Browsing by Subject "Gene Expression Regulation, Viral"
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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 Identification of Smaller Noncoding RNAs Produced by Mycobacterium Tuberculosis in Infected Macrophages That Regulate Mtb Growth and Survival(2021-12-10) Coskun, Fatma Sevde; Hancks, Dustin C.; Conrad, Nicholas; Winter, Sebastian E.; van Oers, Nicolai S. C.It is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb). While much work has focused on the role of different proteins encoded by Mtb in pathogenesis, recent studies have revealed that Mtb also transcribes many noncoding RNAs whose functions remain poorly characterized. A subset includes small RNAs (sRNAs) between the sizes of 50-350 nts. The current study focused on the identification and characterization of miRNA-like sRNAs <50 nts produced by Mtb. A sRNA-centered RNA-sequencing approach was performed and a subset of Mtb-encoded smaller noncoding RNAs (sncRNAs) were identified. Thirty-five distinct Mtb-encoded sncRNAs were discovered, with most being induced in infected eukaryotic cells. Three sncRNAs, sncRNA-1, sncRNA-6, and sncRNA-8, predominated the read counts. They were contained in longer RNA transcripts with stable secondary RNA stem loops and structures like precursor microRNAs. My work established that sncRNA-1 positively regulates two mycobacterial transcripts involved in oleic acid biosynthesis. Loss- and gain- of-function approaches reveal that sncRNA-1 enhances Mtb growth and survival in nutrient-depleted cultures as well as in infected macrophages. Given evidence that RNA processing enzymes were involved in the formation of the sncRNAs, different components of core RNA degradosome were characterized for their ability to process the precursor forms of the sncRNAs. My work revealed that PNPase degrades sncRNA-8 and preliminary evidence suggests that sncRNA-1 is also likely a target, which could be critical in the oleic acid deficient media. Overall, my study reveals that Mtb produces a set of sncRNAs in infected cells, with one modulating mycobacterial gene expression and mycobacterial pathogenicity coupled to oleic acid biogenesis. Future studies will address the functions of other sncRNAs and focus on the identification of sncRNA processing enzymes.