Jeffrey Wilusz - US grants

The overall objective of my laboratory is to elucidate pathways involved in the post-transcriptional regulation of gene expression. We have recently identified a 50kd protein which binds viral pre- mRNAs in a sequence-specific fashion. We have identified binding sites for this protein upstream of the HIV poly(A) signal and downstream of the SV40 polyadenylation signal. This RNA-protein interaction clearly influences the efficiency of 3' end processing, as it can substitute for the downstream element of the SV40 late polyadenylation signal. In addition, the binding of the 50kd protein leads to a site-specific RNA cleavage event near the protein binding site. We will evaluate the functional significance of the 50kd protein with respect to SV40 and HIV mRNA metabolism. Specifically, the role of the protein in 3' end formation, RNA stability, and transcription termination will be evaluated. The relationship of the 50kd protein to another downstream element-specific factor, the hnRNP C proteins, will also be explored. Second, we will purify and characterize the site-specific nuclease activity associated with the 50 kd protein. In addition to relating this information to the function of the 50kd protein in mRNA metabolism, the characterization of this unique activity may be exploitable for future RNA studies such as the "restriction mapping" of spliced families of genes. Finally, we will molecularly clone the 50kd protein cDNA, raise specific antibodies, and biochemically characterize the 50kd protein- RNA interaction. These studies will not only provide important reagents for future work, but will also provide the foundation to construct plausible models for the mechanism of action of this mRNP. This study has pertinence to many health related topics, as post- transcriptional controls play a role in numerous cellular processes. In addition, mutations in the poly(A) signal have been shown to be directly associated with disease (ie thalassemia) in humans. Finally, increasing our knowledge of HIV regulatory elements will provide new avenues for therapeutic control of AIDS.

The long term goal of my research program is to understand mechanisms of constitutive and regulated pre-mRNA 3'end processing. Two types of sequence elements constitute viral and cellular polyadenylation signals. Core elements, consisting of conserved upstream AAUAAA and downstream U-rich motifs, mediate basal levels of 3'end processing. Relatively uncharacterized auxiliary elements, located upstream or downstream of the core elements, may act as enhancers or repressors of 3'processing. Many regulated polyadenylation signals which are used selectively contain either (1) non-consensus sequences in place of the core elements or (2) novel auxiliary elements located upstream or downstream of the core elements. The focus of this proposal will be to elucidate mechanisms of selective polyadenylation of viral pre-mRNAs by identifying functional alternative core motifs and auxiliary elements and characterizing factors which interact with them. First, we will identify and characterize alternative sequence motifs which can serve as core downstream elements by mapping the site of CstF interaction on the unconventional Human Papillomavirus (HPV) 11, 16 and 35 early polyadenylation signals. Second, using a systematic series of mutations, we will identify the core upstream elements of the regulated HPV 11 early polyadenylation signal which lacks a conventional AAUAAA. Third, using a similar series of directed mutations, we will identify novel auxiliary upstream and downstream elements in these three signals. Proteins which specifically bind these elements will be characterized by a UV cross linking approach. Fourth, we will investigate the mechanism of stimulation of 3'end processing by DSEF-1, the only known example of an auxiliary downstream element binding factor. Collectively, these first four studies will systematically characterize nucleic acid elements and protein factors which may play role in selective usage of polyadenylation signals and provide mechanistic insights into the role of these elements in the regulation of HPV 3'end formation. Finally, we will apply the insights gained in these studies to elucidate the mechanism of selective usage of B19 parvovirus poly(A) signals at early and late times post infection. The proposed studies will, therefore, lead to an understanding of a key process in viral gene expression. This information, in turn, will provide an understanding at the molecular level of factors which influence viral host range and pathogenesis, as well as identify potential areas to target viral therapeutics.

posttranscriptional RNA processing; nucleic acid metabolism; tumor necrosis factor alpha; messenger RNA; genetic regulation; polyadenylate; RNA binding protein; nucleic acid sequence; site directed mutagenesis; cell free system;

Regulation of mRNA stability plays a key role in controlling the expression of many growth factors, cytokines and transcription factors. Regulated mRNA turnover, therefore, plays an important role in cell growth, differentiation and development, and can have a significant impact on several disease states, including cancer. The mechanisms and factors that mediate regulated mRNA turnover are, however, generally unclear. We have discovered a novel enzymatic activity in mammalian cells that specifically removes the 5' cap from mRNAs. Furthermore, this decapping activity is highly regulated in our in vitro assay system. The poly (A) tail of the transcript, in conjuction with poly (A) binding protein, specifically represses decapping through a novel, elF4E-independent complex. AU-rich instability elements, which regulate the stability of many short-lived mRNAs in vivo, dramatically stimulate decapping in our in vitro assays. In this application, we will build upon these observations and investigate the underlying mechanisms that regulate decapping of mRNAs in mammalian cells. First, we propose to identify and characterize the novel human decapping enzyme. Second, we will investigate the mechanism of poly(A)-mediated repression of decapping. Finally, we will address the factors and mechanisms involved in the stimulation of decapping by AU-rich instability elements. In total, these studies should provide pivotal insights into this important area of post-transcriptional regulation of gene expression in mammalian cells.

Sensitive, quantitative and efficient data imaging/analysis is vital for all facets of scientific research. This proposal requests funds to purchase a Molecular Dynamics Typhoon 8600 Variable Mode Imager Workstation. This imaging workstation will allow multi-color fluorescence, phosphorimaging and chemiluminescence detection in a single high throughput system to serve the needs of 16 research groups located in the Dept. of Microbiology & Mol. Genetics, many of which will use the instrument on a daily basis. The areas of research supported by this instrument are broad, including replication, transcription, mRNA processing, signal transduction, cell signaling and pathogen- host interactions. Since the users of this imaging system will be moving to a new off-site research facility, no comparable instrumentation will be available in their new building (ICPH) that is located in a new research incubator area called Science Park in an economically disadvantaged section of Newark, NJ. This move makes purchase of this device a key component of the successful relocation of these productive research programs, and would represent an important contribution to the development of the Newark Science Park Initiative. The equipment will be supervised by Dr. J. Wilusz and maintained by departmental staff as a key component of a new multi-user imaging facility in the new ICPH building. These individuals have a proven rack-record in maintaining high-end equipment servicing departmental researchers. Duties of Imaging Facility Staff will include operation and instruction on use of the device, monitoring usage and developing future sharing agreements for other Science Park Researchers. Training in ImageQuant and Excel software will be provided by an on-staff IT Specialist to ensure the device will be used to its full potential by all research groups. It is anticipated that the Typhoon Imager will not only increase research productivity of the identified user group, but will also allow the development of novel experimental approaches that would not be possible without the instrument.

Cyclooxygenases are the key and rate-limiting enzymes in the production of prostaglandins. Prostaglandins play an important role in many diverse biological processes. The goal of this project is to uncover the sequences and factors involved in the regulation of formation and function of the unusually large 3' untranslated region (UTR) of the human cyclooxygenase-2 (COX-2) gene. This ~ 2.5 kb 3' UTR likely contains elements that regulate gene expression by several post-transcriptional mechanisms. COX-2 is a highly regulated gene, and previous studies have shown that transcriptional activation alone cannot account for the sustained induction of the mRNA under abnormal conditions. Therefore, understanding all of the post-transcriptional molecular mechanisms involved in COX-2 gene regulation is critical. First, auxiliary regulatory elements will be identified that play a role in the alternative polyadenylation of COX-2, the process that determines the size of the 3' UTR. Second, protein factor(s) that recognize and bind these auxiliary regulatory elements will be identified. Finally, turnover of the COX-2 mRNA regulated by its 3' UTR will be investigated.

These studies will lead to new insights into mechanisms of gene regulation, with broad applicability to many areas of biology. These investigations will be excellent training for two women who are graduate students in the lab, thereby enhancing the representation of women in experimental science. In addition, the Principal Investigator strives to serve as a role model for women in science. Given the location of the laboratory in an inner-city environment, these investigations will also serve as an outstanding training opportunity for undergraduates and minorities.

DESCRIPTION (provided by applicant): Our overall goal is to elucidate the pathways and mechanisms involved in regulated mRNA stability and post-transcriptional control in general. These processes play a very important role in controlling gene expression related to cell growth and differentiation. Our approach is three-fold. First, we will expand our studies on nucleophosmin, a protein that is deposited on mRNAs as a novel polyadenylation mark, and determine its role in the coordination of gene expression. This work will involve the identification of auxiliary proteins involved in the polyadenylation mark, a determination of the requirements and mechanism of nucleophosmin deposition, the delineation of mRNA targets and a determination of the function(s) of nucleophosmin in post-transcriptional control. Second, we will perform a series of in vivo and in vitro assays to elucidate the full impact of CUG-BP, a regulator of poly(A) tail shortening in human cells, along with its target deadenylase enzyme PARN on the control of gene expression via the modulation of mRNA stability. Finally, we have identified a novel function of for a cytoplasmic Lsm complex in mRNA stability. The goal of the third aim of this proposal is to determine the underlying mechanism for Lsm-mediated stabilization of targeted mRNAs. In summary, these studies should provide new insights into the mechanisms and regulation of mRNA stability as well as post-transcriptional control in general. Given the impact of RNA biology on cellular growth control, these studies may well provide significant insights into the molecular basis of disease (e.g. cancer, myotonic dystrophy) and possibly improved strategies for molecular therapeutics.

[unreadable] DESCRIPTION (provided by applicant): Mosquitoes are vectors of multiple viral pathogens, a large number of which are RNA viruses. Strategies for viral control may target the vector itself or the interaction between the virus and the vector. This application is based upon the hypothesis that RNA viruses have evolved specific mechanisms for evading the mRNA turnover machineries of the mosquito cell and therefore mRNA decay factors may represent a novel target for therapeutics. Our current knowledge of mRNA decay in insects is minimal thus the primary goals of this proposal must be to elucidate the factors and pathways involved in mRNA turnover in the Aedes mosquito and characterize their regulation. Using an in vitro approach that we have successfully adapted to mosquito cell extracts along with in vivo assays, the goal of Aim I is to characterize the processes of mRNA deadenylation, decapping and decay in mosquitoes. In Aim II we will use this knowledge to gain insights into mechanisms of regulated mRNA decay mediated by togavirus 3' untranslated regions that we have observed. The final aim of the application will address the underlying mechanisms responsible for how non- polyadenylated viral RNAs avoid degradation upon entry into the mosquito cell. In summary, this information will provide fundamental insights into insect molecular biology and virus-host interactions that will provide the groundwork for novel avenues of arbovirus control. [unreadable] [unreadable] [unreadable]

The cellular RNA decay machinery routinely polices the cell and effectively removes unwanted RNAs. We have recently discovered that alphaviruses, including VEE, possess specific RNA stabilizing elements in their 3' UTR that recruit a cellular factor and block the deadenylation/decay of viral transcripts, thereby promoting an efficient and productive infection. Based on these data, we hypothesize that many if not all RNA viruses that encode capped and polyadenylated transcripts have evolved mechanisms to selectively suppress the cellular mRNA decay machinery as a prerequisite for efficient virus gene expression. The goal of Aim I of this proposal is to test this hypothesis by assessing whether we can extend our observations on viral suppression of the cellular RNA deadenylation/decay machinery to additional alphaviruses (WEE and EEE) as well as negative sense RNA viruses (Ebola, Marburg and Nipah). In addition to expanding our knowledge of molecular host-virus interactions, these studies will also test whether these agents employ a similar strategy as the alphaviruses for mediating viral RNA stability. If these RNA viruses use a single (as preliminary data indicate is the case for alphaviruses) or limited number of strategies to suppress the cellular mRNA decay machinery, this represents a novel and attractive target for antiviral therapeutics. To this end, the goal of Aim 2 is to optimize our established in vitro assays for measuring viral mRNA stability to allow for the rapid and effective screening of chemical compound libraries. The final aim of this proposal is to perform a chemical library screen to identify and validate candidate lead compounds that overcome viral suppression of the cellular RNA decay machinery. These compounds would represent attractive candidates for further development as antiviral therapeutics that may very well have a broad spectrum activity against a variety of viruses of biodefense significance. This research project fits within the RMRCE Integrated Research Focus on Viral Therapeutics, and will interact directly with RPs 3.1, 3.2, 3.5, 3.7 and 3.8, and utilize the resources of Core E. The goals of this project should add significant expertise to and synergize well with the RMRCE Viral Therapeutics Focus Group, particularly due to the innovative basic virology questions being asked and the novel approach to develop antivirals that could target multiple families of RNA viruses of interest to the Group.

Intellectual Merit: 'DNA codes for mRNA codes for Protein' is the well-established model of gene expression in cells. Recent evidence, however, suggests that the regulation of gene expression in our cells has many additional nuances that were not anticipated by this simple model. The amount of mRNA in the cytoplasm is known to be a product of both the rate of production of the mRNA (transcription) as well as the degradation rate of the mRNA. The underlying hypothesis for this research project is that there is a way for the status of the cytoplasmic mRNA to be communicated back to the nucleus and influence the rate of transcription. In other words, if the rate of degradation of a specific mRNA in the cytoplasm is increased, a signal can be sent back to the nucleus to increase the rate of synthesis (transcription), thus maintaining a steady-state level of the mRNA in the cytoplasm. Preliminary evidence has been obtained to suggest that this may well be the case and the goal of this research project is to formally test this hypothesis in proof of concept studies. The ability of an mRNA to send signals back to the nucleus to regulate its own synthesis is a novel idea that has fundamental bearing on how genetic information in cells is expressed. Thus a full understanding of this process has important implications in a variety of biological disciplines.

Broader Impacts: The research project brings together an interdisciplinary team that will result in the cross-training of both graduate students and postdoctoral level researchers. Such interdisciplinary training will provide an important skillset to these trainees for future career success. The project will strive to recruit underrepresented populations into the research endeavors to enhance scientific diversity. Data from the project will be incorporated into an advanced undergraduate/graduate course to give a broad set of students exposure to cutting-edge research. Outreach activities including posting on internet sites and meeting with high school students at science fairs, will raise public scientific literacy. Finally, the data, technology and models that are developed will be deposited in easily accessible databases and disseminated widely to promote the interdisciplinary incorporation of the potentially transformative ideas of the project.

? DESCRIPTION (provided by applicant): Induced pluripotent stem cells (iPSCs) could be a panacea for a wide range of human diseases. Both the generation of iPSCs and their differentiation into various lineages require massive and coordinated changes in gene expression. When cells are pushed from the adult state into pluripotency a large scale induction of pluripotency genes is required, but also a large scale down-regulation of genes expressed in the differentiated parent cells. Similarly, the more natural process of differentiation requires down-regulation of pluripotency factors with concomitant increased expression of tissue-specific genes. These changes occur at the transcriptional level, but also post-transcriptionally, particularly at the level of mRNA decay. Importantly, the contributions of post-transcriptional events to the achievement and maintenance of pluripotency are poorly characterized. We propose to study three post- transcriptional RNA regulons that likely contribute to the achievement and/or maintenance of pluripotency. First, we determined that a large number of Zinc Finger Protein (ZNF) mRNAs are stabilized in iPSCs. As these ZNF transcription factors have vital roles in development, and perhaps in controlling retrotransposition, understanding their regulation is essential. We will examine a potential mechanism by which miRNAs target repeated domains in the ORF of ZNF mRNAs to induce decay. We will also investigate why these ZNF mRNAs have unusually short poly(A) tails and uncover how this contributes to their expression. Second, we found that methylation of mRNAs, and particularly ZNF mRNAs, is correlated with increased stability in iPSCs. We will uncover the mechanisms by which this modification influences gene expression in pluripotent cells. Finally, we have shown that many mRNAs encoding transcription factors important for embryonic development contain C-rich elements and are significantly destabilized in iPSCs. We will identify the factors responsible for this regulation and decipher their mechanism of action. Overall, these experiments will give much needed insights into post- transcriptional control in stem cells that may lead to improved reprogramming and a better understanding of the differences between pluripotent cells and their fully differentiated relatives.

The Flaviviridae are a family of positive-sense RNA viruses that contain numerous important human pathogens. Many of the molecular mechanisms that underlie how these RNA viruses cause cytopathology and disease are not clearly described. The cellular mRNA decay machinery, in particularly the 5'-3' pathway mediated by the exoribonuclease Xrn1, plays a major role in regulating the abundance and quality of gene expression in the cell. Understudied, but nevertheless very important aspects of flavivirus-host interactions, include how viral RNAs are protected from degradation by the cellular mRNA decay machinery and what are the implications of the viral RNA stabilization strategies on the regulation of cellular mRNA stability. We have recently observed that flaviviruses repress the activity of Xrn1 through trapping the enzyme using unique structured regions of the viral RNA. Interestingly, it is the 5' UTR IRES region that is responsible for this Xrn1 repression in Hepatitis C virus and Bovine Viral Diarrhea virus. These observations serve as the foundation for this proposal to gain in-depth mechanistic insights into molecular mechanisms of Xrn1 repression and regulation that are disrupted by flavivirus RNAs. In Aim 1, we will identify the sequence/structural requirements of IRES-mediated Xrn1 repression and determine whether this is a common property of other viral IRES elements. The goal of Aim 2 is understand at a mechanistic level why the repression of Xrn1 by flavivirus RNAs results in the apparent shut down of the entire 5'-3' mRNA decay pathway ? not just the exonucleolytic digestion step. Uncovering the interplay and feedback regulation of the decay factors in the 5'-3' RNA decay pathway will provide novel insights into how the cell normally regulates this decay pathway and integrate it into the overall process of gene expression. In the third aim we will expand our studies on Xrn1 stalling and investigate whether it is an approach used by the cell to remodel cellular transcripts. In the final Aim, we will characterize key biological aspects of Xrn1 repression from the perspective of both the cell and the virus. A key focus of this part will be on the dysregulation of cellular mRNA stability by flaviviruses that results in dramatic changes in cellular gene expression that could play a significant role in HCV-mediated oncogenesis.

PROJECT SUMMARY / ABSTRACT Zika virus is an emerging flavivirus that presents a significant risk to pregnant women and is associated with Guillian-Barre syndrome. Why the virus elicits these unforeseen pathologies is unclear, but is undoubtedly related to unique aspects of Zika virus-host interactions ? a highly understudied area of investigation to date. Previous work from our laboratory has demonstrated that RNAs from other insect-borne flaviviruses contain a highly structured segment in their 3' UTR that stalls and represses the cellular exoribonuclease Xrn1. Repression of this major cellular RNA decay enzyme causes significant dysregulation of global cellular gene expression, changes that are likely very relevant to virus-induced cytopathology and pathogenesis. The first goal of this project is to demonstrate the stalling and repression of Xrn1 by Zika virus RNAs that we hypothesize should occur based on modeling with the RNAs of other flaviviruses. Importantly, we will then analyze the implications that Xrn1 repression has on Zika virus infection of cell types that are biologically relevant to fetal tissues that are particularly sensitive to virus infection in utero. Second, we will explore the possible functions of the non-coding ~400 base RNA (sfRNA) that is generated by Xrn 1 stalling and accumulates to high levels during infection. This study will for the first time explore the roles of the Zika virus 3' non-coding RNA region in virus-host interactions. We hypothesize that the Zika sfRNA likely accumulates to high levels in infected cells and may serve as a sponge to disrupt the function and/or usurp the activity of key cellular RNA binding proteins. These data should provide foundational insights for understanding Zika virus pathogenesis as well as identify novel targets for antiviral drug development or virus attenuation for vaccine production.

SARS-CoV-2 must cap and methylate its mRNAs to ensure their stability, translatability, and avoid detection by host innate immune mechanism as non-self transcripts. The process of RNA capping, therefore, is pivotal to the success of a SARS-CoV-2 infection. It also represents a key contributor to the molecular mechanisms of pathogenesis as well as a very attractive target for the development of antiviral therapeutics. However, there are three key knowledge gaps that have slowed progress in our understanding of this important area of coronavirus molecular biology that will be addressed in this proposal. First, the identity of the guanylyltransferase (GTase), the centerpiece of the viral RNA capping machinery that transfers GTP to the 5' end of the nascent transcript, is unknown. We will use a two-pronged strategy of complementary molecular and biochemical approaches to address this glaring gap in our understanding of SARS-CoV-2 mRNA capping mechanisms, laying the foundation for the development of capping-targeted antivirals. Second, while RNA capping is a regulated process and uncapped RNAs play an influential role in the biology of other positive sense RNA viral infections, it is not known if RNA capping is a regulated or a default event in coronaviruses. We will determine if uncapped RNAs are produced by SARS-CoV-2 in order to establish the foundation for a role of regulated capping and non-coding viral transcripts in SARS-CoV-2 infections. Finally, every cellular mRNA that begins with a terminal adenosine has that residue 2'O methylated at the ribose ring as well as N6 methylated on the adenosine base (m6Am). The strong conservation of this m6Am modification indicates its importance in cell biology, an assertation recently confirmed with data suggesting that the modification increases translatability and facilitates recognition of the transcript as `self'. Interestingly, SARS-CoV-2 and other coronaviruses all initiate their transcripts with an A residue, but it is not known whether that A residue contains an m6A modification. In the final part of this project, we will determine the m6A modification status of the terminal 5' A residue of SARS-CoV-2 mRNAs and investigate the role that the modification (or lack thereof) plays in the biology of coronaviral transcripts. Collectively these studies will provide important new insights into the molecular biology of SARS-CoV-2 and open up avenues for the development of broad-spectrum anti- coronaviral therapeutics.