PNPT1 Release from Mitochondria during Apoptosis Triggers Decay of Poly(A) RNAs.

Widespread mRNA decay, an unappreciated feature of apoptosis, enhances cell death and depends on mitochondrial outer membrane permeabilization (MOMP), TUTases, and DIS3L2. Which RNAs are decayed and the decay-initiating event are unknown. Here, we show extensive decay of mRNAs and poly(A) noncoding (nc)RNAs at the 3' end, triggered by the mitochondrial intermembrane space 3'-to-5' exoribonuclease PNPT1, released during MOMP. PNPT1 knockdown inhibits apoptotic RNA decay and reduces apoptosis, while ectopic expression of PNPT1, but not an RNase-deficient mutant, increases RNA decay and cell death. The 3' end of PNPT1 substrates thread through a narrow channel. Many non-poly(A) ncRNAs contain 3'-secondary structures or bind proteins that may block PNPT1 activity. Indeed, mutations that disrupt the 3'-stem-loop of a decay-resistant ncRNA render the transcript susceptible, while adding a 3'-stem-loop to an mRNA prevents its decay. Thus, PNPT1 release from mitochondria during MOMP initiates apoptotic decay of RNAs lacking 3'-structures.

ARS2 instructs early transcription termination-coupled RNA decay by recruiting ZC3H4 to nascent transcripts.

The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif (SLiM) in the transcription restriction factor ZC3H4. This interaction recruits ZC3H4 to chromatin to elicit RNAPII termination, independent of other early termination pathways defined by the cleavage and polyadenylation (CPA) and Integrator (INT) complexes. We find that ZC3H4, in turn, forms a direct connection to the nuclear exosome targeting (NEXT) complex, hereby facilitating rapid degradation of the nascent RNA. Hence, ARS2 instructs the coupled transcription termination and degradation of the transcript onto which it is bound. This contrasts with ARS2 function at CPA-instructed termination sites where the protein exclusively partakes in RNA suppression via post-transcriptional decay.

Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression.

Transposable elements (TEs) are widespread genetic parasites known to be kept under tight transcriptional control. Here, we describe a functional connection between the mouse-orthologous "nuclear exosome targeting" (NEXT) and "human silencing hub" (HUSH) complexes, involved in nuclear RNA decay and the epigenetic silencing of TEs, respectively. Knocking out the NEXT component ZCCHC8 in embryonic stem cells results in elevated TE RNA levels. We identify a physical interaction between ZCCHC8 and the MPP8 protein of HUSH and establish that HUSH recruits NEXT to chromatin at MPP8-bound TE loci. However, while NEXT and HUSH both dampen TE RNA expression, their activities predominantly affect shorter non-polyadenylated and full-length polyadenylated transcripts, respectively. Indeed, our data suggest that the repressive action of HUSH promotes a condition favoring NEXT RNA decay activity. In this way, transcriptional and post-transcriptional machineries synergize to suppress the genotoxic potential of TE RNAs.

Minding the message: tactics controlling RNA decay, modification, and translation in virus-infected cells.

With their categorical requirement for host ribosomes to translate mRNA, viruses provide a wealth of genetically tractable models to investigate how gene expression is remodeled post-transcriptionally by infection-triggered biological stress. By co-opting and subverting cellular pathways that control mRNA decay, modification, and translation, the global landscape of post-transcriptional processes is swiftly reshaped by virus-encoded factors. Concurrent host cell-intrinsic countermeasures likewise conscript post-transcriptional strategies to mobilize critical innate immune defenses. Here we review strategies and mechanisms that control mRNA decay, modification, and translation in animal virus-infected cells. Besides settling infection outcomes, post-transcriptional gene regulation in virus-infected cells epitomizes fundamental physiological stress responses in health and disease.

Functional interaction between the RNA exosome and the sirtuin deacetylase Hst3 maintains transcriptional homeostasis.

Eukaryotic cells maintain an optimal level of mRNAs through unknown mechanisms that balance RNA synthesis and degradation. We found that inactivation of the RNA exosome leads to global reduction of nascent mRNA transcripts, and that this defect is accentuated by loss of deposition of histone variant H2A.Z. We identify the mRNA for the sirtuin deacetylase Hst3 as a key target for the RNA exosome that mediates communication between RNA degradation and transcription machineries. These findings reveal how the RNA exosome and H2A.Z function together to control a deacetylase, ensuring proper levels of transcription in response to changes in RNA degradation.

Dysregulated Expression of the Nuclear Exosome Targeting Complex Component Rbm7 in Nonhematopoietic Cells Licenses the Development of Fibrosis.

Fibrosis is an incurable disorder of unknown etiology. Segregated-nucleus-containing atypical monocytes (SatMs) are critical for the development of fibrosis. Here we examined the mechanisms that recruit SatMs to pre-fibrotic areas. A screen based on cytokine expression in the fibrotic lung revealed that the chemokine Cxcl12, which is produced by apoptotic nonhematopoietic cells, was essential for SatM recruitment. Analyses of lung tissues at fibrosis onset showed increased expression of Rbm7, a component of the nuclear exosome targeting complex. Rbm7 deletion suppressed bleomycin-induced fibrosis and at a cellular level, suppressed apoptosis of nonhematopoietic cells. Mechanistically, Rbm7 bound to noncoding (nc)RNAs that form subnuclear bodies, including Neat1 speckles. Dysregulated expression of Rbm7 resulted in the nuclear degradation of Neat1 speckles, the dispersion of the DNA repair protein BRCA1, and the triggering of apoptosis. Thus, Rbm7 in epithelial cells plays a critical role in the development of fibrosis by regulating ncRNA decay and thereby the production of chemokines that recruit SatMs.

Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.

RNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5'-3') mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3'-5') is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.

Structure-Mediated RNA Decay by UPF1 and G3BP1.

Post-transcriptional mechanisms regulate the stability and, hence, expression of coding and noncoding RNAs. Sequence-specific features within the 3' untranslated region (3' UTR) often direct mRNAs for decay. Here, we characterize a genome-wide RNA decay pathway that reduces the half-lives of mRNAs based on overall 3' UTR structure formed by base pairing. The decay pathway is independent of specific single-stranded sequences, as regulation is maintained in both the original and reverse complement orientation. Regulation can be compromised by reducing the overall structure by fusing the 3' UTR with an unstructured sequence. Mutating base-paired RNA regions can also compromise this structure-mediated regulation, which can be restored by re-introducing base-paired structures of different sequences. The decay pathway requires the RNA-binding protein UPF1 and its associated protein G3BP1. Depletion of either protein increased steady-state levels of mRNAs with highly structured 3' UTRs as well as highly structured circular RNAs. This structure-dependent mechanism therefore enables cells to selectively regulate coding and noncoding RNAs.

Obstacles to Scanning by RNase E Govern Bacterial mRNA Lifetimes by Hindering Access to Distal Cleavage Sites.

The diversity of mRNA lifetimes in bacterial cells is difficult to reconcile with the relaxed cleavage site specificity of RNase E, the endonuclease most important for governing mRNA degradation. This enzyme has generally been thought to locate cleavage sites by searching freely in three dimensions. However, our results now show that its access to such sites in 5'-monophosphorylated RNA is hindered by obstacles-such as bound proteins or ribosomes or coaxial small RNA (sRNA) base pairing-that disrupt the path from the 5' end to those sites and prolong mRNA lifetimes. These findings suggest that RNase E searches for cleavage sites by scanning linearly from the 5'-terminal monophosphate along single-stranded regions of RNA and that its progress is impeded by structural discontinuities encountered along the way. This discovery has major implications for gene regulation in bacteria and suggests a general mechanism by which other prokaryotic and eukaryotic regulatory proteins can be controlled.

Nuclear TARBP2 Drives Oncogenic Dysregulation of RNA Splicing and Decay.

Post-transcriptional regulation of RNA stability is a key step in gene expression control. We describe a regulatory program, mediated by the RNA binding protein TARBP2, that controls RNA stability in the nucleus. TARBP2 binding to pre-mRNAs results in increased intron retention, subsequently leading to targeted degradation of TARBP2-bound transcripts. This is mediated by TARBP2 recruitment of the m6A RNA methylation machinery to its target transcripts, where deposition of m6A marks influences the recruitment of splicing regulators, inhibiting efficient splicing. Interactions between TARBP2 and the nucleoprotein TPR then promote degradation of these TARBP2-bound transcripts by the nuclear exosome. Additionally, analysis of clinical gene expression datasets revealed a functional role for TARBP2 in lung cancer. Using xenograft mouse models, we find that TARBP2 affects tumor growth in the lung and that this is dependent on TARBP2-mediated destabilization of ABCA3 and FOXN3. Finally, we establish ZNF143 as an upstream regulator of TARBP2 expression.

Endoribonucleolytic Cleavage of m6A-Containing RNAs by RNase P/MRP Complex.

N6-methyladenosine (m6A) is the most abundant internal modification in RNAs and plays regulatory roles in a variety of biological and physiological processes. Despite its important roles, the molecular mechanism underlying m6A-mediated gene regulation is poorly understood. Here, we show that m6A-containing RNAs are subject to endoribonucleolytic cleavage via YTHDF2 (m6A reader protein), HRSP12 (adaptor protein), and RNase P/MRP (endoribonucleases). We demonstrate that HRSP12 functions as an adaptor to bridge YTHDF2 and RNase P/MRP, eliciting rapid degradation of YTHDF2-bound RNAs. Transcriptome-wide analyses show that m6A RNAs that are preferentially targeted for endoribonucleolytic cleavage have an HRSP12-binding site and a RNase P/MRP-directed cleavage site upstream and downstream of the YTHDF2-binding site, respectively. We also find that a subset of m6A-containing circular RNAs associates with YTHDF2 in an HRSP12-dependent manner and is selectively downregulated by RNase P/MRP. Thus, our data expand the known functions of RNase P/MRP to endoribonucleolytic cleavage of m6A RNAs.

Stresses that Raise Np4A Levels Induce Protective Nucleoside Tetraphosphate Capping of Bacterial RNA.

Present in all realms of life, dinucleoside tetraphosphates (Np4Ns) are generally considered signaling molecules. However, only a single pathway for Np4N signaling has been delineated in eukaryotes, and no receptor that mediates the influence of Np4Ns has ever been identified in bacteria. Here we show that, under disulfide stress conditions that elevate cellular Np4N concentrations, diverse Escherichia coli mRNAs and sRNAs acquire a cognate Np4 cap. Purified E. coli RNA polymerase and lysyl-tRNA synthetase are both capable of adding such 5' caps. Cap removal by either of two pyrophosphatases, ApaH or RppH, triggers rapid RNA degradation in E. coli. ApaH, the predominant decapping enzyme, functions as both a sensor and an effector of disulfide stress, which inactivates it. These findings suggest that the physiological changes attributed to elevated Np4N concentrations in bacteria may result from widespread Np4 capping, leading to altered RNA stability and consequent changes in gene expression.

Improved RNA stability estimation through Bayesian modeling reveals most Salmonella transcripts have subminute half-lives.

RNA decay is a crucial mechanism for regulating gene expression in response to environmental stresses. In bacteria, RNA-binding proteins (RBPs) are known to be involved in posttranscriptional regulation, but their global impact on RNA half-lives has not been extensively studied. To shed light on the role of the major RBPs ProQ and CspC/E in maintaining RNA stability, we performed RNA sequencing of Salmonella enterica over a time course following treatment with the transcription initiation inhibitor rifampicin (RIF-seq) in the presence and absence of these RBPs. We developed a hierarchical Bayesian model that corrects for confounding factors in rifampicin RNA stability assays and enables us to identify differentially decaying transcripts transcriptome-wide. Our analysis revealed that the median RNA half-life in Salmonella in early stationary phase is less than 1 min, a third of previous estimates. We found that over half of the 500 most long-lived transcripts are bound by at least one major RBP, suggesting a general role for RBPs in shaping the transcriptome. Integrating differential stability estimates with cross-linking and immunoprecipitation followed by RNA sequencing (CLIP-seq) revealed that approximately 30% of transcripts with ProQ binding sites and more than 40% with CspC/E binding sites in coding or 3' untranslated regions decay differentially in the absence of the respective RBP. Analysis of differentially destabilized transcripts identified a role for ProQ in the oxidative stress response. Our findings provide insights into posttranscriptional regulation by ProQ and CspC/E, and the importance of RBPs in regulating gene expression.

Telomere-specific regulation of TERRA and its impact on telomere stability.

TERRA is a class of telomeric repeat-containing RNAs that are expressed from telomeres in multiple organisms. TERRA transcripts play key roles in telomere maintenance and their physiological levels are essential to maintain the integrity of telomeric DNA. Indeed, deregulated TERRA expression or its altered localization can impact telomere stability by multiple mechanisms including fueling transcription-replication conflicts, promoting resection of chromosome ends, altering the telomeric chromatin, and supporting homologous recombination. Therefore, a fine-tuned control of TERRA is important to maintain the integrity of the genome. Several studies have reported that different cell lines express substantially different levels of TERRA. Most importantly, TERRA levels markedly vary among telomeres of a given cell type, indicating the existence of telomere-specific regulatory mechanisms which may help coordinate TERRA functions. TERRA molecules contain distinct subtelomeric sequences, depending on their telomere of origin, which may instruct specific post-transcriptional modifications or mediate distinct functions. In addition, all TERRA transcripts share a repetitive G-rich sequence at their 3' end which can form DNA:RNA hybrids and fold into G-quadruplex structures. Both structures are involved in TERRA functions and can critically affect telomere stability. In this review, we examine the mechanisms controlling TERRA levels and the impact of their telomere-specific regulation on telomere stability. We compare evidence obtained in different model organisms, discussing recent advances as well as controversies in the field. Furthermore, we discuss the importance of DNA:RNA hybrids and G-quadruplex structures in the context of TERRA biology and telomere maintenance.

RNA degradation in human mitochondria: the journey is not finished.

Mitochondria are vital organelles present in almost all eukaryotic cells. Although most of the mitochondrial proteins are nuclear-encoded, mitochondria contain their own genome, whose proper expression is necessary for mitochondrial function. Transcription of the human mitochondrial genome results in the synthesis of long polycistronic transcripts that are subsequently processed by endonucleases to release individual RNA molecules, including precursors of sense protein-encoding mRNA (mt-mRNA) and a vast amount of antisense noncoding RNAs. Because of mitochondrial DNA (mtDNA) organization, the regulation of individual gene expression at the transcriptional level is limited. Although transcription of most protein-coding mitochondrial genes occurs with the same frequency, steady-state levels of mature transcripts are different. Therefore, post-transcriptional processes are important for regulating mt-mRNA levels. The mitochondrial degradosome is a complex composed of the RNA helicase SUV3 (also known as SUPV3L1) and polynucleotide phosphorylase (PNPase, PNPT1). It is the best-characterized RNA-degrading machinery in human mitochondria, which is primarily responsible for the decay of mitochondrial antisense RNA. The mechanism of mitochondrial sense RNA decay is less understood. This review aims to provide a general picture of mitochondrial genome expression, with a particular focus on mitochondrial RNA (mtRNA) degradation.

Clearing the slate: RNA turnover to enable cell state switching?

The distribution of mRNA in tissue is determined by the balance between transcription and decay. Understanding the control of RNA decay during development has been somewhat neglected compared with transcriptional control. Here, we explore the potential for mRNA decay to trigger rapid cell state transitions during development, comparing a bistable switch model of cell state conversion with experimental evidence from different developmental systems. We also consider another potential role for large-scale RNA decay that has emerged from studies of stress-induced cell state transitions, in which removal of mRNA unblocks the translation machinery to prioritise the synthesis of proteins that establish the new cell state.

PABP Cooperates with the CCR4-NOT Complex to Promote mRNA Deadenylation and Block Precocious Decay.

Multiple deadenylases are known in vertebrates, the PAN2-PAN3 (PAN2/3) and CCR4-NOT (CNOT) complexes, and PARN, yet their differential functions remain ambiguous. Moreover, the role of poly(A) binding protein (PABP) is obscure, limiting our understanding of the deadenylation mechanism. Here, we show that CNOT serves as a predominant nonspecific deadenylase for cytoplasmic poly(A)+ RNAs, and PABP promotes deadenylation while preventing premature uridylation and decay. PAN2/3 selectively trims long tails (>∼150 nt) with minimal effect on transcriptome, whereas PARN does not affect mRNA deadenylation. CAF1 and CCR4, catalytic subunits of CNOT, display distinct activities: CAF1 trims naked poly(A) segments and is blocked by PABPC, whereas CCR4 is activated by PABPC to shorten PABPC-protected sequences. Concerted actions of CAF1 and CCR4 delineate the ∼27 nt periodic PABPC footprints along shortening tail. Our study unveils distinct functions of deadenylases and PABPC, re-drawing the view on mRNA deadenylation and regulation.

Terminal Uridylyltransferases Execute Programmed Clearance of Maternal Transcriptome in Vertebrate Embryos.

During the maternal-to-zygotic transition (MZT), maternal RNAs are actively degraded and replaced by newly synthesized zygotic transcripts in a highly coordinated manner. However, it remains largely unknown how maternal mRNA decay is triggered in early vertebrate embryos. Here, through genome-wide profiling of RNA abundance and 3' modification, we show that uridylation is induced at the onset of maternal mRNA clearance. The temporal control of uridylation is conserved in vertebrates. When the homologs of terminal uridylyltransferases TUT4 and TUT7 (TUT4/7) are depleted in zebrafish and Xenopus, maternal mRNA clearance is significantly delayed, leading to developmental defects during gastrulation. Short-tailed mRNAs are selectively uridylated by TUT4/7, with the highly uridylated transcripts degraded faster during the MZT than those with unmodified poly(A) tails. Our study demonstrates that uridylation plays a crucial role in timely mRNA degradation, thereby allowing the progression of early development.

Progression of the pluripotent epiblast depends upon the NMD factor UPF2.

Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that degrades RNAs harboring in-frame stop codons in specific contexts. Loss of NMD factors leads to embryonic lethality in organisms spanning the phylogenetic scale, but the mechanism remains unknown. Here, we report that the core NMD factor, UPF2, is required for expansion of epiblast cells within the inner cell mass of mice in vivo. We identify NMD target mRNAs in mouse blastocysts - both canonical and alternatively processed mRNAs - including those encoding cell cycle arrest and apoptosis factors, raising the possibility that NMD is essential for embryonic cell proliferation and survival. In support, the inner cell mass of Upf2-null blastocysts rapidly regresses with outgrowth and is incompetent for embryonic stem cell derivation in vitro. In addition, we uncovered concordant temporal- and lineage-specific regulation of NMD factors and mRNA targets, indicative of a shift in NMD magnitude during peri-implantation development. Together, our results reveal developmental and molecular functions of the NMD pathway in the early embryo.

Dynamically regulated transcription factors are encoded by highly unstable mRNAs in the Drosophila larval brain.

The level of each RNA species depends on the balance between its rates of production and decay. Although previous studies have measured RNA decay across the genome in tissue culture and single-celled organisms, few experiments have been performed in intact complex tissues and organs. It is therefore unclear whether the determinants of RNA decay found in cultured cells are preserved in an intact tissue, and whether they differ between neighboring cell types and are regulated during development. To address these questions, we measured RNA synthesis and decay rates genome wide via metabolic labeling of whole cultured Drosophila larval brains using 4-thiouridine. Our analysis revealed that decay rates span a range of more than 100-fold, and that RNA stability is linked to gene function, with mRNAs encoding transcription factors being much less stable than mRNAs involved in core metabolic functions. Surprisingly, among transcription factor mRNAs there was a clear demarcation between more widely used transcription factors and those that are expressed only transiently during development. mRNAs encoding transient transcription factors are among the least stable in the brain. These mRNAs are characterized by epigenetic silencing in most cell types, as shown by their enrichment with the histone modification H3K27me3. Our data suggest the presence of an mRNA destabilizing mechanism targeted to these transiently expressed transcription factors to allow their levels to be regulated rapidly with high precision. Our study also demonstrates a general method for measuring mRNA transcription and decay rates in intact organs or tissues, offering insights into the role of mRNA stability in the regulation of complex developmental programs.