Browsing by Subject "Alternative Splicing"
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Item Analysis of Interrelationships Among NAD+, PARP1, ADP-Ribosylation, and Splicing in Murine Embryonic Stem Cells(2022-05) Jones, Aarin; Banaszynski, Laura; DeBerardinis, Ralph J.; Wang, Yingfei; Kraus, W. LeeThe differentiation of embryonic stem cells (ESC) into a lineage-committed state is a dynamic process involving changes in epigenetic modifications, gene expression, RNA processing, and cellular metabolism. Previous studies have implicated poly(ADP-ribose) polymerase 1 (PARP1), an abundant nuclear enzyme that plays key roles in a variety of nuclear processes, in ESC self-renewal and lineage commitment. Given the diverse molecular functions of PARP1, I sought to determine the potential regulatory role of PARP1 in determining ESC state. PARP1 functions both as an enzyme, through its NAD+-dependent ADP-ribosyltransferase catalytic activity, and as a structural protein, through its NAD+-independent nucleic acid binding activity. I observed a dramatic induction of PARP1 catalytic activity during the early stages of mESC differentiation (e.g., within 12 hours of LIF removal) leading me to query the regulation and outcome of PARP1-mediated ADP-ribosylation in mESCs. NAD+ is synthesized through three main pathways - De novo, Salvage, and Preiss-Handler - and is constrained within cellular compartments. I found that both pathway usage and subcellular localization were dynamic during differentiation in a PARP1-dependent manner, with transition from De novo to Salvage pathway usage and increases in nuclear NAD+ levels upon differentiation feeding PARP1 catalytic activity. Using an NAD+ analog-sensitive PARP (asPARP) chemical biology approach, I characterized the PARP1-mediated ADP-ribosylated proteome during mESC differentiation. PARP1-modified proteins in mESCs are enriched for biological processes related to stem cell maintenance, transcriptional regulation, and RNA processing. The PARP1 substrates include core spliceosome components, such as U2AF35 and U2AF65, whose splicing functions are modulated by PARP1-mediated site-specific ADP-ribosylation. In addition, I observed a genome-wide dysregulation of splicing events upon loss of PARP1 in transcriptomic analysis. These results demonstrate a role for the NAD+-PARP1 axis in the maintenance of mESC cell state, specifically in the splicing program during differentiation.Item Characterization and Development of Strategies for Altering Protein Expression in JSL1 Cells(2007-08-08) Senitko, Annette Nelson; Lynch, Kristen W.Alternative splicing is a common mechanism for regulating gene expression in eukaryotic cells. This process of differentially including or excluding variable exons provides a means for increasing proteome complexity. Alternative gene splicing occurs in a cell specific manner and may be influenced by changes in the extracellular environment. Despite the importance of this method for regulating gene expression, little is known about the factors involved in regulating its function. The T cell tyrosine phosphatase CD45 provides a valuable model for investigating the factors involved in regulating alternative splicing. The CD45 gene contains three variable exons whose splicing is regulated in response to T cell activation. Studies of this gene have revealed the presence of an exonic silencer sequence within variable exon 4 that is capable of influencing exon skipping under both resting and stimulated conditions. Biochemical assays have shown that the regulatory protein hnRNP L binds to this silencer sequence and results in basal exon repression during resting conditions and undergoes modifications which further influence exon skipping upon stimulation. Furthermore, in vitro assays indicate that upon stimulation, an additional regulatory protein, PSF, binds to the regulatory complex associated with the silencer sequence. Although these studies have provided novel information regarding the regulation of splicing, biochemical assays are unable to fully mimic the signaling pathways inside a cell, thus creating a need for a cell culture system. A Jurkat derived cell line, JSL1 cells, has been identified as being able to recapitulate the signal induced alternative splicing of the CD45 gene as seen in primary human T cells. This cell line presents a cell based system for evaluating the factors involved in splicing. However, in order to conduct in vivo experiments one must be able to modify protein expression. JSL1 cells present limitations due to difficulties in being able to alter protein expression. A strong promoter, EF1-α, has been employed to drive the expression of candidate proteins in JSL1 cells. Transient transfections and stable cell lines expressing cDNAs driven by this promoter have shown little if any overexpression of candidate proteins normally expressed at high levels within the cell; however, significant overexpression has been achieved with the transfection of at least one protein that exists at a lower concentration. Initial experiments indicate that stably expressed flag-tagged proteins, driven by the EF1-α promoter, may be easily purified from JSL1 cells during resting and stimulated conditions and analyzed. Such data suggests that this promoter may afford more flexibility in altering and analyzing protein expression in JSL1 cells, thereby facilitating the investigation of signaling pathways involved in regulating alternative splicing. Furthermore, strategies for regulating protein expression, through the use of a Tet-suppressor system, are in initial stages of being developed and hold the potential for providing an additional tool for evaluating the factors involved in regulating alternative splicing.Item Formation of an Exon-Defined A Complex Spliceosome Intermediate Results in CD45 Exon Repression(2007-12-17) House, Amy Elizabeth; Lynch, Kristen W.Alternative splicing is a common means for genetic regulation in higher eukaryotes, and variability in splicing patterns results in a significant increase in protein diversity. Of particular interest is how the splicing machinery is regulated to allow for alternative splicing while maintaining accurate splicing fidelity. CD45 is a trans-membrane tyrosine phosphatase that is expressed on the surface of hematopoetic cells. The CD45 gene contains three variable exons that are partially repressed in na?T cells and are preferentially skipped upon T cell activation. Importantly, it has been shown that the alternative splicing of CD45 pre-mRNA affects the resultant protein's function within the cell emphasizing the importance of this RNA processing event. As such, CD45 provides an excellent model system in which to study signal-responsive alternative splicing. Pre-mRNA splicing is catalyzed by the spliceosome, a macromolecular complex that assembles de novo on a pre-mRNA template in a stepwise and highly dynamic process. The inherent dynamic nature of the spliceosome suggests the potential for regulation at numerous points along the assembly pathway. Previously, a 60-nucleotide exonic silencer (ESS1) was identified within variable exon 4 of the CD45 gene. ESS1 is bound by at least three hnRNP proteins (hnRNP L, PTB, hnRNP E2) of which hnRNP L is the main functional component. Subsequent work showed that ESS1 does not regulate CD45 exon 4 splicing by the typical mechanism of preventing exon definition. Instead, spliceosome assembly is stalled after the exon has been recognized by the spliceosome such that both U1 and U2 are bound to the splice sites suggesting that the stalled complex represents an A-like exon-defined complex (AEC). Additionally, hnRNP L is sufficient to block spliceosome assembly at the AEC. Further biochemical analysis of the AEC suggests that the stall in assembly may be due to altered interactions between the spliceosomal snRNAs and/or proteins and the pre-mRNA substrate. Overall, this inhibition represents a new mechanism for splicing regulation, and suggests that the formation of an AEC intermediate is an important transition in the spliceosome assembly pathway.Item HNRNP-L Interacts with the Signal-Responsive Alternative Splicing Regulatory Element of CD45(2005-08-11) Rothrock, Caryn Robin; Lynch, Kristen W.Alternative splicing is emerging as a common mechanism for altering protein expression in response to extracellular cues. Of particular interest is how cell-signaling pathways modify mRNA isoform expression. The T cell tyrosine phosphatase CD45 provides a useful model system to explore the regulation of cell-signaling induced alternative splicing. CD45 has three variable exons that are differentially excluded in T cells upon antigen challenge or activation. An exonic splicing silencer element, named ESS1, is necessary for regulation of inclusion or exclusion of CD45 exon 4 in the mature mRNA. This sequence is also sufficient to confer T cell activation induced repression of a heterologous exon. ESS1 contains a sequence motif that is present in CD45 variable exons 5 and 6, as well as exons of other signal responsive genes. Point mutations in conserved nucleotides within this motif abolish T cell activation-induced skipping of CD45 variable exons 4, 5, and 6. Biochemical purification of RNA binding proteins from nuclear extract identifies a number of hnRNP family members that bind ESS1 RNA sequence in a specific manner. Mass spectrometry identifies three of these, including p65 as heterogeneous ribonucleoprotein L (hnRNP-L), p55 as Polypyrimidine Tract Binding Protein (PTB or hnRNP-I), and p40 as heterogeneous ribonucleoprotein E2 (hnRNP-E2). Recombinant hnRNP-L, PTB, and hnRNP-E2 bind specificity to wild type ESS1 RNA, as tested by mobility shift and UV crosslinking assays. Mutations in the ESS1 sequence, which prevent exon skipping in vivo, reduce recombinant hnRNP-L binding in vitro. Recombinant PTB and hnRNP-E2 do not appear to have reduced binding to mutant ESS1 sequence. HnRNP-L appears to be the main sequence-specific binding component of ESS1 RNA, and may be involved in repressing CD45 variable exon 4 in response to T cell activation.Item Regulation of Human Telomerase Alternative Splicing(2014-06-10) Wong, Sze; Fontoura, Beatriz; Wright, Woodring E.; Shay, Jerry W.; Conrad, Nicholas; Martinez, ElisabethTelomerase adds TTAGGG repeats onto chromosome ends (telomeres). Since telomerase is expressed in ~90% of all human cancer cells while being absent in most somatic tissues, telomerase is an almost universal cancer therapeutic target. Yet, the clinical application of an effective telomerase inhibitor is still lacking. The pre-mRNA of the catalytic subunit of human telomerase (hTERT) may be alternatively spliced into 22 different isoforms. Only a small fraction of hTERT transcripts are spliced into the full length isoform, the form capable of being translated into function hTERT with reverse transcriptase activity. If telomerase activity is partially regulated at the level of RNA splicing, then telomerase activity may be modulated to increase the amount of nonfunctional transcripts and decrease the amount of full-length hTERT and this would be a novel anti-cancer therapeutic approach. A hTERT minigene was created to understand the cis-regulatory elements governing hTERT splicing. A 1.1kb region of 38 bp repeats (block 6) ~2 kb from the exon/intron junctions is essential for the exclusion of exons 7 and 8. Block 6 repeats suggested that RNA:RNA pairing may regulate splicing of hTERT. Mutations within the repeat sequence that abolish exon skipping were corrected by compensatory mutations in the pre-mRNA. To identify trans-acting regulators of hTERT alternative splicing, the minigene was modified into a dual-luciferase reporter for a selected 528 RNA-binding proteins/splicing factors siRNA screen. Our initial validation focused on 45 factors with enzymatic activity and resulted in CDC-Like Kinase 1 (CLK-1) as a potential hTERT splicing modulator. Knock-down of CLK-1 altered hTERT splicing, resulting in reduction in telomerase activity and telomere shortening. CLK-1 belongs to the Clk family of dual-specificity nuclear kinases that can auto-phosphorylate SR proteins. TG003, a chemical CLK-1/4 inhibitor, results in less full length hTERT splicing and a decrease in cancer cell telomerase activity. This approach provides a platform to identify additional hTERT splicing regulators that may be suitable drug targets to increase or decrease telomerase activity. Altogether, these results demonstrate the potential of manipulating hTERT splicing as a target for both chemotherapy and regenerative medicine.Item [Southwestern News](2005-04-14) Siegfried, AmandaItem [UT Southwestern Medical Center News](2013-04-22) Lyda, Alex