Browsing by Subject "Heterogeneous-Nuclear Ribonucleoproteins"
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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 The Regulation of hTERT by Alternative Splicing(2017-07-27) Yuan, Laura Yu; Yu, Hongtao; Shay, Jerry W.; Wright, Woodring E.; Fontoura, Beatriz; Cobb, Melanie H.Telomeres are non-coding DNA hexameric repeats (TTAGGG in mammals) located at the ends of linear chromosomes that, along with their associated proteins, protect against the loss of genomic material during cell division and prevent the recognition of chromosome ends as double-strand breaks. Human telomeres shorten with continued cell proliferation but are maintained by human telomerase reverse transcriptase (hTERT), an enzyme that synthesizes telomeric repeats using an RNA template. The regulation of telomerase has been studied at many levels--from epigenetic and transcriptional regulation to the alternative splicing of hTERT pre-mRNA into catalytically inactive splice variants. Our hypothesis is that if the regulation of telomerase reverse transcriptase splicing is necessary for telomere length homeostasis, altering telomerase splicing to decrease the production of full-length hTERT and will result in decreased telomerase activity and subsequently telomere shortening. We focused our efforts on identifying splicing factors are involved in hTERT splicing and characterized the role of two splicing factors, NOVA1 and PTBP1, in regulation of hTERT splicing in non-small cell lung cancer cells. We show that these splicing factors are important for full-length hTERT, telomerase activity and telomere length maintenance in vitro. Xenograft studies suggest that NOVA1 is also important for tumor growth in vivo. We found that these splicing factors are able to directly interact with hTERT in a region our group previously identified to be important for hTERT splicing. Altogether, our work suggests that splicing factors are important for hTERT regulation and telomerase activity in cancer. Since telomerase activity is undetectable in most somatic tissues but is increased in the vast majority of human cancers, dependence on telomerase represents a key vulnerability in cancer tissues which could be therapeutically targetable.Item Regulation of Pumilio RNA Binding Proteins by Long Noncoding RNA NORAD(August 2021) Elguindy, Mahmoud Mohamed; Zhu, Hao; Buszczak, Michael; Sabari, Benjamin; Mendell, Joshua T.The mammalian genome is extensively transcribed and encodes thousands of long noncoding RNAs (lncRNAs). Defining the mechanism of action of lncRNAs has been a critical challenge and priority for understanding their biological functions. An important example of this is the lncRNA NORAD, which is a highly conserved lncRNA that is required for maintaining genome stability in mammals. Work in this thesis begins by examining the physiologic function and molecular mechanism of NORAD in human cells. We clearly demonstrate that NORAD localizes predominantly to the cytoplasm where it functions by binding to and inhibiting PUMILIO (PUM1 and PUM2) RNA binding proteins (RBPs). Thorough dissection of the functional domains of NORAD establish the essentiality of PUM binding to NORAD for its function in maintaining genome stability. We further examine the mechanism by which NORAD is able to efficiently sequester and regulate PUM proteins. Through a multidisciplinary approach involving biochemical, microscopy, and mutational analysis experiments, we uncover how NORAD sequesters a super-stoichiometric amount of PUM proteins into novel liquid-like membraneless organelles through a multivalency-induced phase separation mechanism. Moreover, we provide a molecular understanding of the properties of NORAD that promote PUM phase separation, revealing new principles of lncRNA function and demonstrating the physiologic importance of RNA-driven phase separation as a regulatory mechanism in mammalian biology. Our findings, together with the widespread repetitive architecture of lncRNAs, suggest that the phase separation principles we establish may be broadly utilized for lncRNA-mediated regulation.