Browsing by Subject "Ribonucleoproteins, Small Nuclear"
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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 Enzymatic Disassembly of Promoter Bound 7SK snRNP Drives Transcription Elongation of HIV and Cellular Genes(2015-11-19) McNamara, Ryan Philip; Cobb, Melanie H.; D'Orso, Iván; Conrad, Nicholas; Kahn, JeffreyGene expression of the human immunodeficiency virus (HIV) and cellular primary responsive genes (PRG’s) is regulated at the step of transcription elongation. Shortly after transcription initiation, RNA Polymerase II (Pol II) pauses and it only enters into productive elongation after inducible transcription factors (TF’s) recruit the P-TEFb kinase to phosphorylate Pol II in response to stimuli. To ensure tight regulation of this process, the majority of P-TEFb is held in a catalytically inactive form, reversibly bound to the 7SK small nuclear ribonucleoprotein (snRNP). In the absence of stimuli, the 7SK snRNP resides in both the nucleoplasm and promoter regions. However, an understanding of how TF’s capture P-TEFb from the 7SK snRNP at the promoter and the mechanism and purpose of localizing the 7SK snRNP to promoters has been largely unexplored. It was therefore my goal to biochemically and functionally characterize this pathway through the use of both the HIV encoded TF Tat and cellular TF’s such as nuclear factor kappa b (NF-κB). Detailed throughout this dissertation, I present the novel findings that HIV Tat and NF-κB function to recruit the PPM1G phosphatase to their targeted promoters, which dephosphorylates P-TEFb and triggers its release from the 7SK snRNP. Additionally, this extraction of P-TEFb from the 7SK snRNP occurs at promoters through the transcriptional regulator KAP1, which physically tethers the snRNP to promoters genome wide. Recruitment of the 7SK snRNP complex occurs after transcription initiation, allowing P-TEFb to be directly positioned for rapid extraction by TF’s upon stimuli and transferred onto the paused Pol II. The enzymatic uncoupling of P-TEFb from the promoter bound 7SK snRNP enables rapid Pol II elongation and gene expression in response to stimuli (for PRG’s) or in the presence of Tat (for HIV). Ultimately, these findings indicate that inducible transcription programs can rapidly respond to environmental cues through the localized positioning of elongation factors at promoters. Moreover, these findings illustrate that HIV has evolved to hijack a cellular gene expression program, thus leading to viral takeover of the host and progression of AIDS.