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.

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.

Upf1 ATPase-dependent mRNP disassembly is required for completion of nonsense- mediated mRNA decay.

Cellular mRNAs exist in messenger ribonucleoprotein (mRNP) complexes, which undergo transitions during the lifetime of the mRNAs and direct posttranscriptional gene regulation. A final posttranscriptional step in gene expression is the turnover of the mRNP, which involves degradation of the mRNA and recycling of associated proteins. How tightly associated protein components are released from degrading mRNPs is unknown. Here, we demonstrate that the ATPase activity of the RNA helicase Upf1 allows disassembly of mRNPs undergoing nonsense-mediated mRNA decay (NMD). In the absence of Upf1 ATPase activity, partially degraded NMD mRNA intermediates accumulate in complex with NMD factors and concentrate in processing bodies. Thus, disassembly and completion of turnover of mRNPs undergoing NMD requires ATP hydrolysis by Upf1. This uncovers a previously unappreciated and potentially regulated step in mRNA decay and raises the question of how other mRNA decay pathways release protein components of substrate mRNPs.

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.

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.

mRNA decapping factors and the exonuclease Xrn2 function in widespread premature termination of RNA polymerase II transcription.

We report a function of human mRNA decapping factors in control of transcription by RNA polymerase II. Decapping proteins Edc3, Dcp1a, and Dcp2 and the termination factor TTF2 coimmunoprecipitate with Xrn2, the nuclear 5'-3' exonuclease "torpedo" that facilitates transcription termination at the 3' ends of genes. Dcp1a, Xrn2, and TTF2 localize near transcription start sites (TSSs) by ChIP-seq. At genes with 5' peaks of paused pol II, knockdown of decapping or termination factors Xrn2 and TTF2 shifted polymerase away from the TSS toward upstream and downstream distal positions. This redistribution of pol II is similar in magnitude to that caused by depletion of the elongation factor Spt5. We propose that coupled decapping of nascent transcripts and premature termination by the "torpedo" mechanism is a widespread mechanism that limits bidirectional pol II elongation. Regulated cotranscriptional decapping near promoter-proximal pause sites followed by premature termination could control productive pol II elongation.

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.

Translational coregulation of 5'TOP mRNAs by TIA-1 and TIAR.

The response of cells to changes in their environment often requires coregulation of gene networks, but little is known about how this can occur at the post-transcriptional level. An important example of post-transcriptional coregulation is the selective translational regulation in response to growth conditions of mammalian mRNAs that encode protein biosynthesis factors and contain hallmark 5'-terminal oligopyrimidine tracts (5'TOP). However, the responsible trans-factors and the mechanism by which they coregulate 5'TOP mRNAs have remained elusive. Here we identify stress granule-associated TIA-1 and TIAR proteins as key factors in human 5'TOP mRNA regulation, which upon amino acid starvation assemble onto the 5' end of 5'TOP mRNAs and arrest translation at the initiation step, as evidenced by TIA-1/TIAR-dependent 5'TOP mRNA translation repression, polysome release, and accumulation in stress granules. This requires starvation-mediated activation of the GCN2 (general control nonderepressible 2) kinase and inactivation of the mTOR (mammalian target of rapamycin) signaling pathway. Our findings provide a mechanistic explanation to the long-standing question of how the network of 5'TOP mRNAs are coregulated according to amino acid availability, thereby allowing redirection of limited resources to mount a nutrient deprivation response. This presents a fundamental example of how a group of mRNAs can be translationally coregulated in response to changes in the cellular environment.

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.

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.

The control of mRNA decapping and P-body formation.

mRNA decapping is a critical step in eukaryotic cytoplasmic mRNA turnover. Cytoplasmic mRNA decapping is catalyzed by Dcp2 in conjunction with its coactivator Dcp1 and is stimulated by decapping enhancer proteins. mRNAs associated with the decapping machinery can assemble into cytoplasmic mRNP granules called processing bodies (PBs). Evidence suggests that PB-associated mRNPs are translationally repressed and can be degraded or stored for subsequent translation. However, whether mRNP assembly into a PB is important for translational repression, decapping, or decay has remained controversial. Here, we discuss the regulation of decapping machinery recruitment to specific mRNPs and how their assembly into PBs is governed by the relative rates of translational repression, mRNP multimerization, and mRNA decay.

Structural and functional control of the eukaryotic mRNA decapping machinery.

The regulation of mRNA degradation is critical for proper gene expression. Many major pathways for mRNA decay involve the removal of the 5' 7-methyl guanosine (m(7)G) cap in the cytoplasm to allow for 5'-to-3' exonucleolytic decay. The most well studied and conserved eukaryotic decapping enzyme is Dcp2, and its function is aided by co-factors and decapping enhancers. A subset of these factors can act to enhance the catalytic activity of Dcp2, while others might stimulate the remodeling of proteins bound to the mRNA substrate that may otherwise inhibit decapping. Structural studies have provided major insights into the mechanisms by which Dcp2 and decapping co-factors activate decapping. Additional mRNA decay factors can function by recruiting components of the decapping machinery to target mRNAs. mRNA decay factors, decapping factors, and mRNA substrates can be found in cytoplasmic foci named P bodies that are conserved in eukaryotes, though their function remains unknown. In addition to Dcp2, other decapping enzymes have been identified, which may serve to supplement the function of Dcp2 or act in independent decay or quality control pathways. This article is part of a Special Issue entitled: RNA Decay mechanisms.

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.

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.

RNA decapping inside and outside of processing bodies.

Decapping is a central step in eukaryotic mRNA turnover. Recent studies have identified several factors involved in catalysis and regulation of decapping. These include the following: an mRNA decapping complex containing the proteins Dcp1 and Dcp2; a nucleolar decapping enzyme, X29, involved in the degradation of U8 snoRNA and perhaps of other capped nuclear RNAs; and a decapping 'scavenger' enzyme, DcpS, that hydrolyzes the cap structure resulting from complete 3'-to-5' degradation of mRNAs by the exosome. Several proteins that stimulate mRNA decapping by the Dcp1:Dcp2 complex co-localize with Dcp1 and Dcp2, together with Xrn1, a 5'-to-3' exonuclease, to structures in the cytoplasm called processing bodies. Recent evidence suggests that the processing bodies may constitute specialized cellular compartments of mRNA turnover, which suggests that mRNA and protein localization may be integral to mRNA decay.

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.

A competition between stimulators and antagonists of Upf complex recruitment governs human nonsense-mediated mRNA decay.

The nonsense-mediated decay (NMD) pathway subjects mRNAs with premature termination codons (PTCs) to rapid decay. The conserved Upf1-3 complex interacts with the eukaryotic translation release factors, eRF3 and eRF1, and triggers NMD when translation termination takes place at a PTC. Contrasting models postulate central roles in PTC-recognition for the exon junction complex in mammals versus the cytoplasmic poly(A)-binding protein (PABP) in other eukaryotes. Here we present evidence for a unified model for NMD, in which PTC recognition in human cells is mediated by a competition between 3' UTR-associated factors that stimulate or antagonize recruitment of the Upf complex to the terminating ribosome. We identify cytoplasmic PABP as a human NMD antagonizing factor, which inhibits the interaction between eRF3 and Upf1 in vitro and prevents NMD in cells when positioned in proximity to the termination codon. Surprisingly, only when an extended 3' UTR places cytoplasmic PABP distally to the termination codon does a downstream exon junction complex enhance NMD, likely through increasing the affinity of Upf proteins for the 3' UTR. Interestingly, while an artificial 3' UTR of >420 nucleotides triggers NMD, a large subset of human mRNAs contain longer 3' UTRs but evade NMD. We speculate that these have evolved to concentrate NMD-inhibiting factors, such as PABP, in spatial proximity of the termination codon.