Biology subjects

Ule, J.

Publications and source records attributed to Ule, J..

6 recordsLinked to original sources

bioRxiv2018

The key protein of endosomal mRNP transport binds translational landmark sites of cargo mRNAs

RNA-binding proteins (RBPs) determine spatiotemporal gene expression by mediating active transport and local translation of cargo mRNAs. Here, we cast a transcriptome-wide view on the transported mRNAs and cognate RBP binding sites during endosomal messenger ribonucleoprotein (mRNP) transport in Ustilago maydis. Using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), we compare the key transport RBP Rrm4 and the newly identified endosomal mRNP component Grp1 that is crucial to coordinate hyphal growth. Both RBPs bind predominantly in the 3 untranslated region of thousands of shared cargo mRNAs, often in close proximity. Intriguingly, Rrm4 precisely binds at stop codons, which constitute landmark sites of translation, suggesting an intimate connection of mRNA transport and translation. Towards uncovering the code of recognition, we identify UAUG as specific binding motif of Rrm4 that is bound by its third RRM domain. Altogether, we provide first insights into the positional organisation of co-localising RBPs on individual cargo mRNAs.

cell biology↗

bioRxiv2018

P-TEFb activation by RBM7 shapes a pro-survival transcriptional response to genotoxic stress

Cellular DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill-defined. Given the centrality of RNA polymerase II (Pol II) promoter-proximal pause release in transcriptional control, we evaluated its importance in DDR. Here we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates Pol II elongation and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb). This is mediated by genotoxic stress-enhanced binding of RBM7 to 7SK snRNA (7SK), the scaffold of the 7SK small nuclear ribonucleoprotein (7SK snRNP) which inhibits P-TEFb. In turn, P-TEFb relocates from 7SK snRNP to chromatin to induce transcription of short units including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with RBM7 or P-TEFb provokes cellular hypersensitivity to DNA damage-inducing agents through activation of apoptotic program. By alleviating the inhibition of P-TEFb, RBM7 thus facilitates Pol II elongation to enable a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult. Our work uncovers a new paradigm in stress-dependent control of Pol II pause release, and offers the promise for designing novel anti-cancer interventions using RBM7 and P-TEFb antagonists in combination with DNA-damaging chemotherapeutics.

biochemistry↗

bioRxiv2018

Transcriptome-wide profiling of mammalian spliceosome and branchpoints with iCLIP

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we employed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs), to map human spliceosome engagement with snRNAs and pre-mRNAs. This identified over 50,000 branchpoints (BPs) that have canonical sequence and structural features. Moreover, it revealed 7 binding peaks around BPs and splice sites, each precisely overlapping with binding profiles of specific splicing factors. We show how the binding patterns of these RBPs are affected by the position and strength of BPs. For example, strong or proximally located BPs preferentially bind SF3 rather than U2AF complex. Notably, these effects are partly neutralized during spliceosomal assembly in a way that depends on the core spliceosomal protein PRPF8. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.

genomics↗

bioRxiv2018

Heteromeric RNP assembly at LINEs controls lineage-specific RNA processing

It is challenging for RNA processing machineries to select exons within long intronic regions. We find that intronic LINE repeat sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs). This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites in LINEs. Both RBPs repress splicing and 3 end processing within and around LINEs, as demonstrated in cultured human cells and mouse brain. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are confined to deep intronic regions. These RBPs insulate both LINEs and surrounding regions from RNA processing. Upon evolutionary divergence, gradual loss of insulation diversifies the roles of LINEs. Older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for assembly of repressive RBPs, and contribute to evolution of new, lineage-specific transcripts in mammals.

genomics↗

bioRxiv2017

Data Science Issues in Understanding Protein-RNA Interactions

An interplay of experimental and computational methods is required to achieve a comprehensive understanding of protein-RNA interactions. Crosslinking and immunoprecipitation (CLIP) identifies endogenous interactions by sequencing RNA fragments that co-purify with a selected RBP under stringent conditions. Here we focus on approaches for the analysis of resulting data and appraise the methods for peak calling, visualisation, analysis and computational modelling of protein-RNA binding sites. We advocate a combined assessment of cDNA complexity and specificity for data quality control. Moreover, we demonstrate the value of analysing sequence motif enrichment in peaks assigned from CLIP data, and of visualising RNA maps, which examine the positional distribution of peaks around regulated landmarks in transcripts. We use these to assess how variations in CLIP data quality, and in different peak calling methods, affect the insights into regulatory mechanisms. We conclude by discussing future opportunities for the computational analysis of protein-RNA interaction experiments.

bioinformatics↗

bioRxiv2017

Post-transcriptional remodelling is temporally deregulated during motor neurogenesis in human ALS models

Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate regulators of RNA-processing that are ubiquitously expressed throughout development. To understand the molecular impact of ALS-causing mutations on early neuronal development and disease, we performed transcriptomic analysis of differentiated human control and VCP-mutant induced pluripotent stem cells (iPSCs) during motor neurogenesis. We identify intron retention (IR) as the predominant splicing change affecting early stages of wild-type neural differentiation, targeting key genes involved in the splicing machinery. Importantly, IR occurs prematurely in VCP-mutant cultures compared with control counterparts; these events are also observed in independent RNAseq datasets from SOD1- and FUS-mutant motor neurons (MNs). Together with related effects on 3UTR length variation, these findings implicate alternative RNA-processing in regulating distinct stages of lineage restriction from iPSCs to MNs, and reveal a temporal deregulation of such processing by ALS mutations. Thus, ALS-causing mutations perturb the same post-transcriptional mechanisms that underlie human motor neurogenesis.\n\nHIGHLIGHTSO_LIIntron retention is the main mode of alternative splicing in early differentiation.\nC_LIO_LIThe ALS-causing VCP mutation leads to premature intron retention.\nC_LIO_LIIncreased intron retention is seen with multiple ALS-causing mutations.\nC_LIO_LITranscriptional programs are unperturbed despite post-transcriptional defects.\nC_LI\n\neTOC BLURBLuisier et al. identify post-transcriptional changes underlying human motor neurogenesis: extensive variation in 3 UTR length and intron retention (IR) are the early predominant modes of splicing. The VCP mutation causes IR to occur prematurely during motor neurogenesis and these events are validated in other ALS-causing mutations, SOD1 and FUS.

neuroscience↗