Browsing by Subject "RNA, Small Untranslated"
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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.Item Post-Transcriptional Regulation of Virulence Genes by GlmY and GlmZ in Enterohemorrhagic E. Coli(2014-04-14) Gruber, Charley C.; Hooper, Lora V.; Sperandio, Vanessa; Gardner, Kevin H.; Conrad, NicholasEnterohemorrhagic E. coli O157:H7 (EHEC) is a major cause of foodborne illness and hemolytic uremic syndrome (HUS) throughout the world. One of the major virulence factors in this pathogen is a type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE). EHEC uses this proteinaceous needle to inject effector proteins into host cells to hijack various host cellular processes, as well as the translocation and insertion of the translocated intimin receptor (Tir) into the host cell membrane. The bacterial adhesin intimin then binds to Tir, allowing EHEC to tightly adhere to the host cell’s membrane (109). Tir also indirectly recruits another bacterial effector EspFu, which induces actin polymerization. This causes the formation of the characteristic pedestal that cups the bacterial cell. This process from the assembly of the needle apparatus to the formation of the pedestal must be tightly regulated both transcriptionally and post-transcriptionally. The two two-component systems QseEF and QseBC have been previously shown to regulate various virulence genes. We established that these systems both regulate the transcription of the small RNA (sRNA) glmY. GlmY is known to stabilize another sRNA, GlmZ, which activates the translation of glucosamine synthetase (GlmS) (72). Here we show that GlmY and GlmZ are also important players in the post-transcriptional regulation of virulence genes in EHEC. The transcription factor QseF is required for the expression of EspFu and thus pedestal formation. This defect can be complemented by overexpression of either GlmY or GlmZ and is not at the transcriptional level. Instead, the expression of espFu requires a processing event that is QseF dependent. We have shown that GlmZ also post-transcriptionally regulates two of the operons of the LEE, LEE4 and LEE5. Both of these operons are transcribed from a single promoter, but there is a processing event that separates the first gene of the operon from the rest that requires the endoribonuclease RNase E. Overexpression of either sRNA results in the downregulation of the latter fragment of both the LEE4 and LEE5 operons. In the case of LEE4, this is through direct binding of GlmZ to a region within the LEE4 mRNA. We also investigated the global role of GlmY and GlmZ in EHEC through microarrays and RNA sequencing of the knockout strains. Aside from the LEE, GlmZ also regulates curli which are used to facilitate bacteria attachment to host cells. These data show that GlmZ has been co-opted into being an important regulator of virulence genes in EHEC.Item The Roles of Small and Long Non-Coding RNAs in Regulating Gene Expression(2014-01-23) Xue, Zhihong; McKnight, Steven L.; Liu, Yi; Liu, Qinghua; Conrad, NicholasRecent studies have revealed that a large proportion of a eukaryotic genome is transcribed into non-coding RNAs (ncRNAs). Based on size, these RNAs can be classified as small non-coding RNAs (sRNAs) and large non-coding RNAs (lncRNAs). The ncRNA regulatory networks control various levels of gene expression and play significant roles in diverse biological processes. Argonaute proteins, the core proteins in RNAi pathways, are required for the biogenesis of some sRNAs, including the PIWI-interacting RNAs and some microRNAs. How Argonautes mediate maturation of sRNAs independent of their slicer activity was not clear.The maturation of the Neurospora miRNA-like sRNA, milR-1, requires the Argonaute protein QDE-2, Dicer, and exonuclease QIP. Here, I reconstituted this Argonaute-dependent sRNA biogenesis pathway in vitro, and demonstrated that QDE-2 mediates milR-1 maturation by recruiting exosome and QIP, and by determining the size of milR-1. QIP first separates the QDE-2-bound duplex milR-1 precursor and then mediates 3’ to 5’ trimming and maturation of milR-1 precursor together with exosome using a hand-over mechanism. Our results establish a biochemical mechanism of an Argonaute-dependent sRNA biogenesis pathway and critical roles of exosome in sRNA processing. Natural antisense RNAs, which are mostly lncRNAs, are widely found in eukaryotic organisms and have been implicated in diverse physiological processes. The physiological importance of antisense RNAs and how they regulate sense RNAs are not clear. frequency (frq) encodes a core component of the Neurospora circadian oscillator. Here, I demonstrated that the simultaneous transcription of qrf, the long non-coding frq antisense RNA, represses frq transcription by inducing RNA polymerase II collision-triggered premature transcription termination and chromatin modifications. The expression of frq also inhibits the expression of qrf and surprisingly, drives the antiphasic rhythm of the qrf transcripts in the dark. The mutual inhibition of frq and qrf transcription forms a double negative feedback loop that is required for robust and sustained circadian rhythmicity. Our results establish antisense transcription as an essential feature in a eukaryotic circadian system and demonstrate the importance and mechanism of antisense RNA action. Together, the studies described in this dissertation shed light on the mechanisms of gene expression regulated by sRNAs and lncRNAs.