UV damage induces production of mitochondrial DNA fragments with specific length profiles.

UV light is a potent mutagen that induces bulky DNA damage in the form of cyclobutane pyrimidine dimers (CPDs). Photodamage and other bulky lesions occurring in nuclear genomes can be repaired through nucleotide excision repair (NER), where incisions on both sides of a damaged site precede the removal of a single-stranded oligonucleotide containing the damage. Mitochondrial genomes (mtDNAs) are also susceptible to damage from UV light, but current evidence suggests that the only way to eliminate bulky mtDNA damage is through mtDNA degradation. Damage-containing oligonucleotides excised during NER can be captured with antidamage antibodies and sequenced (XR-seq) to produce high-resolution maps of active repair locations following UV exposure. We analyzed previously published datasets from Arabidopsis thaliana, Saccharomyces cerevisiae, and Drosophila melanogaster to identify reads originating from the mtDNA (and plastid genome in A. thaliana). In A. thaliana and S. cerevisiae, the mtDNA-mapping reads have unique length distributions compared to the nuclear-mapping reads. The dominant fragment size was 26 nt in S. cerevisiae and 28 nt in A. thaliana with distinct secondary peaks occurring in regular intervals. These reads also show a nonrandom distribution of di-pyrimidines (the substrate for CPD formation) with TT enrichment at positions 7-8 of the reads. Therefore, UV damage to mtDNA appears to result in production of DNA fragments of characteristic lengths and positions relative to the damaged location. The mechanisms producing these fragments are unclear, but we hypothesize that they result from a previously uncharacterized DNA degradation pathway or repair mechanism in mitochondria.

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.

UV damage induces production of mitochondrial DNA fragments with specific length profiles.

UV light is a potent mutagen that induces bulky DNA damage in the form of cyclobutane pyrimidine dimers (CPDs). In eukaryotic cells, photodamage and other bulky lesions occurring in nuclear genomes (nucDNAs) can be repaired through nucleotide excision repair (NER), where dual incisions on both sides of a damaged site precede the removal of a single-stranded oligonucleotide containing the damage. Mitochondrial genomes (mtDNAs) are also susceptible to damage from UV light, but current views hold that the only way to eliminate bulky DNA damage in mtDNAs is through mtDNA degradation. Damage-containing oligonucleotides excised during NER can be captured with anti-damage antibodies and sequenced (XR-seq) to produce high resolution maps of active repair locations following UV exposure. We analyzed previously published datasets from Arabidopsis thaliana, Saccharomyces cerevisiae, and Drosophila melanogaster to identify reads originating from the mtDNA (and plastid genome in A. thaliana). In A. thaliana and S. cerevisiae, the mtDNA-mapping reads have unique length distributions compared to the nuclear-mapping reads. The dominant fragment size was 26 nt in S. cerevisiae and 28 nt in A. thaliana with distinct secondary peaks occurring in 2-nt (S. cerevisiae) or 4-nt (A. thaliana) intervals. These reads also show a nonrandom distribution of di-pyrimidines (the substrate for CPD formation) with TT enrichment at positions 7-8 of the reads. Therefore, UV damage to mtDNA appears to result in production of DNA fragments of characteristic lengths and positions relative to the damaged location. We hypothesize that these fragments may reflect the outcome of a previously uncharacterized mechanism of NER-like repair in mitochondria or a programmed mtDNA degradation pathway.

Dynamic "Cap"-abilities of P-bodies and the XRN1-EDC4 axis.

RNA turnover regulates the quality and quantity of cellular gene expression through a coordinated cavalcade of enzymes, factors, and phase transitions. In this issue, Brothers et al reveal the importance of balanced communication between the Xrn1 exonuclease and the EDC4 decapping factor to coordinate P-body dynamics and maintain cellular fitness.

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.

Aedes aegypti miRNA-33 modulates permethrin induced toxicity by regulating VGSC transcripts.

Aedes aegypti is a major vector of Zika, dengue, and other arboviruses. Permethrin adulticidal spraying, which targets the voltage-gated sodium channel (VGSC), is commonly done to reduce local mosquito populations and protect humans from exposure to arbovirus pathogens transmitted by this dangerous pest. Permethrin resistance, however, is a growing problem and understanding its underlying molecular basis may identify avenues to combat it. We identified a single G:C polymorphism in pre-miR-33 that was genetically associated with permethrin resistance; resulting isoforms had structural differences that may affect DICER-1/pre-miRNA processing rates. We then assessed the effects of overexpression of pre-miR-33 isoforms on permethrin toxicological phenotypes, VGSC transcript abundance and protein levels for two genetically related mosquito strains. One strain had its naturally high permethrin resistance levels maintained by periodic treatment, and the other was released from selection. VGSC protein levels were lower in the permethrin resistant strain than in the related permethrin-susceptible strain. Overexpression of the G-pre-miR-33 isoform reduced VGSC expression levels in both strains. To further elucidate changes in gene expression associated with permethrin resistance, exome-capture gDNA deep sequencing, genetic association mapping and subsequent gene set enrichment analysis revealed that transport genes, in particular, were selected in resistant versus susceptible mosquitoes. Collectively, these data indicate that miR-33 regulates VGSC expression as part of a nuanced system of neuronal regulation that contributes to a network of heritable features determining permethrin resistance.

The interface between coronaviruses and host cell RNA biology: Novel potential insights for future therapeutic intervention.

Coronaviruses, including SARS-Cov-2, are RNA-based pathogens that interface with a large variety of RNA-related cellular processes during infection. These processes include capping, polyadenylation, localization, RNA stability, translation, and regulation by RNA binding proteins or noncoding RNA effectors. The goal of this article is to provide an in-depth perspective on the current state of knowledge of how various coronaviruses interact with, usurp, and/or avoid aspects of these cellular RNA biology machineries. A thorough understanding of how coronaviruses interact with RNA-related posttranscriptional processes in the cell should allow for new insights into aspects of viral pathogenesis as well as identify new potential avenues for the development of anti-coronaviral therapeutics. This article is categorized under: RNA in Disease and Development > RNA in Disease.

Mind the Gapmer: Implications of Co-transcriptional Cleavage by Antisense Oligonucleotides.

While gapmers efficiently knock down as well as terminate transcription of nascent lncRNAs and mRNAs, Lee and Mendell (2020) and Lai et al. (2020) also demonstrate that Pol II termination is not observed with gapmers targeting the 3' terminal portions of the transcript.

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.

RNA regulatory processes in RNA virus biology.

Numerous post-transcriptional RNA processes play a major role in regulating the quantity, quality and diversity of gene expression in the cell. These include RNA processing events such as capping, splicing, polyadenylation and modification, but also aspects such as RNA localization, decay, translation, and non-coding RNA-associated regulation. The interface between the transcripts of RNA viruses and the various RNA regulatory processes in the cell, therefore, has high potential to significantly impact virus gene expression, regulation, cytopathology and pathogenesis. Furthermore, understanding RNA biology from the perspective of an RNA virus can shed considerable light on the broad impact of these post-transcriptional processes in cell biology. Thus the goal of this article is to provide an overview of the richness of cellular RNA biology and how RNA viruses use, usurp and/or avoid the associated machinery to impact the outcome of infection. This article is categorized under: RNA in Disease and Development > RNA in Disease.

Engineered viral RNA decay intermediates to assess XRN1-mediated decay.

Both RNA synthesis and decay must be balanced within a cell to achieve proper gene expression. Additionally, modulation of RNA decay specifically offers the cell an opportunity to rapidly reshape the transcriptome in response to specific stimuli or cues. Therefore, it is critical to understand the underlying mechanisms through which RNA decay contribute to gene expression homeostasis. Cell-free reconstitution approaches have been used successfully to reveal mechanisms associated with numerous post-transcriptional RNA processes. Historically, it has been difficult to examine all aspects of RNA decay in such an in vitro setting due, in part, to limitations on the ability to resolve larger RNAs through denaturing polyacrylamide gels. Thus, in vitro systems to study RNA decay rely on smaller, less biologically relevant RNA fragments. Herein, we present an approach to more confidently examine RNA decay parameters of large mRNA size transcripts through the inclusion of an engineered XRN1-resistant reporter RNA (xrRNA). By placing a 67 nucleotide xrRNA near the 3' end of any in vitro transcribed RNA with variable size or sequence context, investigators can observe the accumulation of the xrRNA as a readout of exoribonuclease-mediated 5'-3' decay. This approach may allow in vitro RNA decay assays to include full biologically relevant mRNA/mRNPs, extending their utility and allow improved experimental design considerations to promote biologically relevant outcomes.

Sequences encoding C2H2 zinc fingers inhibit polyadenylation and mRNA export in human cells.

The large C2H2-Zinc Finger (C2H2-ZNF) gene family has rapidly expanded in primates through gene duplication. There is consequently considerable sequence homology between family members at both the nucleotide and amino acid level, allowing for coordinated regulation and shared functions. Here we show that multiple C2H2-ZNF mRNAs experience differential polyadenylation resulting in populations with short and long poly(A) tails. Furthermore, a significant proportion of C2H2-ZNF mRNAs are retained in the nucleus. Intriguingly, both short poly(A) tails and nuclear retention can be specified by the repeated elements that encode zinc finger motifs. These Zinc finger Coding Regions (ZCRs) appear to restrict polyadenylation of nascent RNAs and at the same time impede their export. However, the polyadenylation process is not necessary for nuclear retention of ZNF mRNAs. We propose that inefficient polyadenylation and export may allow C2H2-ZNF mRNAs to moonlight as non-coding RNAs or to be stored for later use.

Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5'→3' Xrn Exoribonuclease Activity.

The RNA3 species of the beet necrotic yellow vein virus (BNYVV), a multipartite positive-stranded RNA phytovirus, contains the 'core' nucleotide sequence required for its systemic movement in Beta macrocarpa. Within this 'core' sequence resides a conserved "coremin" motif of 20 nucleotides that is absolutely essential for long-distance movement. RNA3 undergoes processing steps to yield a noncoding RNA3 (ncRNA3) possessing "coremin" at its 5' end, a mandatory element for ncRNA3 accumulation. Expression of wild-type (wt) or mutated RNA3 in Saccharomyces cerevisiae allows for the accumulation of ncRNA3 species. Screening of S.cerevisiae ribonuclease mutants identified the 5'-to-3' exoribonuclease Xrn1 as a key enzyme in RNA3 processing that was recapitulated both in vitro and in insect cell extracts. Xrn1 stalled on ncRNA3-containing RNA substrates in these decay assays in a similar fashion as the flavivirus Xrn1-resistant structure (sfRNA). Substitution of the BNYVV-RNA3 'core' sequence by the sfRNA sequence led to the accumulation of an ncRNA species in yeast in vitro but not in planta and no viral long distance occurred. Interestingly, XRN4 knockdown reduced BNYVV RNA accumulation suggesting a dual role for the ribonuclease in the viral cycle.

The Interplay between the RNA Decay and Translation Machinery in Eukaryotes.

RNA decay plays a major role in regulating gene expression and is tightly networked with other aspects of gene expression to effectively coordinate post-transcriptional regulation. The goal of this work is to provide an overview of the major factors and pathways of general messenger RNA (mRNA) decay in eukaryotic cells, and then discuss the effective interplay of this cytoplasmic process with the protein synthesis machinery. Given the transcript-specific and fluid nature of mRNA stability in response to changing cellular conditions, understanding the fundamental networking between RNA decay and translation will provide a foundation for a complete mechanistic understanding of this important aspect of cell biology.

Identification of phlebovirus and arenavirus RNA sequences that stall and repress the exoribonuclease XRN1.

Regulated mRNA decay plays a vital role in determining both the level and quality of cellular gene expression. Viral RNAs must successfully evade this host RNA decay machinery to establish a productive infection. One way for RNA viruses to accomplish this is to target the cellular exoribonuclease XRN1, because this enzyme is accessible in the cytoplasm and plays a major role in mRNA decay. Members of the Flaviviridae use RNA structures in their 5'- or 3'-untranslated regions to stall and repress XRN1, effectively stabilizing viral RNAs while also causing significant dysregulation of host cell mRNA stability. Here, we use a series of biochemical assays to demonstrate that the 3'-terminal portion of the nucleocapsid (N) mRNA of Rift Valley fever virus, a phlebovirus of the Bunyaviridae family, also can effectively stall and repress XRN1. The region responsible for impeding XRN1 includes a G-rich portion that likely forms a G-quadruplex structure. The 3'-terminal portions of ambisense-derived transcripts of multiple arenaviruses also stalled XRN1. Therefore, we conclude that RNAs from two additional families of mammalian RNA viruses stall and repress XRN1. This observation. emphasizes the importance and commonality of this viral strategy to interfere with the 5'-to-3'-exoribonuclease component of the cytoplasmic RNA decay machinery.

Trick or TREAT: A Scary-Good New Approach for Single-Molecule mRNA Decay Analysis.

In this issue of Molecular Cell, Horvathova et al. (2017) have developed a powerful approach to single-molecule assessment of RNA decay in living cells by exploiting the ability of flavivirus RNA structural elements to trap XRN1 decay intermediates in dual-labeled reporter constructs.

Metabolic labeling and recovery of nascent RNA to accurately quantify mRNA stability.

Changes in the rate of mRNA decay are closely coordinated with transcriptional changes and together these events have profound effects on gene expression during development and disease. Traditional approaches to assess mRNA decay have relied on inhibition of transcription, which can alter mRNA decay rates and confound interpretation. More recently, metabolic labeling combined with chemical modification and fractionation of labeled RNAs has allowed the isolation of nascent transcripts and the subsequent calculation of mRNA decay rates. This approach has been widely adopted for measuring mRNA half-lives on a global scale, but has proven challenging to use for analysis of single genes. We present a series of normalization and quality assurance steps to be used in combination with 4-thiouridine pulse labeling of cultured eukaryotic cells. Importantly, we demonstrate how the relative amount of 4sU-labeled nascent RNA influences accurate quantification. The approach described facilitates reproducible measurement of individual mRNA half-lives using 4-thiouridine and could be adapted for use with other nucleoside analogs.

The CELF1 RNA-Binding Protein Regulates Decay of Signal Recognition Particle mRNAs and Limits Secretion in Mouse Myoblasts.

We previously identified several mRNAs encoding components of the secretory pathway, including signal recognition particle (SRP) subunit mRNAs, among transcripts associated with the RNA-binding protein CELF1. Through immunoprecipitation of RNAs crosslinked to CELF1 in myoblasts and in vitro binding assays using recombinant CELF1, we now provide evidence that CELF1 directly binds the mRNAs encoding each of the subunits of the SRP. Furthermore, we determined the half-lives of the Srp transcripts in control and CELF1 knockdown myoblasts. Our results indicate CELF1 is a destabilizer of at least five of the six Srp transcripts and that the relative abundance of the SRP proteins is out of balance when CELF1 is depleted. CELF1 knockdown myoblasts exhibit altered secretion of a luciferase reporter protein and are impaired in their ability to migrate and close a wound, consistent with a defect in the secreted extracellular matrix. Importantly, similar defects in wound healing are observed when SRP subunit imbalance is induced by over-expression of SRP68. Our studies support the existence of an RNA regulon containing Srp mRNAs that is controlled by CELF1. One implication is that altered function of CELF1 in myotonic dystrophy may contribute to changes in the extracellular matrix of affected muscle through defects in secretion.