Assigning function to an unexplored Integrator module.

In this issue of Molecular Cell, Razew et al.1 and Sabath et al.2 assign function to an unexplored module of the Integrator (INT) complex, expanding the toolbox of this genome-wide attenuator of RNA polymerase II (RNAPII) transcription.

Protocol for generating customizable and reproducible plots of sequencing coverage data using the seqNdisplayR package.

The widespread usage of next-generation sequencing methods for functional genomics studies requires standardized tools for consistent visualization of the associated data. Here, we present seqNdisplayR, an R package for plotting standard sequencing data coverage within a genomic region of interest in a customizable and reproducible manner. We describe steps for installing software, preparing data files, choosing options, and plotting data. This tool is readily available for users with no prior experience with R through the "Shiny app" interface. For complete details on the use and execution of this protocol, please refer to Lykke-Andersen et al.,1 Gockert et al.,2 and Rouviere et al.3.

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.

Control of non-productive RNA polymerase II transcription via its early termination in metazoans.

Transcription establishes the universal first step of gene expression where RNA is produced by a DNA-dependent RNA polymerase. The most versatile of eukaryotic RNA polymerases, RNA polymerase II (Pol II), transcribes a broad range of DNA including protein-coding and a variety of non-coding transcription units. Although Pol II can be configured as a durable enzyme capable of transcribing hundreds of kilobases, there is reliable evidence of widespread abortive Pol II transcription termination shortly after initiation, which is often followed by rapid degradation of the associated RNA. The molecular details underlying this phenomenon are still vague but likely reflect the action of quality control mechanisms on the early Pol II complex. Here, we summarize current knowledge of how and when such promoter-proximal quality control is asserted on metazoan Pol II.

ARS2/SRRT: at the nexus of RNA polymerase II transcription, transcript maturation and quality control.

ARS2/SRRT is an essential eukaryotic protein that has emerged as a critical factor in the sorting of functional from non-functional RNA polymerase II (Pol II) transcripts. Through its interaction with the Cap Binding Complex (CBC), it associates with the cap of newly made RNAs and acts as a hub for competitive exchanges of protein factors that ultimately determine the fate of the associated RNA. The central position of the protein within the nuclear gene expression machinery likely explains why its depletion causes a broad range of phenotypes, yet an exact function of the protein remains elusive. Here, we consider the literature on ARS2/SRRT with the attempt to garner the threads into a unifying working model for ARS2/SRRT function at the nexus of Pol II transcription, transcript maturation and quality control.

Co-translational assembly and localized translation of nucleoporins in nuclear pore complex biogenesis.

mRNA translation is coupled to multiprotein complex assembly in the cytoplasm or to protein delivery into intracellular compartments. Here, by combining systematic RNA immunoprecipitation and single-molecule RNA imaging in yeast, we have provided a complete depiction of the co-translational events involved in the biogenesis of a large multiprotein assembly, the nuclear pore complex (NPC). We report that binary interactions between NPC subunits can be established during translation, in the cytoplasm. Strikingly, the nucleoporins Nup1/Nup2, together with a number of nuclear proteins, are instead translated at nuclear pores, through a mechanism involving interactions between their nascent N-termini and nuclear transport receptors. Uncoupling this co-translational recruitment further triggers the formation of cytoplasmic foci of unassembled polypeptides. Altogether, our data reveal that distinct, spatially segregated modes of co-translational interactions foster the ordered assembly of NPC subunits and that localized translation can ensure the proper delivery of proteins to the pore and the nucleus.

Integrator is a genome-wide attenuator of non-productive transcription.

Termination of RNA polymerase II (RNAPII) transcription in metazoans relies largely on the cleavage and polyadenylation (CPA) and integrator (INT) complexes originally found to act at the ends of protein-coding and small nuclear RNA (snRNA) genes, respectively. Here, we monitor CPA- and INT-dependent termination activities genome-wide, including at thousands of previously unannotated transcription units (TUs), producing unstable RNA. We verify the global activity of CPA occurring at pA sites indiscriminately of their positioning relative to the TU promoter. We also identify a global activity of INT, which is largely sequence-independent and restricted to a ~3-kb promoter-proximal region. Our analyses suggest two functions of genome-wide INT activity: it dampens transcriptional output from weak promoters, and it provides quality control of RNAPII complexes that are unfavorably configured for transcriptional elongation. We suggest that the function of INT in stable snRNA production is an exception from its general cellular role, the attenuation of non-productive transcription.

The RNA exosome shapes the expression of key protein-coding genes.

The ribonucleolytic exosome complex is central for nuclear RNA degradation, primarily targeting non-coding RNAs. Still, the nuclear exosome could have protein-coding (pc) gene-specific regulatory activities. By depleting an exosome core component, or components of exosome adaptor complexes, we identify ∼2900 transcription start sites (TSSs) from within pc genes that produce exosome-sensitive transcripts. At least 1000 of these overlap with annotated mRNA TSSs and a considerable portion of their transcripts share the annotated mRNA 3' end. We identify two types of pc-genes, both employing a single, annotated TSS across cells, but the first type primarily produces full-length, exosome-sensitive transcripts, whereas the second primarily produces prematurely terminated transcripts. Genes within the former type often belong to immediate early response transcription factors, while genes within the latter are likely transcribed as a consequence of their proximity to upstream TSSs on the opposite strand. Conversely, when genes have multiple active TSSs, alternative TSSs that produce exosome-sensitive transcripts typically do not contribute substantially to overall gene expression, and most such transcripts are prematurely terminated. Our results display a complex landscape of sense transcription within pc-genes and imply a direct role for nuclear RNA turnover in the regulation of a subset of pc-genes.

Mapping domains of ARS2 critical for its RNA decay capacity.

ARS2 is a conserved protein centrally involved in both nuclear RNA productive and destructive processes. To map features of ARS2 promoting RNA decay, we utilized two different RNA reporters, one of which depends on direct ARS2 tethering for its degradation. In both cases, ARS2 triggers a degradation phenotype aided by its interaction with the poly(A) tail exosome targeting (PAXT) connection. Interestingly, C-terminal amino acids of ARS2, responsible for binding the RNA 5'cap binding complex (CBC), become dispensable when ARS2 is directly tethered to the reporter RNA. In contrast, the Zinc-finger (ZnF) domain of ARS2 is essential for the decay of both reporters and consistently co-immunoprecipitation analyses reveal a necessity of this domain for the interaction of ARS2 with the PAXT-associated RNA helicase MTR4. Taken together, our results map the domains of ARS2 underlying two essential properties of the protein: its RNP targeting ability and its capacity to recruit the RNA decay machinery.

A SUMO-dependent feedback loop senses and controls the biogenesis of nuclear pore subunits.

While the activity of multiprotein complexes is crucial for cellular metabolism, little is known about the mechanisms that collectively control the expression of their components. Here, we investigate the regulations targeting the biogenesis of the nuclear pore complex (NPC), the macromolecular assembly mediating nucleocytoplasmic exchanges. Systematic analysis of RNA-binding proteins interactomes, together with in vivo and in vitro assays, reveal that a subset of NPC mRNAs are specifically bound by Hek2, a yeast hnRNP K-like protein. Hek2-dependent translational repression and protein turnover are further shown to finely tune the levels of NPC subunits. Strikingly, mutations or physiological perturbations altering pore integrity decrease the levels of the NPC-associated SUMO protease Ulp1, and trigger the accumulation of sumoylated versions of Hek2 unable to bind NPC mRNAs. Our results support the existence of a quality control mechanism involving Ulp1 as a sensor of NPC integrity and Hek2 as a repressor of NPC biogenesis.

Tma108, a putative M1 aminopeptidase, is a specific nascent chain-associated protein in Saccharomyces cerevisiae.

The discovery of novel specific ribosome-associated factors challenges the assumption that translation relies on standardized molecular machinery. In this work, we demonstrate that Tma108, an uncharacterized translation machinery-associated factor in yeast, defines a subpopulation of cellular ribosomes specifically involved in the translation of less than 200 mRNAs encoding proteins with ATP or Zinc binding domains. Using ribonucleoparticle dissociation experiments we established that Tma108 directly interacts with the nascent protein chain. Additionally, we have shown that translation of the first 35 amino acids of Asn1, one of the Tma108 targets, is necessary and sufficient to recruit Tma108, suggesting that it is loaded early during translation. Comparative genomic analyses, molecular modeling and directed mutagenesis point to Tma108 as an original M1 metallopeptidase, which uses its putative catalytic peptide-binding pocket to bind the N-terminus of its targets. The involvement of Tma108 in co-translational regulation is attested by a drastic change in the subcellular localization of ATP2 mRNA upon Tma108 inactivation. Tma108 is a unique example of a nascent chain-associated factor with high selectivity and its study illustrates the existence of other specific translation-associated factors besides RNA binding proteins.

Sumoylation of the THO complex regulates the biogenesis of a subset of mRNPs.

Assembly of messenger ribonucleoparticles (mRNPs) is a pivotal step in gene expression, but only a few molecular mechanisms contributing to its regulation have been described. Here, through a comprehensive proteomic survey of mRNP assembly, we demonstrate that the SUMO pathway specifically controls the association of the THO complex with mRNPs. We further show that the THO complex, a key player in the interplay between gene expression, mRNA export and genetic stability, is sumoylated on its Hpr1 subunit and that this modification regulates its association with mRNPs. Altered recruitment of the THO complex onto mRNPs in sumoylation-defective mutants does not affect bulk mRNA export or genetic stability, but impairs the expression of acidic stress-induced genes and, consistently, compromises viability in acidic stress conditions. Importantly, inactivation of the nuclear exosome suppresses the phenotypes of the hpr1 non-sumoylatable mutant, showing that SUMO-dependent mRNP assembly is critical to allow a specific subset of mRNPs to escape degradation. This article thus provides the first example of a SUMO-dependent mRNP-assembly event allowing a refined tuning of gene expression, in particular under specific stress conditions.

Multiple crosstalks between mRNA biogenesis and SUMO.

mRNA metabolism involves the orchestration of multiple nuclear events, including transcription, processing (e.g., capping, splicing, polyadenylation), and quality control. This leads to the accurate formation of messenger ribonucleoparticles (mRNPs) that are finally exported to the cytoplasm for translation. The production of defined sets of mRNAs in given environmental or physiological situations relies on multiple regulatory mechanisms that target the mRNA biogenesis machineries. Among other regulations, post-translational modification by the small ubiquitin-like modifier SUMO, whose prominence in several cellular processes has been largely demonstrated, also plays a key role in mRNA biogenesis. Analysis of the multiple available SUMO proteomes and functional validations of an increasing number of sumoylated targets have revealed the key contribution of SUMO-dependent regulation in nuclear mRNA metabolism. While sumoylation of transcriptional activators and repressors is so far best documented, SUMO contribution to other stages of mRNA biogenesis is also emerging. Modification of mRNA metabolism factors by SUMO determine their subnuclear targeting and biological activity, notably by regulating their molecular interactions with nucleic acids or protein partners. In particular, sumoylation of DNA-bound transcriptional regulators interfere with their association to target sequences or chromatin modifiers. In addition, the recent identification of enzymes of the SUMO pathway within specialized mRNA biogenesis machineries may provide a further level of regulation to their specificity. These multiple crosstalks between mRNA metabolism and SUMO appear therefore as important players in cellular regulatory networks.

Wnt4 participates in the formation of vertebrate neuromuscular junction.

Neuromuscular junction (NMJ) formation requires the highly coordinated communication of several reciprocal signaling processes between motoneurons and their muscle targets. Identification of the early, spatially restricted cues in target recognition at the NMJ is still poorly documented, especially in mammals. Wnt signaling is one of the key pathways regulating synaptic connectivity. Here, we report that Wnt4 contributes to the formation of vertebrate NMJ in vivo. Results from a microarray screen and quantitative RT-PCR demonstrate that Wnt4 expression is regulated during muscle cell differentiation in vitro and muscle development in vivo, being highly expressed when the first synaptic contacts are formed and subsequently downregulated. Analysis of the mouse Wnt4⁻/⁻ NMJ phenotype reveals profound innervation defects including motor axons overgrowing and bypassing AChR aggregates with 30% of AChR clusters being unapposed by nerve terminals. In addition, loss of Wnt4 function results in a 35% decrease of the number of prepatterned AChR clusters while Wnt4 overexpression in cultured myotubes increases the number of AChR clusters demonstrating that Wnt4 directly affects postsynaptic differentiation. In contrast, muscle structure and the localization of several synaptic proteins including acetylcholinesterase, MuSK and rapsyn are not perturbed in the Wnt4 mutant. Finally, we identify MuSK as a Wnt4 receptor. Wnt4 not only interacts with MuSK ectodomain but also mediates MuSK activation. Taken together our data reveal a new role for Wnt4 in mammalian NMJ formation that could be mediated by MuSK, a key receptor in synaptogenesis.