The leader RNA of SARS-CoV-2 sequesters polypyrimidine tract binding protein (PTBP1) and influences pre-mRNA splicing in infected cells.

The large amount of viral RNA produced during infections has the potential to interact with and effectively sequester cellular RNA binding proteins, thereby influencing aspects of post-transcriptional gene regulation in the infected cell. Here we demonstrate that the abundant 5' leader RNA region of SARS-CoV-2 viral RNAs can interact with the cellular polypyrimidine tract binding protein (PTBP1). Interestingly, the effect of a knockdown of PTBP1 protein on cellular gene expression is also mimicked during SARS-CoV-2 infection, suggesting that this protein may be functionally sequestered by viral RNAs. Consistent with this model, the alternative splicing of mRNAs that is normally controlled by PTBP1 is dysregulated during SARS-CoV-2 infection. Collectively, these data suggest that the SARS-CoV-2 leader RNA sequesters the cellular PTBP1 protein during infection, resulting in significant impacts on the RNA biology of the host cell. These alterations in post-transcriptional gene regulation may play a role in SARS-CoV-2 mediated molecular pathogenesis.

A plant-infecting subviral RNA associated with poleroviruses produces a subgenomic RNA which resists exonuclease XRN1 in vitro.

Subviral agents are nucleic acids which lack the features for classification as a virus. Tombusvirus-like associated RNAs (tlaRNAs) are subviral positive-sense, single-stranded RNAs that replicate autonomously, yet depend on a coinfecting virus for encapsidation and transmission. TlaRNAs produce abundant subgenomic RNA (sgRNA) upon infection. Here, we investigate how the well-studied tlaRNA, ST9, produces sgRNA and its function. We found ST9 is a noncoding RNA, due to its lack of protein coding capacity. We used resistance assays with eukaryotic Exoribonuclease-1 (XRN1) to investigate sgRNA production via incomplete degradation of genomic RNA. The ST9 3' untranslated region stalled XRN1 very near the 5' sgRNA end. Thus, the XRN family of enzymes drives sgRNA accumulation in ST9-infected tissue by incomplete degradation of ST9 RNA. This work suggests tlaRNAs are not just parasites of viruses with compatible capsids, but also mutually beneficial partners that influence host cell RNA biology.

YTHDF2 destabilizes m6A-modified neural-specific RNAs to restrain differentiation in induced pluripotent stem cells.

N6-methyladenosine (m6A) is an abundant post-transcriptional modification that can impact RNA fate via interactions with m6A-specific RNA binding proteins. Despite accumulating evidence that m6A plays an important role in modulating pluripotency, the influence of m6A reader proteins in pluripotency is less clear. Here, we report that YTHDF2, an m6A reader associated with mRNA degradation, is highly expressed in induced pluripotent stem cells (iPSCs) and down-regulated during neural differentiation. Through RNA sequencing, we identified a group of m6A-modified transcripts associated with neural development that are directly regulated by YTDHF2. Depletion of YTHDF2 in iPSCs leads to stabilization of these transcripts, loss of pluripotency, and induction of neural-specific gene expression. Collectively, our results suggest YTHDF2 functions to restrain expression of neural-specific mRNAs in iPSCs and facilitate their rapid and coordinated up-regulation during neural induction. These effects are both achieved by destabilization of the targeted transcripts.

Zika virus noncoding sfRNAs sequester multiple host-derived RNA-binding proteins and modulate mRNA decay and splicing during infection.

Insect-borne flaviviruses produce a 300-500-base long noncoding RNA, termed subgenomic flavivirus RNA (sfRNA), by stalling the cellular 5'-3'-exoribonuclease 1 (XRN1) via structures located in their 3' UTRs. In this study, we demonstrate that sfRNA production by Zika virus represses XRN1 analogous to what we have previously shown for other flaviviruses. Using protein-RNA reconstitution and a stringent RNA pulldown assay with human choriocarcinoma (JAR) cells, we demonstrate that the sfRNAs from both dengue type 2 and Zika viruses interact with a common set of 21 RNA-binding proteins that contribute to the regulation of post-transcriptional processes in the cell, including splicing, RNA stability, and translation. We found that four of these sfRNA-interacting host proteins, DEAD-box helicase 6 (DDX6) and enhancer of mRNA decapping 3 (EDC3) (two RNA decay factors), phosphorylated adaptor for RNA export (a regulator of the biogenesis of the splicing machinery), and apolipoprotein B mRNA-editing enzyme catalytic subunit 3C (APOBEC3C, a nucleic acid-editing deaminase), inherently restrict Zika virus infection. Furthermore, we demonstrate that the regulations of cellular mRNA decay and RNA splicing are compromised by Zika virus infection as well as by sfRNA alone. Collectively, these results reveal the large extent to which Zika virus-derived sfRNAs interact with cellular RNA-binding proteins and highlight the potential for widespread dysregulation of post-transcriptional control that likely limits the effective response of these cells to viral infection.

Standing your ground to exoribonucleases: Function of Flavivirus long non-coding RNAs.

Members of the Flaviviridae (e.g., Dengue virus, West Nile virus, and Hepatitis C virus) contain a positive-sense RNA genome that encodes a large polyprotein. It is now also clear most if not all of these viruses also produce an abundant subgenomic long non-coding RNA. These non-coding RNAs, which are called subgenomic flavivirus RNAs (sfRNAs) or Xrn1-resistant RNAs (xrRNAs), are stable decay intermediates generated from the viral genomic RNA through the stalling of the cellular exoribonuclease Xrn1 at highly structured regions. Several functions of these flavivirus long non-coding RNAs have been revealed in recent years. The generation of these sfRNAs/xrRNAs from viral transcripts results in the repression of Xrn1 and the dysregulation of cellular mRNA stability. The abundant sfRNAs also serve directly as a decoy for important cellular protein regulators of the interferon and RNA interference antiviral pathways. Thus the generation of long non-coding RNAs from flaviviruses, hepaciviruses and pestiviruses likely disrupts aspects of innate immunity and may directly contribute to viral replication, cytopathology and pathogenesis.

Do You Believe in ReincaRNAtion? Herpesviruses Reveal Connection between RNA Decay and Synthesis.

Many viruses degrade host mRNAs to reduce competition for proteins/ribosomes and promote viral gene expression. In this issue of Cell Host & Microbe, Abernathy et al. (2015) demonstrate that a herpesviral RNA endonuclease induces host transcriptional repression that is mediated through the decay factor Xrn1 and evaded by viral genes.

XRN1 stalling in the 5' UTR of Hepatitis C virus and Bovine Viral Diarrhea virus is associated with dysregulated host mRNA stability.

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 growth and pathogenesis.

Stem cell RNA epigenetics: m(6)arking your territory.

Modifications of mRNAs can have a profound effect on cellular function and differentiation. In this issue of Cell Stem Cell, Batista et al. (2014) describe fundamental parameters of N(6)-methyl-adenosine modification of mRNAs in embryonic stem cells and provide strong evidence that modification plays a role in exit from pluripotency toward differentiation.

Determinants and implications of mRNA poly(A) tail size--does this protein make my tail look big?

While the phenomenon of polyadenylation has been well-studied, the dynamics of poly(A) tail size and its impact on transcript function and cell biology are less well-appreciated. The goal of this review is to encourage readers to view the poly(A) tail as a dynamic, changeable aspect of a transcript rather than a simple static entity that marks the 3' end of an mRNA. This could open up new angles of regulation in the post-transcriptional control of gene expression throughout development, differentiation and cancer.

Changes in cellular mRNA stability, splicing, and polyadenylation through HuR protein sequestration by a cytoplasmic RNA virus.

The impact of RNA viruses on the posttranscriptional regulation of cellular gene expression is unclear. Sindbis virus causes a dramatic relocalization of the cellular HuR protein from the nucleus to the cytoplasm in infected cells. This is to the result of the expression of large amounts of viral RNAs that contain high-affinity HuR binding sites in their 3' UTRs effectively serving as a sponge for the HuR protein. Sequestration of HuR by Sindbis virus is associated with destabilization of cellular mRNAs that normally bind HuR and rely on it to regulate their expression. Furthermore, significant changes can be observed in nuclear alternative polyadenylation and splicing events on cellular pre-mRNAs as a result of sequestration of HuR protein by the 3' UTR of transcripts of this cytoplasmic RNA virus. These studies suggest a molecular mechanism of virus-host interaction that probably has a significant impact on virus replication, cytopathology, and pathogenesis.

A DIStinctively novel exoribonuclease that really likes U.

Regulated degradation plays a major role in determining the levels of both non-coding (miRNA) and coding (mRNA) transcripts. Thus, insights into the factors and pathways that influence this process have broad, interdisciplinary implications. New findings by Malecki et al (2013), Lubas et al (2013), and Chang et al (2013) identify the protein Dis3L2 as a major player in the 3'­5' exonucleolytic decay of transcripts. Furthermore, they demonstrate a strong connection between terminal uridylation of the RNA substrate and enzymatic activity.

Lsm proteins and Hfq: Life at the 3' end.

The bacterial Hfq protein is a versatile modulator of RNA function and is particularly important for regulation mediated by small non-coding RNAs. Hfq is a bacterial Sm protein but bears more similarity to the eukaryotic Sm-like (Lsm) family of proteins than the prototypical Sm proteins. Hfq and Lsm proteins share the ability to chaperone RNA-RNA and RNA/protein interactions and an interesting penchant for protecting the 3' end of a transcript from exonucleolytic decay while encouraging degradation through other pathways. Our view of Lsm function in eukaryotes has historically been informed by studies of Hfq structure and function but mutational analyses and structural studies of Lsm sub-complexes have given important insights as well. Here, we aim to compare and contrast the roles of these evolutionarily related complexes and to highlight areas for future investigation.

Picornavirus modification of a host mRNA decay protein.

UNLABELLED: Due to the limited coding capacity of picornavirus genomic RNAs, host RNA binding proteins play essential roles during viral translation and RNA replication. Here we describe experiments suggesting that AUF1, a host RNA binding protein involved in mRNA decay, plays a role in the infectious cycle of picornaviruses such as poliovirus and human rhinovirus. We observed cleavage of AUF1 during poliovirus or human rhinovirus infection, as well as interaction of this protein with the 5' noncoding regions of these viral genomes. Additionally, the picornavirus proteinase 3CD, encoded by poliovirus or human rhinovirus genomic RNAs, was shown to cleave all four isoforms of recombinant AUF1 at a specific N-terminal site in vitro. Finally, endogenous AUF1 was found to relocalize from the nucleus to the cytoplasm in poliovirus-infected HeLa cells to sites adjacent to (but distinct from) putative viral RNA replication complexes. IMPORTANCE: This study derives its significance from reporting how picornaviruses like poliovirus and human rhinovirus proteolytically cleave a key player (AUF1) in host mRNA decay pathways during viral infection. Beyond cleavage of AUF1 by the major viral proteinase encoded in picornavirus genomes, infection by poliovirus results in the relocalization of this host cell RNA binding protein from the nucleus to the cytoplasm. The alteration of both the physical state of AUF1 and its cellular location illuminates how small RNA viruses manipulate the activities of host cell RNA binding proteins to ensure a faithful intracellular replication cycle.

Dephosphorylation of HuR protein during alphavirus infection is associated with HuR relocalization to the cytoplasm.

We have demonstrated previously that the cellular HuR protein binds U-rich elements in the 3' untranslated region (UTR) of Sindbis virus RNA and relocalizes from the nucleus to the cytoplasm upon Sindbis virus infection in 293T cells. In this study, we show that two alphaviruses, Ross River virus and Chikungunya virus, lack the conserved high-affinity U-rich HuR binding element in their 3' UTRs but still maintain the ability to interact with HuR with nanomolar affinities through alternative binding elements. The relocalization of HuR protein occurs during Sindbis infection of multiple mammalian cell types as well as during infections with three other alphaviruses. Interestingly, the relocalization of HuR is not a general cellular reaction to viral infection, as HuR protein remained largely nuclear during infections with dengue and measles virus. Relocalization of HuR in a Sindbis infection required viral gene expression, was independent of the presence of a high-affinity U-rich HuR binding site in the 3' UTR of the virus, and was associated with an alteration in the phosphorylation state of HuR. Sindbis virus-induced HuR relocalization was mechanistically distinct from the movement of HuR observed during a cellular stress response, as there was no accumulation of caspase-mediated HuR cleavage products. Collectively, these data indicate that virus-induced HuR relocalization to the cytoplasm is specific to alphavirus infections and is associated with distinct posttranslational modifications of this RNA-binding protein.

The 3' untranslated region of the rabies virus glycoprotein mRNA specifically interacts with cellular PCBP2 protein and promotes transcript stability.

Viral polymerase entry and pausing at intergenic junctions is predicted to lead to a defined polarity in the levels of rhabdovirus gene expression. Interestingly, we observed that the rabies virus glycoprotein mRNA is differentially over-expressed based on this model relative to other transcripts during infection of 293T cells. During infection, the rabies virus glycoprotein mRNA also selectively interacts with the cellular poly(rC)-binding protein 2 (PCBP2), a factor known to influence mRNA stability. Reporter assays performed both in electroporated cells and in a cell-free RNA decay system indicate that the conserved portion of the 3' UTR of the rabies virus glycoprotein mRNA contains an RNA stability element. PCBP2 specifically interacts with reporter transcripts containing this 72 base 3' UTR sequence. Furthermore, the PCBP2 interaction is directly associated with the stability of reporter transcripts. Therefore, we conclude that PCBP2 specifically and selectively interacts with the rabies virus glycoprotein mRNA and that this interaction may contribute to the post-transcriptional regulation of glycoprotein expression.

Sindbis virus usurps the cellular HuR protein to stabilize its transcripts and promote productive infections in mammalian and mosquito cells.

How viral transcripts are protected from the cellular RNA decay machinery and the importance of this protection for the virus are largely unknown. We demonstrate that Sindbis virus, a prototypical single-stranded arthropod-borne alphavirus, uses U-rich 3' UTR sequences in its RNAs to recruit a known regulator of cellular mRNA stability, the HuR protein, during infections of both human and vector mosquito cells. HuR binds viral RNAs with high specificity and affinity. Sindbis virus infection induces the selective movement of HuR out of the mammalian cell nucleus, thereby increasing the available cytoplasmic HuR pool. Finally, knockdown of HuR results in a significant increase in the rate of decay of Sindbis virus RNAs and diminishes viral yields in both human and mosquito cells. These data indicate that Sindbis virus and likely other alphaviruses usurp the HuR protein to avoid the cellular mRNA decay machinery and maintain a highly productive infection.

Systematic analysis of cis-elements in unstable mRNAs demonstrates that CUGBP1 is a key regulator of mRNA decay in muscle cells.

BACKGROUND: Dramatic changes in gene expression occur in response to extracellular stimuli and during differentiation. Although transcriptional effects are important, alterations in mRNA decay also play a major role in achieving rapid and massive changes in mRNA abundance. Moreover, just as transcription factor activity varies between different cell types, the factors influencing mRNA decay are also cell-type specific. PRINCIPAL FINDINGS: We have established the rates of decay for over 7000 transcripts expressed in mouse C2C12 myoblasts. We found that GU-rich (GRE) and AU-rich (ARE) elements are over-represented in the 3'UTRs of short-lived mRNAs and that these mRNAs tend to encode factors involved in cell cycle and transcription regulation. Stabilizing elements were also identified. By comparing mRNA decay rates in C2C12 cells with those previously measured for pluripotent and differentiating embryonic stem (ES) cells, we identified several groups of transcripts that exhibit cell-type specific decay rates. Further, whereas in C2C12 cells the impact of GREs on mRNA decay appears to be greater than that of AREs, AREs are more significant in ES cells, supporting the idea that cis elements make a cell-specific contribution to mRNA stability. GREs are recognized by CUGBP1, an RNA-binding protein and instability factor whose function is affected in several neuromuscular diseases. We therefore utilized RNA immunoprecipitation followed by microarray (RIP-Chip) to identify CUGBP1-associated transcripts. These mRNAs also showed dramatic enrichment of GREs in their 3'UTRs and encode proteins linked with cell cycle, and intracellular transport. Interestingly several CUGBP1 substrate mRNAs, including those encoding the myogenic transcription factors Myod1 and Myog, are also bound by the stabilizing factor HuR in C2C12 cells. Finally, we show that several CUGBP1-associated mRNAs containing 3'UTR GREs, including Myod1, are stabilized in cells depleted of CUGBP1, consistent with the role of CUGBP1 as a destabilizing factor. CONCLUSIONS: Taken together, our results systematically establish cis-acting determinants of mRNA decay rates in C2C12 myoblast cells and demonstrate that CUGBP1 associates with GREs to regulate decay of a wide range of mRNAs including several that are critical for muscle development.

Virus-mediated mRNA decay by hyperadenylation.

Degradation of cellular mRNAs during Kaposi's sarcoma-associated herpesvirus infection is associated with hyperadenylation of transcripts and a relocalization of cytoplasmic poly(A)-binding proteins to the nucleus.

The RNA-binding protein CUGBP1 regulates stability of tumor necrosis factor mRNA in muscle cells: implications for myotonic dystrophy.

Type I myotonic dystrophy (DM1) is caused by a triplet repeat expansion in the 3'-untranslated region (UTR) of the dystrophia myotonia protein kinase (DMPK) gene. Pathogenesis is closely linked with production of a toxic RNA from the mutant allele, which interferes with function of several RNA-binding proteins, including CUGBP1. Here we show that expression of a mutant DMPK 3'-UTR containing 960 CUG repeats is sufficient to increase expression and stability of an mRNA encoding the potent proinflammatory cytokine, tumor necrosis factor (TNF). CUGBP1 specifically recognizes sequences within the TNF 3'-UTR that are dissimilar from its canonical UG-rich binding site. Depletion of CUGBP1 from mouse myoblasts results in increased abundance of TNF mRNA through stabilization of the transcript. Moreover, activation of the protein kinase C pathway by treatment with phorbol ester, which has been shown previously to result in CUGBP1 phosphorylation, also causes TNF mRNA stabilization. Our results suggest that the elevated serum TNF seen in DM1 patients may be derived from muscle where it is induced by expression of toxic DMPK RNA. Importantly, overexpression of this potent cytokine could contribute to the muscle wasting and insulin resistance that are characteristic of this debilitating disease.