Browsing by Subject "RNA Processing, Post-Transcriptional"
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Item HP1BP3, A Chromatin Retention Factor for Co-Transcriptional MicroRNA Processing(2016-06-27) Liu, Haoming; Tu, Benjamin; Liu, Qinghua; Roth, Michael G.; Orth, KimRNA interference (RNAi) is a post-transcriptional gene silencing mechanism found in all eukaryotic organisms. It is characterized by a family of small non-coding RNAs, either endogenous (in the case of microRNAs) or exogenous (in the case of siRNAs), that inhibits gene expression post-transcriptionally. MicroRNAs (miRNAs) are a family of ~21-nt cellular RNAs that govern numerous pathological and physiological processes by mediating translational repression and deadenylation/decay of cognate mRNA. Dysregulation of miRNA expression have been associated with various types of cancer and developmental diseases. Typically, primary (pri-)miRNA transcripts are processed by Drosha complex into precursor (pre-)miRNAs, and then by cytoplasmic Dicer complex into mature miRNAs. The processing of pri-miRNAs is the most highly regulated step in the miRNA biogenesis pathway. Therefore, understanding the molecular mechanisms of pri-miRNA processing and its regulation represents a very important objective in the miRNA filed. Recent studies suggest that the Drosha-DGCR8 complex can be recruited to chromatin to catalyze co-transcriptional processing of primary microRNAs (pri-miRNAs) in mammalian cells. However, the molecular mechanism of co-transcriptional miRNA processing is poorly understood. Here, we find that HP1BP3, a histone H1-like chromatin protein, specifically associates with the Microprocessor and promotes global miRNA biogenesis in HeLa cells. Accordingly, chromatin immunoprecipitation (ChIP) studies reveal genome-wide co-localization of HP1BP3 & Drosha and HP1BP3-dependent Drosha binding to actively transcribed miRNA loci. Moreover, HP1BP3 exhibits a novel pri-miRNA binding activity and promotes the Drosha-pri-miRNA association in vivo. Knockdown of HP1BP3 compromises pri-miRNA processing by resulting in premature release of pri-miRNA transcripts from the chromatin. Taken together, these studies suggest that HP1BP3 promotes co-transcriptional miRNA processing via chromatin retention of nascent pri-miRNA transcripts. This work expands the functional repertoire of the H1 family of proteins and suggests a new concept of chromatin retention factor for widespread co-transcriptional miRNA processing.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.