Browsing by Subject "RNA Precursors"
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Item Characterization of the Interactions Between Kaposi’s Sarcoma-Associated Herpesvirus ORF57 and Its RNA Partners(2014-07-30) Sei, Emi; Corey, David R.; D'Orso, Iván; Fontoura, Beatriz; Conrad, NicholasKaposi's sarcoma-associated herpesvirus (KSHV; HHV-8) is a human gammaherpesvirus and the etiological agent of Kaposi's sarcoma (KS), the most common AIDS-associated malignancy. Like all herpesviruses, KSHV has evolved mechanisms to modulate both host and viral gene expression. The essential and multifunctional KSHV ORF57 protein has been reported to enhance viral gene expression at multiple levels including transcription, splicing, mRNA export, RNA stability, and translation. At least in some cases, direct interactions between ORF57 and its target RNAs are necessary for ORF57-mediated upregulation of viral gene expression. The work highlighted in this document reveals our current efforts to study the elements driving ORF57's binding specificity. We started by studying a known ORF57 target: the KSHV polyadenylated nuclear (PAN) RNA, a nuclear non-coding transcript of unknown function that is highly expressed during lytic stage. We first devised an in vitro binding assay to identify the regions in PAN RNA that were bound by ORF57. These PAN RNA fragments were also inserted into the 3' UTR of an intronless β-globin reporter to test ORF57 responsiveness in vivo. Our analyses revealed an ORF57 responsive element (ORE) at the 5' end of PAN RNA that we hypothesize functions as a high-affinity binding site to recruit ORF57. Next, we optimized the high-throughput sequencing of RNAs isolated by crosslinking and immunoprecipitation (HITS-CLIP) protocol to identify novel host and viral targets of ORF57 in the context of viral infection. Bioinformatic analysis of potential host ORF57 targets reveals that ORF57 binding is enriched near the 5' end of the transcripts and often close to the first exon-intron junction. Preferential binding at the 5' end is also seen for PAN RNA. However, our data suggests that ORF57 binding to other viral genes can be promiscuous and that, in some cases, binding can occur at multiple sites across the target RNAs. Through these studies, we hope to provide further insight into the requirements for ORF57 binding and potentially shed light into the mechanisms controlling gene expression of this oncogenic virus.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.