Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate ubiquitously expressed regulators of RNA processing. To understand the molecular impact of ALS-causing mutations on neuronal development and disease, we analysed transcriptomes during in vitro differentiation of motor neurons (MNs) from human control and patient-specific VCP mutant induced-pluripotent stem cells (iPSCs). We identify increased intron retention (IR) as a dominant feature of the splicing programme during early neural differentiation. Importantly, IR occurs prematurely in VCP mutant cultures compared with control counterparts. These aberrant IR events are also seen in independent RNAseq data sets from SOD1- and FUS-mutant MNs. The most significant IR is seen in the SFPQ transcript. The SFPQ protein binds extensively to its retained intron, exhibits lower nuclear abundance in VCP mutant cultures and is lost from nuclei of MNs in mouse models and human sporadic ALS. Collectively, we demonstrate SFPQ IR and nuclear loss as molecular hallmarks of familial and sporadic ALS.

Post-transcriptional regulation of cellular mRNA is essential for protein synthesis. Here we describe the importance of mRNA translational repression and mRNA subcellular location for protein expression during B lymphocyte activation and the DNA damage response. Cytoplasmic RNA granules are formed upon cell activation with mitogens, including stress granules that contain the RNA binding protein Tia1. Tia1 binds to a subset of transcripts involved in cell stress, including p53 mRNA, and controls translational silencing and RNA granule localization. DNA damage promotes mRNA relocation and translation in part due to dissociation of Tia1 from its mRNA targets. Upon DNA damage, p53 mRNA is released from stress granules and associates with polyribosomes to increase protein synthesis in a CAP-independent manner. Global analysis of cellular mRNA abundance and translation indicates that this is an extended ATM-dependent mechanism to increase protein expression of key modulators of the DNA damage response.Sequestering mRNA in cytoplasmic stress granules is a mechanism for translational repression. Here the authors find that p53 mRNA, present in stress granules in activated B lymphocytes, is released upon DNA damage and is translated in a CAP-independent manner.

Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. Our results unveil a non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins.

MicroRNAs (miRNAs) interact with 3'-untranslated regions of messenger RNAs to restrict expression of most protein-coding genes during normal development and cancer. RNA-binding proteins (RBPs) can control the biogenesis, stability and activity of miRNAs. Here we identify RBM38 in a genetic screen for RBPs whose expression controls miRNA access to target mRNAs. RBM38 is induced by p53 and its ability to modulate miRNA-mediated repression is required for proper p53 function. In contrast, RBM38 shows lower propensity to block the action of the p53-controlled miR-34a on SIRT1. Target selectivity is determined by the interaction of RBM38 with uridine-rich regions near miRNA target sequences. Furthermore, in large cohorts of human breast cancer, reduced RBM38 expression by promoter hypermethylation correlates with wild-type p53 status. Thus, our results indicate a novel layer of p53 gene regulation, which is required for its tumour suppressive function.

RNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3'-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases.