Nucleosome positioning stability is a modulator of germline mutation rate variation across the human genome

Nucleosome organization has been suggested to affect local mutation rates in the genome. However, the lack of de novo mutation and high-resolution nucleosome data has limited the investigation of this hypothesis. Additionally, analyses using indirect mutation rate measurements have yielded contradictory and potentially confounding results. Here, we combine data on >300,000 human de novo mutations with high-resolution nucleosome maps and find substantially elevated mutation rates around translationally stable (\u27strong\u27) nucleosomes. We show that the mutational mechanisms affected by strong nucleosomes are low-fidelity replication, insufficient mismatch repair and increased double-strand breaks. Strong nucleosomes preferentially locate within young SINE/LINE transposons, suggesting that when subject to increased mutation rates, transposons are then more rapidly inactivated. Depletion of strong nucleosomes in older transposons suggests frequent positioning changes during evolution. The findings have important implications for human genetics and genome evolution

clipplotr - a comparative visualisation and analysis tool for CLIP data

CLIP technologies are now widely used to study RNA-protein interactions and many datasets are now publicly available. An important first step in CLIP data exploration is the visual inspection and assessment of processed genomic data on selected genes or regions and performing comparisons: either across conditions within a particular project, or incorporating publicly available data. However, the output files produced by data processing pipelines or preprocessed files available to download from data repositories are often not suitable for direct comparison and usually need further processing. Furthermore, to derive biological insight it is usually necessary to visualise CLIP signal alongside other data such as annotations, or orthogonal functional genomic data (e.g. RNA-seq). We have developed a simple, but powerful, command-line tool: clipplotr, which facilitates these visual comparative and integrative analyses with normalisation and smoothing options for CLIP data and the ability to show these alongside reference annotation tracks and functional genomic data. These data can be supplied as input to clipplotr in a range of file formats, which will output a publication quality figure. It is written in R and can both run on a laptop computer independently, or be integrated into computational workflows on a high-performance cluster. Releases, source code and documentation are freely available at: https://github.com/ulelab/clipplotr

SpeCond: a method to detect condition-specific gene expression

Transcriptomic studies routinely measure expression levels across numerous conditions. These datasets allow identification of genes that are specifically expressed in a small number of conditions. However, there are currently no statistically robust methods for identifying such genes. Here we present SpeCond, a method to detect condition-specific genes that outperforms alternative approaches. We apply the method to a dataset of 32 human tissues to determine 2,673 specifically expressed genes. An implementation of SpeCond is freely available as a Bioconductor package at http://www.bioconductor.org/packages/release/bioc/html/SpeCond.html

RNA polymerase II-associated proteins reveal pathways affected in VCP-related amyotrophic lateral sclerosis

Valosin-containing protein (VCP) is a hexameric ATPase associated with diverse cellular activities. Genetic mutations in VCP are associated with several forms of muscular and neuronal degeneration, including amyotrophic lateral sclerosis (ALS). Moreover, VCP mediates UV-induced proteolysis of RNA polymerase II (RNAPII), but little is known about the effects of VCP mutations on the transcriptional machinery. Here, we used silica particle-assisted chromatin enrichment and mass spectrometry to study proteins co-localized with RNAPII in precursor neurons differentiated from VCP-mutant or control induced pluripotent stem cells. Remarkably, we observed diminished RNAPII binding of proteins involved in transcription elongation and mRNA splicing in mutant cells. One of these is SART3, a recycling factor of the splicing machinery, whose knockdown leads to perturbed intron retention in several ALS-associated genes. Additional reduced proteins are RBM45, EIF5A and RNF220, mutations in which are associated with various neurodegenerative disorders and are linked to TDP-43 aggregation. Conversely, we observed increased RNAPII binding of heat shock proteins such as HSPB1. Together, these findings shed light on how transcription and splicing machinery are impaired by VCP mutations, which might contribute to aberrant alternative splicing and proteinopathy in neurodegeneration

RNA polymerase II-associated proteins reveal pathways affected in VCP-related amyotrophic lateral sclerosis

Valosin-containing protein (VCP) is a hexameric ATPase associated with diverse cellular activities. Genetic mutations in VCP are associated with several forms of muscular and neuronal degeneration, including amyotrophic lateral sclerosis (ALS). Moreover, VCP mediates UV-induced proteolysis of RNA polymerase II (RNAPII), but little is known about the effects of VCP mutations on the transcriptional machinery. Here, we used silica particle-assisted chromatin enrichment and mass spectrometry to study proteins co-localized with RNAPII in precursor neurons differentiated from VCP-mutant or control induced pluripotent stem cells. Remarkably, we observed diminished RNAPII binding of proteins involved in transcription elongation and mRNA splicing in mutant cells. One of these is SART3, a recycling factor of the splicing machinery, whose knockdown leads to perturbed intron retention in several ALS-associated genes. Additional reduced proteins are RBM45, EIF5A and RNF220, mutations in which are associated with various neurodegenerative disorders and are linked to TDP-43 aggregation. Conversely, we observed increased RNAPII binding of heat shock proteins such as HSPB1. Together, these findings shed light on how transcription and splicing machinery are impaired by VCP mutations, which might contribute to aberrant alternative splicing and proteinopathy in neurodegeneration.journal articl

Finding cell-specific expression patterns in the early Ciona embryo with single-cell RNA-seq

Single-cell RNA-seq has been established as a reliable and accessible technique enabling new types of analyses, such as identifying cell types and studying spatial and temporal gene expression variation and change at single-cell resolution. Recently, single-cell RNA-seq has been applied to developing embryos, which offers great potential for finding and characterising genes controlling the course of development along with their expression patterns. In this study, we applied single-cell RNA-seq to the 16-cell stage of the Ciona embryo, a marine chordate and performed a computational search for cell-specific gene expression patterns. We recovered many known expression patterns from our single-cell RNA-seq data and despite extensive previous screens, we succeeded in finding new cell-specific patterns, which we validated by in situ and single-cell qPCR

A computationally-enhanced hiCLIP atlas reveals Staufen1-RNA binding features and links 3′ UTR structure to RNA metabolism

The structure of mRNA molecules plays an important role in its interactions with trans-acting factors, notably RNA binding proteins (RBPs), thus contributing to the functional consequences of this interplay. However, current transcriptome-wide experimental methods to chart these interactions are limited by their poor sensitivity. Here we extend the hiCLIP atlas of duplexes bound by Staufen1 (STAU1) ∼10-fold, through careful consideration of experimental assumptions, and the development of bespoke computational methods which we apply to existing data. We present Tosca, a Nextflow computational pipeline for the processing, analysis and visualisation of proximity ligation sequencing data generally. We use our extended duplex atlas to discover insights into the RNA selectivity of STAU1, revealing the importance of structural symmetry and duplex-span-dependent nucleotide composition. Furthermore, we identify heterogeneity in the relationship between transcripts with STAU1-bound 3' UTR duplexes and metabolism of the associated RNAs that we relate to RNA structure: transcripts with short-range proximal 3' UTR duplexes have high degradation rates, but those with long-range duplexes have low rates. Overall, our work enables the integrative analysis of proximity ligation data delivering insights into specific features and effects of RBP-RNA structure interactions