was shown that hnRNP L regulates exon 4 by stalling the U1 and U2 snRNPs in a non-permissive A-like exon-defined spliceosomal complex. Here, we show that, in contrast to its direct repression of exon 4, hnRNP L represses exon 5 by countering the activity of a neighboring splicing enhancer element. As the splice sites flanking exon 4 and 5 are distinct, we directly examined the effect of varying splice site strength on the mechanism of hnRNP L function. Remarkably, binding of hnRNP L to an exon represses strong splice sites but enhances weak splice sites. A model in which hnRNP L stabilizes snRNP-binding can explain both effects in a manner determined by the inherent snRNP-substrate affinity. Overall, these findings demonstrate that context can fundamentally alter the activity of a splicing regulatory protein and can therefore impact our predictions of splicing patterns and mechanisms of splicing regulation.Item SAM Homeostasis Is Regulated by CFIm-Mediated Splicing of MAT2A(August 2021) Scarborough, Anna Maurine; Tu, Benjamin; Mendell, Joshua T.; Green, Carla B.; Conrad, NicholasS-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of MAT2A, the only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) as a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in the detained intron and 3´ UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal a previously undescribed role for CFIm in splicing and SAM metabolism.Item The U6 snRNA m6A Methyltransferase METTL16 Regulates MAT2A Intron Retention Through Co-Transcriptional Splicing(2017-07-19) Pendleton, Kathryn Elizabeth; Corey, David R.; Conrad, Nicholas; Mendell, Joshua T.; Tu, BenjaminMaintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Unlike previously studied intron retention events, we show that splicing induction of the MAT2A retained intron is regulated at the level of co-transcriptional splicing. This induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3´ UTR. Increasing METTL16 occupancy on the MAT2A 3´ UTR is sufficient to induce efficient splicing. We propose that under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal snRNA. These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis.