Sm complex assembly and 5' cap trimethylation promote selective processing of snRNAs by the 3' exonuclease TOE1.

Competing exonucleases that promote 3' end maturation or degradation direct quality control of small non-coding RNAs, but how these enzymes distinguish normal from aberrant RNAs is poorly understood. The Pontocerebellar Hypoplasia 7 (PCH7)-associated 3' exonuclease TOE1 promotes maturation of canonical small nuclear RNAs (snRNAs). Here, we demonstrate that TOE1 achieves specificity toward canonical snRNAs through their Sm complex assembly and cap trimethylation, two features that distinguish snRNAs undergoing correct biogenesis from other small non-coding RNAs. Indeed, disruption of Sm complex assembly via snRNA mutations or protein depletions obstructs snRNA processing by TOE1, and in vitro snRNA processing by TOE1 is stimulated by a trimethylated cap. An unstable snRNA variant that normally fails to undergo maturation becomes fully processed by TOE1 when its degenerate Sm binding motif is converted into a canonical one. Our findings uncover the molecular basis for how TOE1 distinguishes snRNAs from other small non-coding RNAs and explain how TOE1 promotes maturation specifically of canonical snRNAs undergoing proper processing.

Assigning functionality to cysteines by base editing of cancer dependency genes.

Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events affect protein function, however, remains challenging. Here we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation. The resulting atlas, which covers more than 13,800 cysteines on more than 1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in more than 160 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

The 3' exonuclease TOE1 selectively processes snRNAs through recognition of Sm complex assembly and 5' cap trimethylation.

Competing exonucleases that promote 3' end maturation or degradation direct quality control of small non-coding RNAs, but how these enzymes distinguish normal from aberrant RNAs is poorly understood. The Pontocerebellar Hypoplasia 7 (PCH7)-associated 3' exonuclease TOE1 promotes maturation of canonical small nuclear RNAs (snRNAs). Here, we demonstrate that TOE1 achieves specificity towards canonical snRNAs by recognizing Sm complex assembly and cap trimethylation, two features that distinguish snRNAs undergoing correct biogenesis from other small non-coding RNAs. Indeed, disruption of Sm complex assembly via snRNA mutations or protein depletions obstructs snRNA processing by TOE1, and in vitro snRNA processing by TOE1 is stimulated by a trimethylated cap. An unstable snRNA variant that normally fails to undergo maturation becomes fully processed by TOE1 when its degenerate Sm binding motif is converted into a canonical one. Our findings uncover the molecular basis for how TOE1 distinguishes snRNAs from other small non-coding RNAs and explain how TOE1 promotes maturation specifically of canonical snRNAs undergoing proper processing.

The Conserved CNOT1 Interaction Motif of Tristetraprolin Regulates ARE-mRNA Decay Independently of the p38 MAPK-MK2 Kinase Pathway.

The regulation of the mRNA decay activator Tristetraprolin (TTP) by the p38 mitogen-activated protein kinase (MAPK) pathway during the mammalian inflammatory response represents a paradigm for the control of mRNA turnover by signaling. TTP activity is regulated through multiple phosphorylation sites, including an evolutionary conserved serine in its CNOT1 Interacting Motif (CIM) whose phosphorylation disrupts an interaction with CNOT1 of the CCR4-NOT deadenylase complex. Here we present evidence that the TTP CIM recruits the CCR4-NOT deadenylase complex and activates mRNA degradation cooperatively with the conserved tryptophan residues of TTP, previously identified to interact with CNOT9. Surprisingly, the TTP CIM remains unphosphorylated and capable of promoting association with the CCR4-NOT complex and mRNA decay upon activation of p38-MAPK-activated kinase MK2, a well-established regulator of TTP activity. The CIM is instead targeted by other kinases including PKCα. These observations suggest that signaling pathways regulate TTP activity in a cooperative manner and that the p38 MAPK-MK2 kinase pathway relies on the activation of additional kinase pathway(s) to fully control TTP function.

Tailer: a pipeline for sequencing-based analysis of nonpolyadenylated RNA 3' end processing.

Post-transcriptional trimming and tailing of RNA 3' ends play key roles in the processing and quality control of noncoding RNAs (ncRNAs). However, bioinformatic tools to examine changes in the RNA 3' "tailome" are sparse and not standardized. Here we present Tailer, a bioinformatic pipeline in two parts that allows for robust quantification and analysis of tail information from next-generation sequencing experiments that preserve RNA 3' end information. The first part of Tailer, Tailer-processing, uses genome annotation or reference FASTA gene sequences to quantify RNA 3' ends from SAM-formatted alignment files or FASTQ sequence read files produced from sequencing experiments. The second part, Tailer-analysis, uses the output of Tailer-processing to identify statistically significant RNA targets of trimming and tailing and create graphs for data exploration. We apply Tailer to RNA 3' end sequencing experiments from three published studies and find that it accurately and reproducibly recapitulates key findings. Thus, Tailer should be a useful and easily accessible tool to globally investigate tailing dynamics of nonpolyadenylated RNAs and conditions that perturb them.

Competition between maturation and degradation drives human snRNA 3' end quality control.

Polymerases and exonucleases act on 3' ends of nascent RNAs to promote their maturation or degradation but how the balance between these activities is controlled to dictate the fates of cellular RNAs remains poorly understood. Here, we identify a central role for the human DEDD deadenylase TOE1 in distinguishing the fates of small nuclear (sn)RNAs of the spliceosome from unstable genome-encoded snRNA variants. We found that TOE1 promotes maturation of all regular RNA polymerase II transcribed snRNAs of the major and minor spliceosomes by removing posttranscriptional oligo(A) tails, trimming 3' ends, and preventing nuclear exosome targeting. In contrast, TOE1 promotes little to no maturation of tested U1 variant snRNAs, which are instead targeted by the nuclear exosome. These observations suggest that TOE1 is positioned at the center of a 3' end quality control pathway that selectively promotes maturation and stability of regular snRNAs while leaving snRNA variants unprocessed and exposed to degradation in what could be a widespread mechanism of RNA quality control given the large number of noncoding RNAs processed by DEDD deadenylases.

Chromatin Modification and Global Transcriptional Silencing in the Oocyte Mediated by the mRNA Decay Activator ZFP36L2.

Global transcriptional silencing is a highly conserved mechanism central to the oocyte-to-embryo transition. We report the unexpected discovery that global transcriptional silencing in oocytes depends on an mRNA decay activator. Oocyte-specific loss of ZFP36L2 an RNA-binding protein that promotes AU-rich element-dependent mRNA decay prevents global transcriptional silencing and causes oocyte maturation and fertilization defects, as well as complete female infertility in the mouse. Single-cell RNA sequencing revealed that ZFP36L2 downregulates mRNAs encoding transcription and chromatin modification regulators, including a large group of mRNAs for histone demethylases targeting H3K4 and H3K9, which we show are bound and degraded by ZFP36L2. Oocytes lacking Zfp36l2 fail to accumulate histone methylation at H3K4 and H3K9, marks associated with the transcriptionally silent, developmentally competent oocyte state. Our results uncover a ZFP36L2-dependent mRNA decay mechanism that acts as a developmental switch during oocyte growth, triggering wide-spread shifts in chromatin modification and global transcription.

A human microprotein that interacts with the mRNA decapping complex.

Proteomic detection of non-annotated microproteins indicates the translation of hundreds of small open reading frames (smORFs) in human cells, but whether these microproteins are functional or not is unknown. Here, we report the discovery and characterization of a 7-kDa human microprotein we named non-annotated P-body dissociating polypeptide (NoBody). NoBody interacts with mRNA decapping proteins, which remove the 5' cap from mRNAs to promote 5'-to-3' decay. Decapping proteins participate in mRNA turnover and nonsense-mediated decay (NMD). NoBody localizes to mRNA-decay-associated RNA-protein granules called P-bodies. Modulation of NoBody levels reveals that its abundance is anticorrelated with cellular P-body numbers and alters the steady-state levels of a cellular NMD substrate. These results implicate NoBody as a novel component of the mRNA decapping complex and demonstrate potential functionality of a newly discovered microprotein.

Protein-RNA Networks Regulated by Normal and ALS-Associated Mutant HNRNPA2B1 in the Nervous System.

HnRNPA2B1 encodes an RNA binding protein associated with neurodegeneration. However, its function in the nervous system is unclear. Transcriptome-wide crosslinking and immunoprecipitation in mouse spinal cord discover UAGG motifs enriched within ∼2,500 hnRNP A2/B1 binding sites and an unexpected role for hnRNP A2/B1 in alternative polyadenylation. HnRNP A2/B1 loss results in alternative splicing (AS), including skipping of an exon in amyotrophic lateral sclerosis (ALS)-associated D-amino acid oxidase (DAO) that reduces D-serine metabolism. ALS-associated hnRNP A2/B1 D290V mutant patient fibroblasts and motor neurons differentiated from induced pluripotent stem cells (iPSC-MNs) demonstrate abnormal splicing changes, likely due to increased nuclear-insoluble hnRNP A2/B1. Mutant iPSC-MNs display decreased survival in long-term culture and exhibit hnRNP A2/B1 localization to cytoplasmic granules as well as exacerbated changes in gene expression and splicing upon cellular stress. Our findings provide a cellular resource and reveal RNA networks relevant to neurodegeneration, regulated by normal and mutant hnRNP A2/B1. VIDEO ABSTRACT.

Hyperphosphorylation amplifies UPF1 activity to resolve stalls in nonsense-mediated mRNA decay.

Many gene expression factors contain repetitive phosphorylation sites for single kinases, but the functional significance is poorly understood. Here we present evidence for hyperphosphorylation as a mechanism allowing UPF1, the central factor in nonsense-mediated decay (NMD), to increasingly attract downstream machinery with time of residence on target mRNAs. Indeed, slowing NMD by inhibiting late-acting factors triggers UPF1 hyperphosphorylation, which in turn enhances affinity for factors linking UPF1 to decay machinery. Mutational analyses reveal multiple phosphorylation sites contributing to different extents to UPF1 activity with no single site being essential. Moreover, the ability of UPF1 to undergo hyperphosphorylation becomes increasingly important for NMD when downstream factors are depleted. This hyperphosphorylation-dependent feedback mechanism may serve as a molecular clock ensuring timely degradation of target mRNAs while preventing degradation of non-targets, which, given the prevalence of repetitive phosphorylation among central gene regulatory factors, may represent an important general principle in gene expression.

The C-Terminal RGG Domain of Human Lsm4 Promotes Processing Body Formation Stimulated by Arginine Dimethylation.

Processing bodies (PBs) are conserved cytoplasmic aggregations of translationally repressed mRNAs assembled with mRNA decay factors. The aggregation of mRNA-protein (mRNP) complexes into PBs involves interactions between low-complexity regions of protein components of the mRNPs. In Saccharomyces cerevisiae, the carboxy (C)-terminal Q/N-rich domain of the Lsm4 subunit of the Lsm1-7 complex plays an important role in PB formation, but the C-terminal domain of Lsm4 in most eukaryotes is an RGG domain rather than Q/N rich. Here we show that the Lsm4 RGG domain promotes PB accumulation in human cells and that symmetric dimethylation of arginines within the RGG domain stimulates this process. A mutant Lsm4 protein lacking the RGG domain failed to rescue PB formation in cells depleted of endogenous Lsm4, although this mutant protein retained the ability to assemble with Lsm1-7, associate with decapping factors, and promote mRNA decay and translational repression. Mutation of the symmetrically dimethylated arginines within the RGG domain impaired the ability of Lsm4 to promote PB accumulation. Depletion of PRMT5, the primary protein arginine methyltransferase responsible for symmetric arginine dimethylation, including Lsm4, resulted in loss of PBs. We also uncovered the histone acetyltransferase 1 (HAT1)-RBBP7 lysine acetylase complex as an interaction partner of the Lsm4 RGG domain but found no evidence of a role for this complex in PB metabolism. Together, our findings suggest a stimulatory role for posttranslational modifications in PB accumulation and raise the possibility that mRNP dynamics are posttranslationally regulated.

Recruitment of the 4EHP-GYF2 cap-binding complex to tetraproline motifs of tristetraprolin promotes repression and degradation of mRNAs with AU-rich elements.

The zinc finger protein tristetraprolin (TTP) promotes translation repression and degradation of mRNAs containing AU-rich elements (AREs). Although much attention has been directed toward understanding the decay process and machinery involved, the translation repression role of TTP has remained poorly understood. Here we identify the cap-binding translation repression 4EHP-GYF2 complex as a cofactor of TTP. Immunoprecipitation and in vitro pull-down assays demonstrate that TTP associates with the 4EHP-GYF2 complex via direct interaction with GYF2, and mutational analyses show that this interaction occurs via conserved tetraproline motifs of TTP. Mutant TTP with diminished 4EHP-GYF2 binding is impaired in its ability to repress a luciferase reporter ARE-mRNA. 4EHP knockout mouse embryonic fibroblasts (MEFs) display increased induction and slower turnover of TTP-target mRNAs as compared to wild-type MEFs. Our work highlights the function of the conserved tetraproline motifs of TTP and identifies 4EHP-GYF2 as a cofactor in translational repression and mRNA decay by TTP.

DDX6 Orchestrates Mammalian Progenitor Function through the mRNA Degradation and Translation Pathways.

In adult tissues, stem and progenitor cells must balance proliferation and differentiation to maintain homeostasis. How this is done is unclear. Here, we show that the DEAD box RNA helicase, DDX6 is necessary for maintaining adult progenitor cell function. DDX6 loss results in premature differentiation and decreased proliferation of epidermal progenitor cells. To maintain self-renewal, DDX6 associates with YBX1 to bind the stem loops found in the 3' UTRs of regulators of proliferation/self-renewal (CDK1, EZH2) and recruit them to EIF4E to facilitate their translation. To prevent premature differentiation of progenitor cells, DDX6 regulates the 5' UTR of differentiation inducing transcription factor, KLF4 and degrades its transcripts through association with mRNA degradation proteins. Our results demonstrate that progenitor function is maintained by DDX6 complexes through two distinct pathways that include the degradation of differentiation-inducing transcripts and by promoting the translation of self-renewal and proliferation mRNAs.

Target Discrimination in Nonsense-Mediated mRNA Decay Requires Upf1 ATPase Activity.

RNA quality-control pathways get rid of faulty RNAs and therefore must be able to discriminate these RNAs from those that are normal. Here we present evidence that the adenosine triphosphatase (ATPase) cycle of the SF1 helicase Upf1 is required for mRNA discrimination during nonsense-mediated decay (NMD). Mutations affecting the Upf1 ATPase cycle disrupt the mRNA selectivity of Upf1, leading to indiscriminate accumulation of NMD complexes on both NMD target and non-target mRNAs. In addition, two modulators of NMD-translation and termination codon-proximal poly(A) binding protein-depend on the ATPase activity of Upf1 to limit Upf1-non-target association. Preferential ATPase-dependent dissociation of Upf1 from non-target mRNAs in vitro suggests that selective release of Upf1 contributes to the ATPase dependence of Upf1 target discrimination. Given the prevalence of helicases in RNA regulation, ATP hydrolysis may be a widely used activity in target RNA discrimination.

Competition between Decapping Complex Formation and Ubiquitin-Mediated Proteasomal Degradation Controls Human Dcp2 Decapping Activity.

mRNA decapping is a central step in eukaryotic mRNA decay that simultaneously shuts down translation initiation and activates mRNA degradation. A major complex responsible for decapping consists of the decapping enzyme Dcp2 in association with decapping enhancers. An important question is how the activity and accumulation of Dcp2 are regulated at the cellular level to ensure the specificity and fidelity of the Dcp2 decapping complex. Here, we show that human Dcp2 levels and activity are controlled by a competition between decapping complex assembly and Dcp2 degradation. This is mediated by a regulatory domain in the Dcp2 C terminus, which, on the one hand, promotes Dcp2 activation via decapping complex formation mediated by the decapping enhancer Hedls and, on the other hand, targets Dcp2 for ubiquitin-mediated proteasomal degradation in the absence of Hedls association. This competition between Dcp2 activation and degradation restricts the accumulation and activity of uncomplexed Dcp2, which may be important for preventing uncontrolled decapping or for regulating Dcp2 levels and activity according to cellular needs.

Post-transcriptional regulation of satellite cell quiescence by TTP-mediated mRNA decay.

Skeletal muscle satellite cells in their niche are quiescent and upon muscle injury, exit quiescence, proliferate to repair muscle tissue, and self-renew to replenish the satellite cell population. To understand the mechanisms involved in maintaining satellite cell quiescence, we identified gene transcripts that were differentially expressed during satellite cell activation following muscle injury. Transcripts encoding RNA binding proteins were among the most significantly changed and included the mRNA decay factor Tristetraprolin. Tristetraprolin promotes the decay of MyoD mRNA, which encodes a transcriptional regulator of myogenic commitment, via binding to the MyoD mRNA 3' untranslated region. Upon satellite cell activation, p38α/ß MAPK phosphorylates MAPKAP2 and inactivates Tristetraprolin, stabilizing MyoD mRNA. Satellite cell specific knockdown of Tristetraprolin precociously activates satellite cells in vivo, enabling MyoD accumulation, differentiation and cell fusion into myofibers. Regulation of mRNAs by Tristetraprolin appears to function as one of several critical post-transcriptional regulatory mechanisms controlling satellite cell homeostasis.

Identification of elements in human long 3' UTRs that inhibit nonsense-mediated decay.

The nonsense-mediated mRNA decay (NMD) pathway serves an important role in gene expression by targeting aberrant mRNAs that have acquired premature termination codons (PTCs) as well as a subset of normally processed endogenous mRNAs. One determinant for the targeting of mRNAs by NMD is the occurrence of translation termination distal to the poly(A) tail. Yet, a large subset of naturally occurring mRNAs contain long 3' UTRs, many of which, according to global studies, are insensitive to NMD. This raises the possibility that such mRNAs have evolved mechanisms for NMD evasion. Here, we analyzed a set of human long 3' UTR mRNAs and found that many are indeed resistant to NMD. By dissecting the 3' UTR of one such mRNA, TRAM1 mRNA, we identified a cis element located within the first 200 nt that inhibits NMD when positioned in downstream proximity of the translation termination codon and is sufficient for repressing NMD of a heterologous reporter mRNA. Investigation of other NMD-evading long 3' UTR mRNAs revealed a subset that, similar to TRAM1 mRNA, contains NMD-inhibiting cis elements in the first 200 nt. A smaller subset of long 3' UTR mRNAs evades NMD by a different mechanism that appears to be independent of a termination-proximal cis element. Our study suggests that different mechanisms have evolved to ensure NMD evasion of human mRNAs with long 3' UTRs.

hnRNP F complexes with tristetraprolin and stimulates ARE-mRNA decay.

The tristetraprolin (TTP) family of zinc-finger proteins, TTP, BRF1 and BRF2, regulate the stability of a subset of mRNAs containing 3'UTR AU-rich elements (AREs), including mRNAs coding for cytokines, transcription factors, and proto-oncogenes. To better understand the mechanism by which TTP-family proteins control mRNA stability in mammalian cells, we aimed to identify TTP- and BRF1-interacting proteins as potential TTP-family co-factors. This revealed hnRNP F as a prominent interactor of TTP and BRF1. While TTP, BRF1 and hnRNP F are all RNA binding proteins (RBPs), the interaction of hnRNP F with TTP and BRF1 is independent of RNA. Depletion of hnRNP F impairs the decay of a subset of TTP-substrate ARE-mRNAs by a mechanism independent of the extent of hnRNP F binding to the mRNA. Taken together, these findings implicate hnRNP F as a co-factor in a subset of TTP/BRF-mediated mRNA decay and highlight the importance of RBP cooperativity in mRNA regulation.

Protecting the proteome: Eukaryotic cotranslational quality control pathways.

The correct decoding of messenger RNAs (mRNAs) into proteins is an essential cellular task. The translational process is monitored by several quality control (QC) mechanisms that recognize defective translation complexes in which ribosomes are stalled on substrate mRNAs. Stalled translation complexes occur when defects in the mRNA template, the translation machinery, or the nascent polypeptide arrest the ribosome during translation elongation or termination. These QC events promote the disassembly of the stalled translation complex and the recycling and/or degradation of the individual mRNA, ribosomal, and/or nascent polypeptide components, thereby clearing the cell of improper translation products and defective components of the translation machinery.

Regulation of ARE-mRNA Stability by Cellular Signaling: Implications for Human Cancer.

During recent years, it has become clear that regulation of mRNA stability is an important event in the control of gene expression. The stability of a large class of mammalian mRNAs is regulated by AU-rich elements (AREs) located in the mRNA 3' UTRs. mRNAs with AREs are inherently labile but as a response to different cellular cues they can become either stabilized, allowing expression of a given gene, or further destabilized to silence their expression. These tightly regulated mRNAs include many that encode growth factors, proto-oncogenes, cytokines, and cell cycle regulators. Failure to properly regulate their stability can therefore lead to uncontrolled expression of factors associated with cell proliferation and has been implicated in several human cancers. A number of transfactors that recognize AREs and regulate the translation and degradation of ARE-mRNAs have been identified. These transfactors are regulated by signal transduction pathways, which are often misregulated in cancers. This chapter focuses on the function of ARE-binding proteins with an emphasis on their regulation by signaling pathways and the implications for human cancer.