Browsing by Author "Wilusz, Jeffrey, advisor"
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Item Open Access An exploration of viral RNA-mediated strategies to stall and repress the cellular exoribonuclease XRN1(Colorado State University. Libraries, 2018) Charley, Phillida A., author; Wilusz, Jeffrey, advisor; Zabel, Mark, committee member; Perera, Rushika, committee member; Reddy, Anireddy, committee memberThe regulation of mRNA decay plays a vital role in determining both the level and quality control of cellular gene expression in eukaryotes. Since they are likely recognized as foreign/unwanted transcripts, viral RNAs must also successfully navigate around the cellular host RNA decay machinery to establish a productive infection. This bypass of the cellular RNA decay machinery can be accomplished in many ways, including the sequestering of regulatory proteins or inactivating enzymatic components. One attractive way for RNA viruses to undermine the cellular RNA decay machinery is to target the cellular exoribonuclease XRN1 since this enzyme plays a major role in mRNA decay, appears to coordinate transcription rates with RNA decay rates, and is localized to the cytoplasm and thus readily accessible to cytoplasmic RNA viruses. We have previously shown that many members of Flaviviridae (e.g. Dengue, West Nile, Hepatitis C and Bovine Viral Diarrhea viruses) use RNA structures in their 5' or 3' untranslated regions (UTRs) to stall and repress XRN1. This results in the stabilization of viral RNAs while also causing significant dysregulation of cellular RNA stability (and thus dysregulation of overall cellular gene expression). In this dissertation we first extend this observation to another member of the Flaviviridae, Zika virus, by demonstrating that structures in the 3' UTR of the viral genomic RNA can stall and repress XRN1. Significantly, we also demonstrate that the 3' UTR of the N mRNA of the ambisense segment of Rift Valley Fever virus, as well as two other phleboviruses of the Phenuiviridae, also can effectively stall and repress XRN1. This observation establishes XRN1 stalling in an additional family of RNA viruses, in this case in the order Bunyavirales. We have mapped the region responsible for XRN1 stalling to a G-rich core of ~50 nucleotides and provide evidence that the formation of a G-quadruplex is contributing to stalling of XRN1. In addition to phleboviruses, we also detected RNA regions that stall XRN1 in the non-coding regions of two other virus families. The 3' UTRs of all four ambisense transcripts of Junin virus, an arenavirus, stall and repress XRN1. This observation was extended to two additional arenaviruses, suggesting that XRN1 stalling may be a conserved property of the 3' UTRs in the Arenaviridae. Finally, we demonstrate that the non-coding RNA from beet necrotic yellow vein virus RNA segment 3 is produced by XRN1 stalling and requires a conserved sequence called the coremin motif. Collectively, these observations establish XRN1 stalling and repression as a major strategy used by many virus families to effectively interface with the cellular RNA decay machinery during infection. We performed two proof of principle studies to extend the significance of the observation of XRN1 stalling during RNA virus infections. First, since XRN1 stalling may be associated with successful viral gene expression as well as cytopathology, we explored whether we could identify a small molecule compound that could interfere with the knot-like three helix RNA junction structure that stalls XRN1 in the 3' UTR of flaviviruses. We tested several triptycene-based molecules, compounds that have been previously shown to intercalate into three helix junctions and identified four triptycene derivatives that interfere with XRN1 stalling. Lastly, we explored whether there might be a cellular exoribonuclease that could navigate through the well-characterized flavivirus structure that effectively stalls XRN1. Our efforts focused on the mammalian Dom3z/DXO enzyme which contains both 5' decapping and 5'-3' exoribonuclease activity. Interestingly, recombinant Dom3z/DXO enzyme did not stall on RNAs containing the 3' UTR of either Dengue virus or the Rift Valley Fever Virus N mRNA. This may suggest that there is a molecular arms race of sorts between the cell and the virus for supremacy of regulating the 5'-3' decay of RNA during infection.Item Open Access Assessing and understanding the generation and function of RNA decay intermediates in non-insect borne flaviviruses(Colorado State University. Libraries, 2019) Mundell, Cary T., author; Wilusz, Jeffrey, advisor; Geiss, Brian, committee member; Perera, Rushika, committee member; Reddy, Anireddy, committee memberCellular gene expression is an intricate process regulated on many levels that allows the cell to react correctly to stimuli or to maintain homeostasis. RNA viruses must act to preferentially drive production of their own messenger RNAs (mRNAs) and proteins in order to successfully replicate and ensure continued infection. Due to the necessity for RNA viruses to remain in the cytoplasm, regulatory factors that affect host mRNAs likely also affect the transcripts of RNA viruses. RNA decay represents a major pathway of regulation for mRNAs. A multitude of RNA viruses possess unique mechanisms that act to prevent the decay of viral transcripts and allow for successful translation. Members of the viral family Flaviviridae are positive sense, single-stranded RNA viruses that do not possess a poly(A) tail. Therefore, it is highly likely that these transcripts would be marked as deadenylated and shuttled down one of the RNA decay pathways that exist in the cell. Interestingly, members of the genera Flavivirus of the family Flaviviridae possess a conserved structured 3' untranslated region (UTR) that acts to interfere with the decay processes of the major cytoplasmic cellular 5'-3' decay enzyme XRN1. In addition, members of the generas Hepacivirus, Hepatitis C Virus (HCV) and Pestivirus, Bovine Viral Diarrhea Virus (BVDV), possess XRN1 stalling elements within their 5' UTRs. These stalling sites block the action of the exonuclease and generate decay intermediates. The generation of these decay intermediates represses XRN1 activity in the infected cell. Herein we demonstrate a new method for studying RNA decay through the use of XRN1-resistant RNAs (xrRNAs). In this method we utilize the well characterized xrRNA of Dengue Virus Type 2 (DENV2) as a readout to study the decay rates of relatively large RNA constructs. We show that not only is utilizing an xrRNA an effective method for confirming XRN1-mediated decay, but that the accumulation of the readout xrRNA can be utilized to understand changes in the decay kinetics of RNA substrates. We further utilize this method to demonstrate a lack of XRN1 stalling elements within the poliovirus internal ribosomal entry site (IRES) element. We provide evidence that the stalling of XRN1 in the 5' UTR of BVDV is dependent on both the presence of the entire IRES structure and the presence of a stem loop 5' to the IRES element through the analysis of a series of truncations. Finally, we demonstrate one possible role for the HCV and BVDV decay intermediates as the truncated IRES element maintains translatability in an in vitro system. Collectively, these data better define the structural requirements for the novel XRN1 stalling elements located in the 5' UTR of non-insect borne members of the Flaviviridae as well as the potential function of the decay intermediates.Item Open Access Binding of MBNL1 to CUG repeats slows 5'-to-3' RNA decay by XRN2 in a cell culture model of type I myotonic dystrophy(Colorado State University. Libraries, 2017) Zhang, Junzhen, author; Wilusz, Carol J., advisor; Wilusz, Jeffrey, advisor; Duval, Dawn, committee member; Di Pietro, Santiago, committee member; Yao, Tingting, committee memberType I myotonic dystrophy (DM1) is a multi-systemic inherited disease caused by expanded CTG repeats within the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene. The encoded CUG repeat-containing mRNAs are toxic to the cell and accumulate in nuclear foci, where they sequester cellular RNA-binding proteins such as the splicing factor Muscleblind-1 (MBNL1). This leads to widespread changes in gene expression. Currently, there is no treatment or cure for this disease. Targeting CUG repeat-containing mRNAs for degradation is a promising therapeutic avenue for myotonic dystrophy, but we know little about how and where these mutant mRNAs are naturally decayed. We established an inducible C2C12 mouse myoblast model to study decay of reporter mRNAs containing the DMPK 3' UTR with 0 (CUG0) or ~700 (CUG700) CUG repeats and showed that the CUG700 cell line exhibits characteristic accumulation of repeat-containing mRNA in nuclear foci. We utilized qRT-PCR and northern blotting to assess the pathway and rate of decay of these reporter mRNAs following depletion of mRNA decay factors by RNA interference. We have identified four factors that influence decay of the repeat-containing mRNA – the predominantly nuclear 5' 3' exonuclease XRN2, the nuclear exosome containing RRP6, the RNA-binding protein MBNL1, and the nonsense-mediated decay factor, UPF1. We have discovered that the 5' end of the repeat-containing transcript is primarily degraded in the nucleus by XRN2, while the 3' end is decayed by the nuclear exosome. Interestingly, we have shown for the first time that the ribonucleoprotein complex formed by the CUG repeats and MBNL1 proteins represents a barrier for XRN2-mediated decay. We suggest that this limitation in XRN2-mediated decay and the resulting delay in degradation of the repeats and 3' region may play a role in DM1 pathogenesis. Additionally, our results support previous studies suggesting that UPF1 plays a role in initiating the degradation of mutant DMPK transcripts. This work uncovers a new role for MBNL1 in DM1 and other CUG-repeat expansion diseases and identifies the nuclear enzymes involved in decay of the mutant DMPK mRNA. Our model has numerous applications for further dissecting the pathways and factors involved in removing toxic CUG-repeat mRNAs, as well as in identifying and optimizing therapeutics that enhance their turnover.Item Open Access Commandeering of the cellular HuR protein by alphaviruses affects the regulation of host post-transcriptional gene expression(Colorado State University. Libraries, 2013) Barnhart, Michael D., author; Wilusz, Jeffrey, advisor; Wilusz, Carol J., committee member; Laybourn, Paul J., committee memberIt was previously shown that cellular HuR protein binds to a U-rich region in the 3'UTR of Sindbis virus RNA resulting in stabilization of viral transcripts and increased replication efficiency. While the presence of this U-rich region is generally conserved among alphaviruses, a subset lacks a typical U-rich region. The 3'UTR of two alphaviruses - Ross River virus and Chikungunya virus - that do not contain a typical U-rich region were tested for HuR interactions by Electrophoretic Mobility Shift Assay. HuR protein bound these 3'UTRs with nanomolar affinities, similar to what was observed for the U-rich region of Sindbis virus. These observations demonstrate that the critical role for HuR-mediated viral RNA stabilization is likely a conserved property of most, if not all, members of the virus family. By analyzing deletion derivatives, we mapped the novel HuR binding sites in these two viruses to specific regions in their 3'UTR. Next, we uncovered four novel aspects of virus-host interaction and pathogenesis related to the high affinity interaction between the 3'UTR of alphaviruses and the cellular HuR protein. First, HuR protein, which is usually localized predominantly to the nucleus, dramatically accumulates in the cytoplasm during Sindbis virus (SinV) infection. Studies involving the transfection of constructs that express viral 3'UTR RNA fragments indicated that the mechanism of induction of HuR accumulation to the cytoplasm in infected cells is due to the viral RNA acting as a sponge for the protein. Second, HuR interaction with numerous cellular mRNAs was found to be drastically decreased during a SinV infection and was associated with dramatic destabilization of the cellular transcripts as determined by mRNA half-life analysis. Third, we found that the reduced amounts of free HuR during a SinV infection results in the increased targeting of mRNAs by miRNAs. Together, these data indicate that in the process of commandeering the cellular HuR protein for its own use, alphaviruses are also effectively destabilizing numerous cellular mRNAs. Interestingly, many of the cellular mRNAs affected by alphaviruses play key roles in inflammation, innate immune responses and other fundamental cellular processes. Finally, we observed a novel effect of SinV infection on alternative polyadenylation of cellular transcripts. This is likely a direct result of sequestration of the HuR protein in the cytoplasm by the virus, preventing the protein from influencing nuclear polyadenylation site choice. Intriguingly, SinV infection influences the poly(A) site choice of the HuR pre-mRNA, favoring a more translatable isoform to promote the overexpression of this viral host factor. Therefore, the alphaviral-induced alterations in cellular mRNA stability and polyadenylation identified in this thesis may play a very important but underappreciated role in pathogenesis.Item Open Access Global analysis of mRNA decay rates and RNA-binding specificity reveals novel roles for CUGBP1 and PARN deadenylase in muscle cells(Colorado State University. Libraries, 2011) Lee, Jerome Edward, author; Wilusz, Carol J., advisor; Wilusz, Jeffrey, advisor; Garrity, Deborah M., committee member; Curthoys, Norman P., committee memberType I Myotonic Dystrophy (DM1) is characterized by myotonia, cardiac conduction defects, muscle wasting, and insulin resistance. In patient muscle cells expression and function of the RNA-binding proteins CUGBP1 and MBNL1 are disrupted, resulting in altered mRNA metabolism at the levels of splicing and translation. Intriguingly, despite strong evidence for CUGBP1 being a regulator of mRNA turnover in humans and other organisms, the possibility that defects in mRNA decay contribute to DM1 pathogenesis has not been investigated to date. As such, we sought to further characterize the roles of CUGBP1 and its partner, the deadenylase PARN, in mRNA decay in mouse C2C12 muscle cells. The TNF message, which encodes a cytokine known to cause muscle wasting and insulin resistance when over-expressed, was stabilized by depletion of CUGBP1. The normally rapid decay of the TNF mRNA was also disrupted in cells treated with phorbol ester and this coincided with phosphorylation of CUGBP1. These findings provided impetus to undertake a global analysis of mRNA decay rates in muscle cells. Our investigation revealed that GU- and AU-rich sequence elements are enriched in labile transcripts, which encode cell cycle regulators, transcription factors, and RNA-processing proteins. Transcripts specifically bound to CUGBP1 in myoblasts are linked with processes such as mRNA metabolism, protein targeting to the endoplasmic reticulum, cytoskeletal organization, and transcriptional regulation, all of which have implications for muscle cell biology. Consistent with this, CUGBP1 depletion profoundly altered the formation of myotubes during differentiation. Finally we investigated whether PARN, which interacts with CUGBP1 and mediates rapid deadenylation of TNF in HeLa cell extracts, also plays a role in mediating mRNA decay in muscle. We identified 64 mRNA targets whose decay was dependent on PARN. Moreover, deadenylation of the Brf2 mRNA was impaired in PARN knock-down cells supporting that this mRNA is directly and specifically targeted for decay by PARN. Taken together our findings demonstrate that CUGBP1 and PARN are critical regulators of decay for specific sets of transcripts in muscle cells. It seems likely that some or all of the CUGBP1 targets we have identified may be affected in myotonic dystrophy. Defective mRNA turnover could be linked with defects in myogenesis, TNF over-expression, muscle wasting and/or ER stress, all of which have been documented in DM1.Item Open Access Global analysis reveals differential regulation of mRNA decay in human induced pluripotent stem cells(Colorado State University. Libraries, 2013) Neff, Ashley T., author; Wilusz, Jeffrey, advisor; Wilusz, Carol J., advisor; Thamm, Douglas H., committee member; Weil, Michael, committee memberInduced Pluripotent Stem (iPS) cells are able to proliferate indefinitely while maintaining the capacity for unlimited differentiation and these properties are reflected by global changes in gene expression required for reprogramming of differentiated cells. Although the rate of transcription is an important regulator of steady-state mRNA levels, mRNA decay also plays a significant role in modulating the expression of cell-specific genes. The contribution of regulated mRNA decay towards establishing and maintaining pluripotency is largely unknown. To address this, we sought to determine global mRNA decay rates in iPS cells and the genetically-matched fibroblasts (HFFs) they were derived from. Using a microarray based approach, we determined half-lives for 5,481 mRNAs in both cell lines and identified three classes of mRNAs whose decay is differentially regulated in iPS cells compared to HFFs. We found that replication-dependent histone mRNAs are more abundant and more stable in iPS cells, resulting in increased histone protein abundances. This up-regulation of histone expression may facilitate the unique chromatin dynamics of pluripotent cells. A large set of C2H2 ZNF mRNAs are also stabilized in iPS cells compared to HFFs, possibly through reduced expression of miRNAs that target their coding regions. As many of these mRNAs encode transcriptional repressors, stabilization of these transcripts may support the overall increased expression of C2H2 ZNF transcription factors in early embryogenesis. Finally, we found that mRNAs containing C-rich elements in their 3'UTR are destabilized in iPS cells compared to HFFs and many of these mRNAs encode factors important for development. Interestingly, we also identified the Poly(C)-Binding Protein (PCBP) family as differentially regulated in iPS cells and investigated their possible involvement in regulation of the mRNAs in our dataset identified as destabilized in iPS cells and having C-rich 3'UTR elements. Thus, we identified several interesting classes of mRNAs whose decay is differentially regulated in iPS cells compared to HFFs and our results highlight the importance of post-transcriptional control in stem cell gene expression. Coordinated control of mRNA decay is evident in pluripotency and characterization of the mechanisms involved would further contribute to our limited understanding of pluripotent gene expression and possibly identify additional targets for reprogramming.Item Open Access In vitro amplification and enhanced trans-species transmission of chronic wasting disease prions(Colorado State University. Libraries, 2009) Kurt, Timothy Daniel, author; Hoover, Edward A., advisor; Wilusz, Jeffrey, advisorChronic wasting disease (CWD) is a prion disease of deer, elk and moose that is spreading rapidly in North America. Like all prion diseases, CWD is associated with conversion of a normal protein, PrPC, to a protease-resistant conformer, PrPRES (or PrPCWD). Little is known about the mechanisms of prion conversion or how it could lead to the rapid spread of CWD among cervids in nature. In this dissertation, I demonstrate the in vitro conversion of PrPC to PrPCWD via two protocols: non-denaturing amplification and serial protein misfolding cyclic amplification (sPMCA). Serial PMCA using brain substrate from transgenic mice that express cervid PrPC [Tg(CerPrP)1536 mice] produced PrPCWD amplification of >6.5 x 109-fold after six rounds. Efficient in vitro amplification of PrPCWD is a significant step toward potential ante-mortem detection of PrPCWD in CWD-infected animals. Whether CWD presents a threat to non-cervid species is not known. To predict non-cervid susceptibility to CWD, I used sPMCA to amplify PrP CWD in normal brain substrates from several non-cervid species. I show that brain homogenates from several CWD-susceptible species, such as ferrets and hamsters, support amplification of PrPCWD by sPMCA, whereas brain homogenates from CWD-resistant species, such as laboratory mice and transgenic mice expressing human PrPC, do not. Three common rodent species (including prairie voles and field mice) that share the environment with infected cervids supported PrPCWD amplification, whereas several other species (including prairie dogs, cats and coyotes) did not. Analysis of PrP sequences suggests that ability to support amplification of PrPCWD in trans-species sPMCA correlates with the presence of asparagine at position 170 of the substrate species PrP. I then inoculated CWD from deer into prairie voles (Microtus ochrogaster) and found they are somewhat susceptible to CWD. Inoculation of prairie voles with trans-species sPMCA products resulted in shorter and more consistent incubation periods. Furthermore, immunohistochemical analysis revealed an altered pattern of CWD prion deposition in infected voles compared to infected Tg(CerPrP)1536 mice, suggesting a different CWD strain. These results indicate that sPMCA can be used to increase PrPCWD detection sensitivity, predict susceptibility to CWD, accelerate adaptation in non-cervid species, and create new strains of CWD.Item Open Access Influence and regulation of PCBP2 and YTHDF2 RNA-binding proteins during self-renewal and differentiation of human induced pluripotent stem cells(Colorado State University. Libraries, 2019) Heck, Adam M., author; Wilusz, Carol J., advisor; Wilusz, Jeffrey, advisor; Osborne Nishimura, Erin, committee member; Montgomery, Tai, committee member; Zhou, Wen, committee memberEmbryonic stem cells (ESCs) are able to self-renew or differentiate into any cell type in the body, a property known as pluripotency that enables them to initiate early growth and development. However, the ethical implications of harvesting and manipulating ESCs hinders their use in basic research and the clinical applications. Thus, the discovery that somatic cells can be exogenously reprogrammed into induced pluripotent stem cells (iPSCs) offers new and exciting possibilities for gene therapy, personalized medicine and basic research. However, more research is needed into the mechanisms involved in regulating pluripotency in order for iPSCs to reach their full potential in the research lab and clinic. To maintain a state of self-renewal, yet also be able to rapidly differentiate in response to external signals, pluripotent stem cells need to exert tight control over gene expression through transcriptional and post-transcriptional mechanisms. There are several notable transcriptional networks that regulate pluripotency, but the post-transcriptional mechanisms remain poorly characterized. mRNA decay is one form of post-transcriptional regulation that can help to both maintain the steady-state of a transcriptome or facilitate its rapid remodeling. To this end, degradation rates are influenced by the elements contained in an mRNA and the RNA-binding proteins (RBPs) they associate with. Previous reports have indicated the RNA modification N6-methyladenosine (m6A) and C-rich sequence elements (CREs) can affect mRNA decay in pluripotent stem cells. Therefore, we sought to further understand the roles of m6A and CREs in mRNA decay in stem cells by characterizing the expression and mRNA targets of two RBPs that recognize these elements, YTHDF2 and PCBP2, respectively. In this thesis, I report YTHDF2 is differentially regulated in pluripotent and differentiated cells and that YTHDF2 contributes to pluripotency by targeting a group of mRNAs encoding factors important for neural development. The down-regulation of YTHDF2 during neural differentiation is consistent with increased expression of neural factors during this time. Moreover, YTHDF2 expression is regulated at the level of translation via elements located in the first 300 nucleotides of the 3' untranslated region of YTHDF2 mRNA. Based on these results, I propose that stem cells are primed for rapid differentiation by transcribing low levels of mRNAs encoding neural factors that are subsequently targeted for degradation, in part by YTHDF2, until differentiation is induced. On the other hand, PCBP2 is up-regulated upon differentiation of pluripotent stem cells and regulates several mRNAs associated with pluripotency and development, including LIN28B. Notably, expression of long non-coding RNAs (lncRNAs) that contain human endogenous retrovirus element H (HERV-H) is influenced by PCBP2. HERV-H lncRNAs are almost exclusively expressed in stem cells and play a role in maintaining a pluripotent state, although their functions are not fully understood. Intriguingly, some HERV-lncRNAs can also regulate PCBP2 expression, as altering the expression of LINC01356 or LINC00458 effects PCBP2 protein levels. Based on these results, I propose the reciprocal regulation of PCBP2 and HERV-H lncRNAs influences whether stem cells maintain a state of self-renewal or differentiate. Taken together, these findings demonstrate that YTHDF2 and PCBP2 post-transcriptionally regulate gene expression in stem cells and influence pluripotency.Item Open Access Inhibition of the host 5'-3' RNA decay pathway is a novel mechanism by which flaviviruses influence cellular gene expression(Colorado State University. Libraries, 2014) Moon, Stephanie L., author; Wilusz, Jeffrey, advisor; Wilusz, Carol, advisor; Schenkel, Alan, committee member; Curthoys, Norman, committee memberHost gene expression is an intricate process that requires many levels of regulation to allow the cell to react properly to a given stimulus or maintain homeostasis. One mechanism by which RNA viruses perturb host gene expression and potentially favor the allocation of host cell resources for viral proliferation is through interfering with cellular post-transcriptional processes. Furthermore, because viral RNAs must persist in the host cell cytoplasm to allow translation of viral proteins and ultimately viral replication, the same post-transcriptional processes that regulate host messenger RNAs (mRNAs) likely act on viral RNAs as well. The general RNA decay machinery in the cell serves as an important regulatory step for proper gene expression at the post-transcriptional level. Many RNA viruses have evolved unique mechanisms for dealing with the cellular RNA decay machinery to preserve their transcripts and ensure a productive infection. Flaviviruses contain positive-sense, single-stranded RNA genomes that are not polyadenylated. Therefore, these viral RNAs are likely recognized by the host cell as deadenylated, incongruous mRNAs and are likely substrates for the general cellular RNA decay machinery. Remarkably, flaviviruses including the dengue viruses (DENV) and West Nile virus (WNV) produce an abundant non-coding subgenomic RNA (sfRNA) during infection that is generated through incomplete degradation of the viral genome by the host 5'-3' exoribonuclease 1 (XRN1). We demonstrate that human and mosquito XRN1 stalls on highly structured, conserved elements in the 3' untranslated region of flaviviral RNAs, resulting in sfRNA formation. Furthermore, we determined that these sfRNAs act as competitive, reversible inhibitors of XRN1. Infected cells display several signs of sfRNA-dependent XRN1 dysfunction, including the accumulation of uncapped transcripts and an overall stabilization of host mRNAs. Additionally, sfRNA acts as a weak inhibitor of the host cell RNA interference (RNAi) pathway. We propose that sfRNA likely acts as a sponge for Argonaute-2 (AGO2) and DICER, and have determined that siRNA-mediated decay is suppressed in an sfRNA-dependent fashion in flavivirus-infected human cells. This suppression of the RNAi pathway appears to alter host gene expression to a limited extent, and may be especially important for viral replication in the mosquito vector. Other flaviviruses, including hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) do not form an sfRNA from their 3' untranslated regions, but they do contain highly structured 5' untranslated regions. Herein we show that aside from acting as internal ribosome entry sites, the 5' UTRs of HCV and BVDV also stall and inhibit XRN1. Therefore, flaviviruses, pestiviruses and hepaciviruses appear to inhibit a major mRNA decay pathway by suppressing XRN1 activity via highly structured viral RNAs. Consequences of XRN1 suppression during viral infection include the stabilization and upregulation of short-lived transcripts including those encoding oncogenes, angiogenic factors, and pro-inflammatory factors. Furthermore, we present evidence that WNV sfRNA may dysregulate the coordination between mRNA stability and transcription. Therefore, the suppression of XRN1 may potentially act as an important mechanism by which diverse viruses in the Flaviviridae induce pathogenesis by dysregulating cellular gene expression.Item Open Access Nucleophosmin deposition during mRNA 3' end processing influences poly(A) tail length and mRNA export(Colorado State University. Libraries, 2011) Sagawa, Fumihiko, author; Wilusz, Jeffrey, advisor; Wilusz, Carol J., advisor; Reddy, Anireddy S. N., committee member; Thamm, Douglas, committee memberDuring polyadenylation the multi-functional protein nucleophosmin is deposited onto all cellular mRNAs analyzed. Premature termination of poly(A) tail synthesis using cordycepin abrogates deposition of the protein onto the mRNA, indicating natural termination of poly(A) addition is required for nucleophosmin binding. Nucleophosmin appears to be a bona fide member of the complex involved in 3' end processing as it is directly associated with the AAUAAA-binding CPSF-160 protein and can be co-immunoprecipitated with other polyadenylation factors. Furthermore, reduction in the levels of nucleophosmin results in hyperadenylation of mRNAs, consistent with alterations in poly(A) tail chain termination. Finally, knock down of nucleophosmin results in retention of poly(A)+ RNAs in the cell nucleus, indicating that nucleophosmin binding influences mRNA export. Collectively these data suggest that nucleophosmin plays an important role in poly(A) tail length determination and helps network 3' end processing with other aspects of nuclear mRNA maturation.Item Open Access Repeated sequences encoding Cys2His2 zinc finger motifs influence mRNA polyadenylation and localization(Colorado State University. Libraries, 2017) Jalkanen, Aimee L., author; Wilusz, Carol, advisor; Wilusz, Jeffrey, advisor; Bailey, Susan, committee member; Bouma, Gerrit, committee member; Thamm, Douglas, committee memberThe Cysteine2 Histidine2 zinc finger (C2H2-ZNF) proteins are a vast family with over 700 members in primates, many of which are transcription factors with important roles in development, differentiation, cell cycle progression, and tumor suppression. Due to the sheer number of C2H2-ZNF proteins and their roles in modulating expression of other genes, any mechanism for coordinating their expression could have wide-ranging impacts on cell function and phenotype. Previously, a large subset of C2H2-ZNF transcripts were determined to have significant populations with short poly(A) tails. Here, we show that multiple C2H2-ZNF mRNAs accumulate with very short or undetectable poly(A) tails, even when newly transcribed. Furthermore, these C2H2-ZNF mRNAs are restricted to the nucleus. Reporter mRNAs with sequences from the ZNF12 open reading frame (ORF) and/or the 3' untranslated region (3' UTR) have short poly(A) tails and are retained in the nucleus. Deletion analysis suggests that repeated sequence elements in the ZNF12 mRNA that code for zinc finger protein motifs are important in controlling both poly(A) tail length and nuclear localization. Remnants of C2H2-ZNF motif sequences found in the ZNF12 3' UTR are also able to confer short poly(A) tails and nuclear retention. Finally, we use RNA-fluorescence in situ hybridization (RNA-FISH) to reveal that ZNF12 reporter transcripts are found in foci within the nucleus that could represent sites for storage or processing. Overall, our findings suggest repeated sequence elements encoding C2H2-ZNF protein motifs play a dual role as regulatory elements that may coordinate expression of the C2H2-ZNF protein family by controlling post-transcriptional events.Item Open Access SARS-CoV-2 viral RNA biology and its impact on the infected cell(Colorado State University. Libraries, 2023) Altina, Noelia H., author; Wilusz, Jeffrey, advisor; Argueso, Lucas, committee member; Geiss, Brian, committee member; Perera, Rushika, committee member; Yao, Tingting, committee memberThe fine-tuning of the replication and transcription of RNA viruses often requires the interaction of viral RNAs with cellular RNA binding proteins. This project addresses fundamental knowledge gaps on the molecular mechanisms that underlie SARS-CoV-2 gene expression, regulation, and viral RNA-host interactions. After infection, SARS-CoV-2 generates a large set of sub-genomic mRNAs, each containing an identical ~70 base 'leader' region in their 5'UTR (from position 14 to position 75 in RefSeq NC_045512) and a 229 base region at the 3'UTR (from position 29,675 to position 29,903 in RefSeq NC_045512) generated by discontinuous transcription. The accumulation of a considerable amount of these leader/3'UTR regions during the infection represents a possible sink for cellular RNA binding proteins. We demonstrated that PTBP1, a cellular protein involved in the regulation of alternative splicing, binds to the SARS-CoV-2 leader region. SARS-CoV-2 infection critically impacted the splicing of several cellular pre-mRNAs that are normally regulated by PTBP1. Mechanistically, we suggest that SARS-CoV-2 sequesters and influences the re-localization of shuttling splicing factors like PTBP1 from the nucleus to the cytoplasm resulting in significant effects on the host cell splicing machinery leading to changes in cellular mRNA splicing patterns during SARS-CoV-2 infection. Given the current extensive interest in epigenetic methylations of both cellular and viral RNAs, our study also explored the role of post-transcriptional RNA modifications on viral mRNAs. We demonstrated that SARS-CoV-2 can usurp the cellular enzyme, namely PCIF1, to place the m6Am modification at the cap proximal position in its mRNAs. This double methylation is usually found on all host mRNAs that initiate with an adenosine residue, and thus SARS-CoV-2 likely installs this modification on its mRNAs to avoid host immune recognition. Interestingly, we also discovered that capping and m6Am modification are tightly regulated throughout the infection. The highest levels of these 5' end RNA modifications were observed at 12 hours post infection, correlating with the full establishment of viral gene expression in infected cells. These findings indicate that 5' end modification of SARS-CoV-2 transcripts is not simply a default process but rather undergoes unanticipated regulation throughout the infection. Collectively, the data presented provide not only new insights into the complex interactions that SARS-CoV-2 has with the RNA biology of the cell during infection, but also identify attractive potential targets for developing novel anti-coronavirus drugs to treat future emerging coronavirus diseases.Item Open Access Sindbis virus usurps the cellular HUR protein to stabilize its transcripts and promote infections of mammalian and mosquito cells(Colorado State University. Libraries, 2010) Sokoloski, Kevin J., author; Wilusz, Jeffrey, advisor; Wilusz, Carol, advisor; Blair, Carol, committee member; Peersen, Olve, committee member; Quackenbush, Sandra, committee memberMembers of the genus Alphavirus are recognized as significant human pathogens. Infection of vertebrate hosts often results in febrile illness and occasionally severe encephalitis. The archetypical alphavirus is Sindbis virus, which we have utilized in these studies. The genomic and subgenomic RNAs of Sindbis virus strongly resemble cellular mRNAs as they are capped at their 5’ ends and polyadenylated at their 3’ termini. These features allow the viral RNAs to act like cellular mRNAs and make them prime substrates for the cellular mRNA decay machinery. Sindbis virus RNAs are indeed subject to degradation by the cellular mRNA decay machinery in cell culture models of infection. Nevertheless, they decay by a mechanism that is different from the majority of cellular mRNAs as the decay of Sindbis virus transcripts is predominantly deadenylation-independent. As cellular mRNAs are often regulated by elements present in their 3’ untranslated regions (UTR), we hypothesized that these viral 3’UTR elements were functioning similarly to cellular mRNA stability elements resulting in the enhancement of viral infection. The primary goal of the research described in this dissertation was to characterize in mechanistic detail how the Sindbis virus 3’UTR represses deadenylation. To this end we used both cell free extracts and tissue culture systems to assay the effects of the viral 3’UTR on transcript stability. Interestingly, multiple elements were found to be independently repressing deadenylation in mosquito cytoplasmic extracts. Further examination revealed that a major stability determinant was the U-rich element (URE) observed in the 3’UTR of many alphaviruses. The ability to repress deadenylation in our cell free extract system was similarly observed with the UREs of Venezuelan equine, eastern equine, western equine and Semliki Forest viruses. Taken together, these data strongly assert that the repression of deadenylation via the URE is evolutionarily conserved. Prior to this study, the URE had no ascribed function. The repression of deadenylation imparted by the URE correlated with the binding of a cellular 38kDa factor. This 38kDa factor was determined to be the cellular HuR protein. Both the human and mosquito HuR proteins were found to bind with high affinity to the Sindbis virus 3’UTR. Reduction of cellular HuR protein levels using RNAi resulted in an increase in the rate of viral RNA decay. Furthermore, a significant decrease in the titer of progeny virus was observed. A similar effect on viral titer was observed when the predominant HuR binding site, the URE, was deleted from the viral 3’UTR. Taken together these observations identify a novel Alphavirus/ host interface that significantly impacts viral biology. Furthermore these studies have confirmed our hypothesis that the members of genus Alphavirus have indeed evolved RNA stability elements that resemble cellular mRNA stability elements for the purpose of enhancing viral infection. Furthermore these studies identify a potential therapeutic anti-viral target - the cellular HuR protein.Item Open Access The role of cellular RNA decay pathways in Sindbis virus infection(Colorado State University. Libraries, 2009) Garneau, Nicole L., author; Wilusz, Jeffrey, advisor; Wilusz, Carol, advisorSindbis virus is the prototypic species of the Alphavirus genus. Members of this genus can cause febrile illness, arthritic pain and potentially fatal encephalitis. The alphaviral lifecycle generates single-stranded, positive-sense genomic and subgenomic RNAs which are capped on the 5' terminus, contain 5' and 3' untranslated regions (UTRs), and are polyadenylated at the 3' terminus. These characteristics make alphaviral RNAs similar in structure to cellular mRNAs. Such features allow alphaviruses, such as Sindbis, to benefit from the host cell translation process; however, they also could make the viral transcript vulnerable to the cellular mRNA decay enzymes. mRNA decay is a form of post-transcriptional regulation of gene expression found in both mammalian and mosquito hosts of Sindbis virus. The interaction between Sindbis viral RNAs and mRNA decay pathways was investigated in this dissertation. Using a novel in vivo viral RNA decay assay to accurately assess the rate of alphavirus RNA decay during infection, we found a correlation between Sindbis viral RNA stability and viral replication efficiency, demonstrating mRNA decay potentially represents a novel host cell restriction factor. We established that the RNAi pathway likely plays a dominant role in the decay of the viral RNAs during infection in mammalian cells. These data represent a novel demonstration that the RNAi pathway is potentially an effective antiviral response in the mammalian host as it is in the mosquito host. With the development of a highly sensitive method to assess poly(A) tail length, we were able to demonstrate the importance of the viral 3'UTR as a repressor of deadenylation of viral RNAs in vivo. Lastly, we found that Repeat Sequence Element 3 (RSE 3), the third and final in a series of three RSEs within the viral 3'UTR, hinders the processivity of the cellular deadenylases on viral RNAs in vitro, providing the first evidence for a function of this conserved alphaviral genome element. Taken together, these results shed light on the much understudied area of viral RNA decay. Our data support the notion that the interaction between viral RNAs and the cellular RNA decay machinery is very important to the biology of the virus.Item Open Access Zika virus noncoding sfRNA sequesters viral restriction factors involved in RNA splicing and nucleic acid editing(Colorado State University. Libraries, 2019) Ontiveros Valles, Jesús Gustavo, author; Wilusz, Jeffrey, advisor; Geiss, Brian, committee member; Chen, Chaoping, committee memberZIka virus (ZIKV) is a single-stranded positive sense RNA flavivirus that is transmitted primarily by Aedes aegypti. To date, all vector-borne flaviviruses are known to generate stable subgenomic flavivirus RNAs (sfRNA), due to the stalling of the major cytoplasmic 5'-3' exoribonuclease XRN1 at a knot-like three helix junction structure located in viral 3' untranslated regions (UTRs). Formation of sfRNAs not only stalls XRN1, but also represses its function. sfRNA decay intermediates accumulate to high levels in infected cells and studies with other flaviviruses have implicated sfRNAs in cytopathology. Our objective was to characterize the function of ZIKV sfRNAs to gain insight into ZIKV pathogenesis. Specifically, we identified host proteins that interact with ZIKV sfRNA and have begun to evaluate their role in cytopathology and pathogenesis. RNA pull-down experiments revealed that PHAX and SF3B1, critically important RNA splicing factors involved in nuclear-cytoplasmic shuttling, bind sfRNA. Additionally, the cytidine deaminase APOBEC3C was found to bind ZIKV sfRNA. Knockdown and subsequent overexpression of these RNA Binding Proteins (RBPs) identified the nucleic acid deaminase APOBEC3C and the splicing-associated factor PHAX as negative viral restriction factors whose activity may be suppressed and/or altered by sfRNA interaction during ZIKV infection. sfRNA interactions with the splicing factors also resulted in the accumulation of aberrantly spliced transcripts, possibly due to sequestration of the host cell proteins. Thus, in addition to targeting XRN1, sfRNAs appear to interact with a set of RBPs to disrupt cellular mRNA decay regulation as well as other RNA processing events in an effort to compromise multiple steps of RNA metabolism and promote pathogenesis.