Carol Wilusz - Academia.edu (original) (raw)

Papers by Carol Wilusz

Virology, 2015

Productive arbovirus infections require mechanisms to suppress or circumvent the cellular RNA int... more Productive arbovirus infections require mechanisms to suppress or circumvent the cellular RNA interference (RNAi) pathway, a major antiviral response in mosquitoes. In this study, we demonstrate that two flaviviruses, Dengue virus and Kunjin virus, significantly repress siRNA-mediated RNAi in infected human cells as well as during infection of the mosquito vector Culex quinquefasciatus. Arthropod-borne flaviviruses generate a small structured non-coding RNA from the viral 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; UTR referred to as sfRNA. Analysis of infections with a mutant Kunjin virus that is unable to generate appreciable amounts of the major sfRNA species indicated that RNAi suppression was associated with the generation of the non-coding sfRNA. Co-immunoprecipitation of sfRNA with RNAi mediators Dicer and Ago2 suggest a model for RNAi suppression. Collectively, these data help to establish a clear role for sfRNA in RNAi suppression and adds to the emerging impact of viral long non-coding RNAs in modulating aspects of anti-viral immune processes.

Reproduction Fertility and Development, 2007

ABSTRACT The maternal pool of mRNA undergoes major changes during oocyte maturation and early emb... more ABSTRACT The maternal pool of mRNA undergoes major changes during oocyte maturation and early embryonic development. Specific genes are activated or degraded in response to changes in poly-(A) tail length. However, little is known about how the oocyte targets specific transcripts for degradation or translation in a timely manner. The objective of this study was to determine how poly-(A) tail length of different transcripts is affected in bovine oocytes by time of in vitro maturation. Cyclin B1 and GDF-9 32 untranslated regions (UTRs) were cloned into modified p-GEM plasmids containing a poly-(A) tract of 60 or 0 adenosines (A60 or A0, respectively). Each 32 UTR was transcribed in vitro with (A60) or without (A0) a poly-(A) tail to generate UTP32-labeled RNA. Transcriptions producing at least 200 000 counts per min (cpm) per µL were used for subsequent injections into denuded bovine oocytes. Cumulus-oocyte complexes (COCs) recovered from slaughterhouse-derived ovaries (n = 216) were vortexed to remove cumulus cells immediately after aspiration, after 3 h of in vitro maturation, or after 19 h of maturation in a chemically defined medium supplemented with FSH, LH, EGF, and cysteamine. After vortexing, denuded oocytes were injected and snap frozen, or matured in vitro for 1 or 3 h. Eight oocytes were injected with ~0.5 nL (~100 cpm/oocyte) labeled RNA at each time point in 3 replicates. Total RNA was isolated from injected oocyte pools and loaded onto a 5% denaturing acrylamide gel for size separation. Radiolabeled A0 was used as a control point of reference for deadenylation. Gels were dried, and RNA was visualized on a phosphoimager after 24 h exposure to a phosphor screen. Changes in polyadenylation status (transcript size) were evaluated by comparing shifts in bands from gene-specific A60

Nature reviews. Molecular cell biology, 2001

The levels of cellular messenger RNA transcripts can be regulated by controlling the rate at whic... more The levels of cellular messenger RNA transcripts can be regulated by controlling the rate at which the mRNA decays. Because decay rates affect the expression of specific genes, they provide a cell with flexibility in effecting rapid change. Moreover, many clinically relevant mRNAs--including several encoding cytokines, growth factors and proto-oncogenes--are regulated by differential RNA stability. But what are the sequence elements and factors that control the half-lives of mRNAs?

PLoS pathogens, 2015

We demonstrate that both Hepatitis C virus (HCV) and Bovine Viral Diarrhea virus (BVDV) contain r... more We demonstrate that both Hepatitis C virus (HCV) and Bovine Viral Diarrhea virus (BVDV) contain regions in their 5' UTRs that stall and repress the enzymatic activity of the cellular 5'-3' exoribonuclease XRN1, resulting in dramatic changes in the stability of cellular mRNAs. We used biochemical assays, virus infections, and transfection of the HCV and BVDV 5' untranslated regions in the absence of other viral gene products to directly demonstrate the existence and mechanism of this novel host-virus interaction. In the context of HCV infection, we observed globally increased stability of mRNAs resulting in significant increases in abundance of normally short-lived mRNAs encoding a variety of relevant oncogenes and angiogenesis factors. These findings suggest that non-coding regions from multiple genera of the Flaviviridae interfere with XRN1 and impact post-transcriptional processes, causing global dysregulation of cellular gene expression which may promote cell grow...

Methods in Enzymology, 2008

The field of RNA decay has grown extensively over the last few years and numerous decay pathways ... more The field of RNA decay has grown extensively over the last few years and numerous decay pathways have been identified and characterized. This is a truly powerful machinery for both regulation and quality control of gene expression. It is very likely that the transcripts of RNA viruses must successfully confront this arsenal of enzymes and RNA binding factors in order to establish a productive infection. This interface is an understudied branch of virology that needs to be explored if we are to fully comprehend the molecular biology of virus-cell interactions. Research in this area has the potential to increase our understanding of the fundamentals of both mRNA stability and viral biology, perhaps leading to novel antiviral approaches. This chapter discusses methods for examining the half-lives of viral RNAs during natural infection, including purification of the viral transcripts and subsequent analysis of both deadenylation and decay. Additionally, a hybrid selection protocol for identifying viral-specific small RNAs that are generated during infection by the RNAi branch of the cellular RNA decay machinery is described.

Nature communications, 2014

HuR promotes myogenesis by stabilizing the MyoD, myogenin and p21 mRNAs during the fusion of musc... more HuR promotes myogenesis by stabilizing the MyoD, myogenin and p21 mRNAs during the fusion of muscle cells to form myotubes. Here we show that HuR, via a novel mRNA destabilizing activity, promotes the early steps of myogenesis by reducing the expression of the cell cycle promoter nucleophosmin (NPM). Depletion of HuR stabilizes the NPM mRNA, increases NPM protein levels and inhibits myogenesis, while its overexpression elicits the opposite effects. NPM mRNA destabilization involves the association of HuR with the decay factor KSRP as well as the ribonuclease PARN and the exosome. The C terminus of HuR mediates the formation of the HuR-KSRP complex and is sufficient for maintaining a low level of the NPM mRNA as well as promoting the commitment of muscle cells to myogenesis. We therefore propose a model whereby the downregulation of the NPM mRNA, mediated by HuR, KSRP and its associated ribonucleases, is required for proper myogenesis.

The EMBO Journal, 2001

While decapping plays a major role in mRNA turnover in yeast, biochemical evidence for a similar ... more While decapping plays a major role in mRNA turnover in yeast, biochemical evidence for a similar activity in mammalian cells has been elusive. We have now identi®ed a decapping activity in HeLa cytoplasmic extracts that releases 7me GDP from capped transcripts. Decapping is activated in extracts by the addition of 7me GpppG, which speci®cally sequesters cap-binding proteins such as eIF4E and the deadenylase DAN/PARN. Similar to in vivo observations, the presence of a poly(A) tail represses decapping of RNAs in vitro in a poly(A)-binding protein-dependent fashion. AU-rich elements (AREs), which act as regulators of mRNA stability in vivo, are potent stimulators of decapping in vitro. The stimulation of decapping by AREs requires sequence-speci®c ARE-binding proteins. These data suggest that cap recognition and decapping play key roles in mediating mRNA turnover in mammalian cells.

Proceedings of the National Academy of Sciences, 2007

In Caenorhabditis elegans, the small nuclear ribonucleoprotein (snRNP)-associated proteins U1A an... more In Caenorhabditis elegans, the small nuclear ribonucleoprotein (snRNP)-associated proteins U1A and U2B؆ are Ϸ50% identical to each other, and neither bears signature characteristics of mammalian U1A or U2B؆ or the single Drosophila homolog, SNF. We show here that the genes that encode these proteins (rnp-2 and rnp-3) are cotranscribed in an operon, and that ribonucleoprotein RNP-2 is U1 snRNP-associated (U1A) whereas RNP-3 is U2 snRNPassociated (U2B؆). U2B؆ interacts with U2 even in the absence of another U2 snRNP protein, U2A. Like U1A and U2B؆ from yeast, plants, and vertebrates, worm U1A and U2B؆ are more similar to each other than they are to other U1A or U2B؆ proteins, respectively. Even though U1A and U2B؆ interact with different snRNPs, they are functionally redundant; knockout of both is required for a lethal phenotype. Interestingly, U1A associates with U2 RNA when U2B؆ is deleted. Thus, the two members of this gene family normally function as components of different snRNPs but apparently remain capable of performing the function of the other. Redundancy results from the fact that one protein can substitute for the other, even though it normally does not.

Nature Structural & Molecular Biology, 2006

Nucleophosmin (NPM), an abundant, predominantly nucleolar protein that influences numerous cellul... more Nucleophosmin (NPM), an abundant, predominantly nucleolar protein that influences numerous cellular processes, was shown to specifically associate with the bodies of messenger RNAs as a result of the process of 3′-end formation. NPM deposition requires polyadenylation but not the 3′ cleavage event to occur on the transcript. Furthermore, the protein does not associate with RNAs bearing a preformed poly(A) tail or with mRNAs that have undergone cleavage but not polyadenylation. A region within 10 bases upstream of the AAUAAA element is required for NPM association, but deposition of the protein seems to be sequence independent. NPM association with poly(A) + mRNAs was also demonstrated in vivo. NPM, therefore, represents a mark left on transcripts as a result of 3′end processing and may have a role in one or more of a variety of post-transcriptional processes influenced by the polyadenylation event.

Nature Structural & Molecular Biology, 2007

Many orthopoxvirus messenger RNAs have an unusual nontemplated poly(A) tract of 5 to 40 residues ... more Many orthopoxvirus messenger RNAs have an unusual nontemplated poly(A) tract of 5 to 40 residues at the 5¢ end. The precise function of this feature is unknown. Here we show that 5¢ poly(A) tracts are able to repress RNA decay by inhibiting 3¢-to-5¢ exonucleases as well as decapping of RNA substrates. UV cross-linking analysis demonstrated that the Lsm complex associates with the 5¢ poly(A) tract. Furthermore, recombinant Lsm1-7 complex specifically binds 5¢ poly(A) tracts 10 to 21 nucleotides in length, consistent with the length of 5¢ poly(A) required for stabilization. Knockdown of Lsm1 abrogates RNA stabilization by the 5¢ poly(A) tract. We propose that the Lsm complex simultaneously binds the 3¢ and 5¢ ends of these unusual messenger RNAs and thereby prevents 3¢-to-5¢ decay. The implications of this phenomenon for cellular mRNA decay are discussed.

Nature Reviews Molecular Cell Biology, 2007

Molecular and Cellular Biology, 2005

Regulation of mRNA turnover is an important cellular strategy for posttranscriptional control of ... more Regulation of mRNA turnover is an important cellular strategy for posttranscriptional control of gene expression, mediated by the interplay of cis-acting sequences and associated trans-acting factors. Pub1p, an ELAV-like yeast RNA-binding protein with homology to T-cell internal antigen 1 (TIA-1)/TIA-1-related protein (TIAR), is an important modulator of the decay of two known classes of mRNA. Our goal in this study was to determine the range of mRNAs whose stability is dependent on Pub1p, as well as to identify specific transcripts that directly bind to this protein. We have examined global mRNA turnover in isogenic PUB1 and pub1⌬ strains through gene expression analysis and demonstrate that 573 genes exhibit a significant reduction in half-life in a pub1⌬ strain. We also examine the binding specificity of Pub1p using affinity purification followed by microarray analysis to comprehensively distinguish between direct and indirect targets and find that Pub1p significantly binds to 368 cellular transcripts. Among the Pub1p-associated mRNAs, 53 transcripts encoding proteins involved in ribosomal biogenesis and cellular metabolism are selectively destabilized in the pub1⌬ strain. In contrast, genes involved in transporter activity demonstrate association with Pub1p but display no measurable changes in transcript stability. Characterization of two candidate genes, SEC53 and RPS16B, demonstrate that both Pub1p-dependent regulation of stability and Pub1p binding require 3 untranslated regions, which harbor distinct sequence motifs. These results suggest that Pub1p binds to discrete subsets of cellular transcripts and posttranscriptionally regulates their expression at multiple levels.

Journal of Virology, 2008

The positive-sense transcripts of Sindbis virus (SINV) resemble cellular mRNAs in that they posse... more The positive-sense transcripts of Sindbis virus (SINV) resemble cellular mRNAs in that they possess a 5 cap and a 3 poly(A) tail. It is likely, therefore, that SINV RNAs must successfully overcome the cytoplasmic mRNA decay machinery of the cell in order to establish an efficient, productive infection. In this study, we have taken advantage of a temperature-sensitive polymerase to shut off viral transcription, and we demonstrate that SINV RNAs are subject to decay during a viral infection in both C6/36 (Aedes albopictus) and baby hamster kidney cells. Interestingly, in contrast to most cellular mRNAs, the decay of SINV RNAs was not initiated by poly(A) tail shortening in either cell line except when most of the 3 untranslated region (UTR) was deleted from the virus. This block in deadenylation of viral transcripts was recapitulated in vitro using C6/36 mosquito cell cytoplasmic extracts. Two distinct regions of the 319-base SINV 3 UTR, the repeat sequence elements and a U-rich domain, were shown to be responsible for mediating the repression of deadenylation of viral mRNAs. Through competition studies performed in parallel with UV cross-linking and functional assays, mosquito cell factors-including a 38-kDa protein-were implicated in the repression of deadenylation mediated by the SINV 3 UTR. This same 38-kDa protein was also implicated in mediating the repression of deadenylation by the 3 UTR of another alphavirus, Venezuelan equine encephalitis virus. In summary, these data provide clear evidence that SINV transcripts do indeed interface with the cellular mRNA decay machinery during an infection and that the virus has evolved a way to avoid the major deadenylation-dependent pathway of mRNA decay.

Journal of Virology, 2007

Chronic wasting disease (CWD) of cervids is associated with conversion of the normal cervid prion... more Chronic wasting disease (CWD) of cervids is associated with conversion of the normal cervid prion protein, PrP C , to a protease-resistant conformer, PrP CWD . Here we report the use of both nondenaturing amplification and protein-misfolding cyclic amplification (PMCA) to amplify PrP CWD in vitro. Normal brains from deer, transgenic mice expressing cervid PrP C [Tg(cerPrP)1536 mice], and ferrets supported amplification. PMCA using normal Tg(cerPrP)1536 brains as the PrP C substrate produced >6.5 ؋ 10 9 -fold amplification after six rounds. Highly efficient in vitro amplification of PrP CWD is a significant step toward detection of PrP CWD in the body fluids or excreta of CWD-susceptible species.

Genetics and Molecular Biology, 2007

Several mRNAs have been shown to exhibit distinct patterns of poly(A) shortening prior to their d... more Several mRNAs have been shown to exhibit distinct patterns of poly(A) shortening prior to their decay in vivo. In this study, we show that individual transcripts also demonstrate distinct patterns of deadenylation in in vitro systems derived from HeLa and Jurkat T cell cytoplasmic extracts. The major patterns observed were slow/synchronous and fast/asynchronous poly(A) tail shortening. For all RNA substrates tested, PARN was shown to be the enzyme responsible for the deadenylation patterns that were observed. Sequences in the 3' untranslated regions influenced the deadenylation pattern. Using a fragment of the 3'UTR of the c-fos mRNA as a model, the interaction of CUG-BP, the human homolog of EDEN-BP -a protein previously implicated in regulated deadenylation in Xenopus oocyteswas shown to be associated with changes in PARN-mediated deadenylation patterns. Our results suggest that association of CUG-BP with 3'UTR sequences can modulate the activity of the PARN deadenylase in mammalian cell extracts.

Genes & Development, 2008

Cell, 2010

As mRNAs are generated, they are clothed with proteins to form messenger ribonucleoprotein partic... more As mRNAs are generated, they are clothed with proteins to form messenger ribonucleoprotein particles (mRNPs), which are then actively remodeled during various steps of gene expression. Franks et al. (2010) now show that mRNP remodeling is required even for the death of an mRNA.

BioEssays, 2002

Gene expression is an inherently complex process and errors often occur during the transcription ... more Gene expression is an inherently complex process and errors often occur during the transcription and processing of mRNAs. Several surveillance mechanisms have evolved to check the fidelity at each step of mRNA manufacture. Two recent reports describe the identification of a novel pathway in eukaryotes that recognizes and degrades mRNAs that lack a stop codon. The non-stop decay mechanism releases ribosomes stalled at the 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; end of a mRNA and stimulates the exosome to rapidly degrade the transcript.