Translation-coupled mRNA quality control mechanisms.

mRNA surveillance pathways are essential for accurate gene expression and to maintain translation homeostasis, ensuring the production of fully functional proteins. Future insights into mRNA quality control pathways will enable us to understand how cellular mRNA levels are controlled, how defective or unwanted mRNAs can be eliminated, and how dysregulation of these can contribute to human disease. Here we review translation-coupled mRNA quality control mechanisms, including the non-stop and no-go mRNA decay pathways, describing their mechanisms, shared trans-acting factors, and differences. We also describe advances in our understanding of the nonsense-mediated mRNA decay (NMD) pathway, highlighting recent mechanistic findings, the discovery of novel factors, as well as the role of NMD in cellular physiology and its impact on human disease.

Histone H1 regulates non-coding RNA turnover on chromatin in a m6A-dependent manner.

Linker histones are highly abundant chromatin-associated proteins with well-established structural roles in chromatin and as general transcriptional repressors. In addition, it has been long proposed that histone H1 exerts context-specific effects on gene expression. Here, we identify a function of histone H1 in chromatin structure and transcription using a range of genomic approaches. In the absence of histone H1, there is an increase in the transcription of non-coding RNAs, together with reduced levels of m6A modification leading to their accumulation on chromatin and causing replication-transcription conflicts. This strongly suggests that histone H1 prevents non-coding RNA transcription and regulates non-coding transcript turnover on chromatin. Accordingly, altering the m6A RNA methylation pathway rescues the replicative phenotype of H1 loss. This work unveils unexpected regulatory roles of histone H1 on non-coding RNA turnover and m6A deposition, highlighting the intimate relationship between chromatin conformation, RNA metabolism, and DNA replication to maintain genome performance.

A dual role for the RNA helicase DHX34 in NMD and pre-mRNA splicing and its function in hematopoietic differentiation.

The DExD/H-box RNA helicase DHX34 is a nonsense-mediated decay (NMD) factor that together with core NMD factors coregulates NMD targets in nematodes and in vertebrates. Here, we show that DHX34 is also associated with the human spliceosomal catalytic C complex. Mapping of DHX34 endogenous binding sites using cross-linking immunoprecipitation (CLIP) revealed that DHX34 is preferentially associated with pre-mRNAs and locates at exon-intron boundaries. Accordingly, we observed that DHX34 regulates a large number of alternative splicing (AS) events in mammalian cells in culture, establishing a dual role for DHX34 in both NMD and pre-mRNA splicing. We previously showed that germline DHX34 mutations associated to familial myelodysplasia (MDS)/acute myeloid leukemia (AML) predisposition abrogate its activity in NMD. Interestingly, we observe now that DHX34 regulates the splicing of pre-mRNAs that have been linked to AML/MDS predisposition. This is consistent with silencing experiments in hematopoietic stem/progenitor cells (HSPCs) showing that loss of DHX34 results in differentiation blockade of both erythroid and myeloid lineages, which is a hallmark of AML development. Altogether, these data unveil new cellular functions of DHX34 and suggest that alterations in the levels and/or activity of DHX34 could contribute to human disease.

RNA splicing is a key mediator of tumour cell plasticity and a therapeutic vulnerability in colorectal cancer.

Tumour cell plasticity is a major barrier to the efficacy of targeted cancer therapies but the mechanisms that mediate it are poorly understood. Here, we identify dysregulated RNA splicing as a key driver of tumour cell dedifferentiation in colorectal cancer (CRC). We find that Apc-deficient CRC cells have dysregulated RNA splicing machinery and exhibit global rewiring of RNA splicing. We show that the splicing factor SRSF1 controls the plasticity of tumour cells by controlling Kras splicing and is required for CRC invasion in a mouse model of carcinogenesis. SRSF1 expression maintains stemness in human CRC organoids and correlates with cancer stem cell marker expression in human tumours. Crucially, partial genetic downregulation of Srsf1 does not detrimentally affect normal tissue homeostasis, demonstrating that tumour cell plasticity can be differentially targeted. Thus, our findings link dysregulation of the RNA splicing machinery and control of tumour cell plasticity.

Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function.

Shuttling RNA-binding proteins coordinate nuclear and cytoplasmic steps of gene expression. The SR family proteins regulate RNA splicing in the nucleus and a subset of them, including SRSF1, shuttles between the nucleus and cytoplasm affecting post-splicing processes. However, the physiological significance of this remains unclear. Here, we used genome editing to knock-in a nuclear retention signal (NRS) in Srsf1 to create a mouse model harboring an SRSF1 protein that is retained exclusively in the nucleus. Srsf1NRS/NRS mutants displayed small body size, hydrocephalus, and immotile sperm, all traits associated with ciliary defects. We observed reduced translation of a subset of mRNAs and decreased abundance of proteins involved in multiciliogenesis, with disruption of ciliary ultrastructure and motility in cells and tissues derived from this mouse model. These results demonstrate that SRSF1 shuttling is used to reprogram gene expression networks in the context of high cellular demands, as observed here, during motile ciliogenesis.

Concordance Analysis of ALK Gene Fusion Detection Methods in Patients with Non-Small-Cell Lung Cancer from Chile, Brazil, and Peru.

About 4% to 7% of the non-small-cell lung cancer patients have anaplastic lymphoma kinase (ALK) rearrangements, and specific targeted therapies improve patients' outcomes significantly. ALK gene fusions are detected by immunohistochemistry or fluorescent in situ hybridization as gold standards in South America. Next-generation sequencing-based assays are a reliable alternative, able to perform simultaneous detection of multiple events from a single sample. We analyzed 4240 non-small-cell lung cancer samples collected in 37 hospitals from Chile, Brazil, and Peru, where ALK rearrangements were determined as part of their standard of care (SofC) using either immunohistochemistry or fluorescent in situ hybridization. A subset of 1450 samples was sequenced with the Oncomine Focus Assay (OFA), and the concordance with the SofC tests was measured. An orthogonal analysis was performed using a real-time quantitative PCR echinoderm microtubule-associated protein-like 4-ALK fusion detection kit. ALK fusion prevalence is similar for Chile (3.67%; N = 2142), Brazil (4.05%; N = 1013), and Peru (4.59%; N = 675). Although a comparison between OFA and SofC assays showed similar sensitivity, OFA had significantly higher specificity and higher positive predictive value, which opens new opportunities for a more specific determination of ALK gene rearrangements.

Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1, and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.

Novel TRPV1 Channel Agonists With Faster and More Potent Analgesic Properties Than Capsaicin.

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a member of the family of Transient Receptor Potential (TRP) channels that acts as a molecular detector of noxious signals in primary sensory neurons. Activated by capsaicin, heat, voltage and protons, it is also well known for its desensitization, which led to the medical use of topically applied TRPV1 agonist capsaicin for its long-lasting analgesic effects. Here we report three novel small molecules, which were identified using a Structure-Based Virtual Screening for TRPV1 from the ZINC database. The three compounds were tested using electrophysiological assays, which confirmed their capsaicin-like agonist activity. von Frey filaments were used to measure the analgesic effects of the compounds applied topically on tactile allodynia induced by intra-plantar carrageenan. All compounds had anti-nociceptive activity, but two of them showed faster and longer lasting analgesic effects than capsaicin. The present results suggest that TRPV1 agonists different from capsaicin could be used to develop topical analgesics with faster onset and more potent effects.

Identification of a localized nonsense-mediated decay pathway at the endoplasmic reticulum.

Nonsense-mediated decay (NMD) is a translation-dependent RNA quality control mechanism that occurs in the cytoplasm. However, it is unknown how NMD regulates the stability of RNAs translated at the endoplasmic reticulum (ER). Here, we identify a localized NMD pathway dedicated to ER-translated mRNAs. We previously identified NBAS, a component of the Syntaxin 18 complex involved in Golgi-to-ER trafficking, as a novel NMD factor. Furthermore, we show that NBAS fulfills an independent function in NMD. This ER-NMD pathway requires the interaction of NBAS with the core NMD factor UPF1, which is partially localized at the ER in the proximity of the translocon. NBAS and UPF1 coregulate the stability of ER-associated transcripts, in particular those associated with the cellular stress response. We propose a model where NBAS recruits UPF1 to the membrane of the ER and activates an ER-dedicated NMD pathway, thus providing an ER-protective function by ensuring quality control of ER-translated mRNAs.

High Proportion of Potential Candidates for Immunotherapy in a Chilean Cohort of Gastric Cancer Patients: Results of the FORCE1 Study.

Gastric cancer (GC) is a heterogeneous disease. This heterogeneity applies not only to morphological and phenotypic features but also to geographical variations in incidence and mortality rates. As Chile has one of the highest mortality rates within South America, we sought to define a molecular profile of Chilean GCs (ClinicalTrials.gov identifier: NCT03158571/(FORCE1)). Solid tumor samples and clinical data were obtained from 224 patients, with subsets analyzed by tissue microarray (TMA; n = 90) and next generation sequencing (NGS; n = 101). Most demographic and clinical data were in line with previous reports. TMA data indicated that 60% of patients displayed potentially actionable alterations. Furthermore, 20.5% were categorized as having a high tumor mutational burden, and 13% possessed micro-satellite instability (MSI). Results also confirmed previous studies reporting high Epstein-Barr virus (EBV) positivity (13%) in Chilean-derived GC samples suggesting a high proportion of patients could benefit from immunotherapy. As expected, TP53 and PIK3CA were the most frequently altered genes. However, NGS demonstrated the presence of TP53, NRAS, and BRAF variants previously unreported in current GC databases. Finally, using the Kendall method, we report a significant correlation between EBV+ status and programmed death ligand-1 (PDL1)+ and an inverse correlation between p53 mutational status and MSI. Our results suggest that in this Chilean cohort, a high proportion of patients are potential candidates for immunotherapy treatment. To the best of our knowledge, this study is the first in South America to assess the prevalence of actionable targets and to examine a molecular profile of GC patients.

A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes.

The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is greater in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development.

Rapid Depletion of DIS3, EXOSC10, or XRN2 Reveals the Immediate Impact of Exoribonucleolysis on Nuclear RNA Metabolism and Transcriptional Control.

Cell-based studies of human ribonucleases traditionally rely on methods that deplete proteins slowly. We engineered cells in which the 3'→5' exoribonucleases of the exosome complex, DIS3 and EXOSC10, can be rapidly eliminated to assess their immediate roles in nuclear RNA biology. The loss of DIS3 has the greatest impact, causing the substantial accumulation of thousands of transcripts within 60 min. These transcripts include enhancer RNAs, promoter upstream transcripts (PROMPTs), and products of premature cleavage and polyadenylation (PCPA). These transcripts are unaffected by the rapid loss of EXOSC10, suggesting that they are rarely targeted to it. More direct detection of EXOSC10-bound transcripts revealed its substrates to prominently include short 3' extended ribosomal and small nucleolar RNAs. Finally, the 5'→3' exoribonuclease, XRN2, has little activity on exosome substrates, but its elimination uncovers different mechanisms for the early termination of transcription from protein-coding gene promoters.

Post-transcriptional control of miRNA biogenesis.

MicroRNAs (miRNAs) are important regulators of gene expression that bind complementary target mRNAs and repress their expression. Precursor miRNA molecules undergo nuclear and cytoplasmic processing events, carried out by the endoribonucleases DROSHA and DICER, respectively, to produce mature miRNAs that are loaded onto the RISC (RNA-induced silencing complex) to exert their biological function. Regulation of mature miRNA levels is critical in development, differentiation, and disease, as demonstrated by multiple levels of control during their biogenesis cascade. Here, we will focus on post-transcriptional mechanisms and will discuss the impact of cis-acting sequences in precursor miRNAs, as well as trans-acting factors that bind to these precursors and influence their processing. In particular, we will highlight the role of general RNA-binding proteins (RBPs) as factors that control the processing of specific miRNAs, revealing a complex layer of regulation in miRNA production and function.

Structural basis for terminal loop recognition and stimulation of pri-miRNA-18a processing by hnRNP A1.

Post-transcriptional mechanisms play a predominant role in the control of microRNA (miRNA) production. Recognition of the terminal loop of precursor miRNAs by RNA-binding proteins (RBPs) influences their processing; however, the mechanistic basis for how levels of individual or subsets of miRNAs are regulated is mostly unexplored. We previously showed that hnRNP A1, an RBP implicated in many aspects of RNA processing, acts as an auxiliary factor that promotes the Microprocessor-mediated processing of pri-mir-18a. Here, by using an integrative structural biology approach, we show that hnRNP A1 forms a 1:1 complex with pri-mir-18a where both RNA recognition motifs (RRMs) bind to cognate RNA sequence motifs in the terminal loop of pri-mir-18a. Terminal loop binding induces an allosteric destabilization of base-pairing in the pri-mir-18a stem that promotes its downstream processing. Our results highlight terminal loop RNA recognition by RBPs as a potential general principle of miRNA biogenesis and regulation.

The Secretion of miR-200s by a PKCζ/ADAR2 Signaling Axis Promotes Liver Metastasis in Colorectal Cancer.

Most colorectal cancer (CRC)-related deaths are due to liver metastases. PKCζ is a tumor suppressor in CRC with reduced expression in metastasis. Given the importance of microRNAs (miRNAs) in regulating cellular plasticity, we performed an unbiased screening and identified the miR-200 family as the most relevant miRNAs downregulated by PKCζ deficiency. The regulation of the intracellular levels of miR-200 by PKCζ is post-transcriptional and involves their secretion in extracellular vesicles. Here, we identified ADAR2 as a direct substrate of PKCζ in CRC cells. Phosphorylation of ADAR2 regulates its editing activity, which is required to maintain miR-200 steady-state levels, suggesting that the PKCζ/ADAR2 axis regulates miR-200 secretion through RNA editing. Loss of this axis results in epithelial-to-mesenchymal transition (EMT) and increased liver metastases, which can be inhibited in vivo by blocking miR-200 release. Therefore, the PKCζ/ADAR2 axis is a critical regulator of CRC metastases through modulation of miR-200 levels.

Labor induction just after external cephalic version with epidural analgesia at term.

OBJECTIVE: To analyze the benefits of external cephalic version (ECV) with epidural analgesia at term and labor induction just after the procedure. MATERIALS AND METHODS: This is a retrospective observational study with patients who did not want trying a breech vaginal delivery and decided trying an ECV with epidural analgesia at term and wanted labor induction or cesarean section after the procedure. We present the results of 40 ECV with epidural analgesia at term and labor induction or cesarean section just after the ECV. RESULTS: ECV succeeded in 26 out of 40 (65%) patients. Among the 26 successful ECV, 6 delivered by cesarean (23.1%). 20 patients delivered vaginally (76.9%; 50% of all patients). CONCLUSION: Considering that a high number of cesarean deliveries can be avoided, induction of labor after ECV with epidural analgesia at term can be considered after being discussed in selected patient.

Genetic variation and RNA structure regulate microRNA biogenesis.

MiRNA biogenesis is highly regulated at the post-transcriptional level; however, the role of sequence and secondary RNA structure in this process has not been extensively studied. A single G to A substitution present in the terminal loop of pri-mir-30c-1 in breast and gastric cancer patients had been previously described to result in increased levels of mature miRNA. Here, we report that this genetic variant directly affects Drosha-mediated processing of pri-mir-30c-1 in vitro and in cultured cells. Structural analysis of this variant revealed an altered RNA structure that facilitates the interaction with SRSF3, an SR protein family member that promotes pri-miRNA processing. Our results are compatible with a model whereby a genetic variant in pri-mir-30c-1 leads to a secondary RNA structure rearrangement that facilitates binding of SRSF3 resulting in increased levels of miR-30c. These data highlight that primary sequence determinants and RNA structure are key regulators of miRNA biogenesis.

The RNA-binding landscape of RBM10 and its role in alternative splicing regulation in models of mouse early development.

Mutations in the RNA-binding protein, RBM10, result in a human syndromic form of cleft palate, termed TARP syndrome. A role for RBM10 in alternative splicing regulation has been previously demonstrated in human cell lines. To uncover the cellular functions of RBM10 in a cell line that is relevant to the phenotype observed in TARP syndrome, we used iCLIP to identify its endogenous RNA targets in a mouse embryonic mandibular cell line. We observed that RBM10 binds to pre-mRNAs with significant enrichment in intronic regions, in agreement with a role for this protein in pre-mRNA splicing. In addition to protein-coding transcripts, RBM10 also binds to a variety of cellular RNAs, including non-coding RNAs, such as spliceosomal small nuclear RNAs, U2 and U12. RNA-seq was used to investigate changes in gene expression and alternative splicing in RBM10 KO mouse mandibular cells and also in mouse ES cells. We uncovered a role for RBM10 in the regulation of alternative splicing of common transcripts in both cell lines but also identified cell-type specific events. Importantly, those pre-mRNAs that display changes in alternative splicing also contain RBM10 iCLIP tags, suggesting a direct role of RBM10 in these events. Finally, we show that depletion of RBM10 in mouse ES cells leads to proliferation defects and to gross alterations in their differentiation potential. These results demonstrate a role for RBM10 in the regulation of alternative splicing in two cell models of mouse early development and suggests that mutations in RBM10 could lead to splicing changes that affect normal palate development and cause human disease.

The RNA-binding profile of Acinus, a peripheral component of the exon junction complex, reveals its role in splicing regulation.

Acinus (apoptotic chromatin condensation inducer in the nucleus) is an RNA-binding protein (RBP) originally identified for its role in apoptosis. It was later found to be an auxiliary component of the exon junction complex (EJC), which is deposited at exon junctions as a consequence of pre-mRNA splicing. To uncover the cellular functions of Acinus and investigate its role in splicing, we mapped its endogenous RNA targets using the cross-linking immunoprecipitation protocol (iCLIP). We observed that Acinus binds to pre-mRNAs, associating specifically to a subset of suboptimal introns, but also to spliced mRNAs. We also confirmed the presence of Acinus as a peripheral factor of the EJC. RNA-seq was used to investigate changes in gene expression and alternative splicing following siRNA-mediated depletion of Acinus in HeLa cells. This analysis revealed that Acinus is preferentially required for the inclusion of specific alternative cassette exons and also controls the faithful splicing of a subset of introns. Moreover, a large number of splicing changes can be related to Acinus binding, suggesting a direct role of Acinus in exon and intron definition. In particular, Acinus regulates the splicing of DFFA/ICAD transcript, a major regulator of DNA fragmentation. Globally, the genome-wide identification of RNA targets of Acinus revealed its role in splicing regulation as well as its involvement in other cellular pathways, including cell cycle progression. Altogether, this study uncovers new cellular functions of an RBP transiently associated with the EJC.

The RNA helicase DHX34 functions as a scaffold for SMG1-mediated UPF1 phosphorylation.

Nonsense-mediated decay (NMD) is a messenger RNA quality-control pathway triggered by SMG1-mediated phosphorylation of the NMD factor UPF1. In recent times, the RNA helicase DHX34 was found to promote mRNP remodelling, leading to activation of NMD. Here we demonstrate the mechanism by which DHX34 functions in concert with SMG1. DHX34 comprises two distinct structural units, a core that binds UPF1 and a protruding carboxy-terminal domain (CTD) that binds the SMG1 kinase, as shown using truncated forms of DHX34 and electron microscopy of the SMG1-DHX34 complex. Truncation of the DHX34 CTD does not affect binding to UPF1; however, it compromises DHX34 binding to SMG1 to affect UPF1 phosphorylation and hence abrogate NMD. Altogether, these data suggest the existence of a complex comprising SMG1, UPF1 and DHX34, with DHX34 functioning as a scaffold for UPF1 and SMG1. This complex promotes UPF1 phosphorylation leading to functional NMD.