clipplotr-a comparative visualization and analysis tool for CLIP data.

CLIP technologies are now widely used to study RNA-protein interactions and many data sets 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 visualize a 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 normalization 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.

m6A-ELISA, a simple method for quantifying N6-methyladenosine from mRNA populations.

N6-methyladenosine (m6A) is a widely studied and abundant RNA modification. The m6A mark regulates the fate of RNAs in various ways, which in turn drives changes in cell physiology, development, and disease pathology. Over the last decade, numerous methods have been developed to map and quantify m6A sites genome-wide through deep sequencing. Alternatively, m6A levels can be quantified from a population of RNAs using techniques such as liquid chromatography-mass spectrometry or thin layer chromatography. However, many methods for quantifying m6A levels involve extensive protocols and specialized data analysis, and often only a few samples can be handled in a single experiment. Here, we developed a simple method for determining relative m6A levels in mRNA populations from various sources based on an enzyme-linked immunosorbent-based assay (m6A-ELISA). We have optimized various steps of m6A-ELISA, such as sample preparation and the background signal resulting from the primary antibody. We validated the method using mRNA populations from budding yeast and mouse embryonic stem cells. The full protocol takes less than a day, requiring only 25 ng of mRNA. The m6A-ELISA protocol is quick, cost-effective, and scalable, making it a valuable tool for determining relative m6A levels in samples from various sources that could be adapted to detect other mRNA modifications.

Interrogation of RNA-protein interaction dynamics in bacterial growth.

Characterising RNA-protein interaction dynamics is fundamental to understand how bacteria respond to their environment. In this study, we have analysed the dynamics of 91% of the Escherichia coli expressed proteome and the RNA-interaction properties of 271 RNA-binding proteins (RBPs) at different growth phases. We find that 68% of RBPs differentially bind RNA across growth phases and characterise 17 previously unannotated proteins as bacterial RBPs including YfiF, a ncRNA-binding protein. While these new RBPs are mostly present in Proteobacteria, two of them are orthologs of human mitochondrial proteins associated with rare metabolic disorders. Moreover, we reveal novel RBP functions for proteins such as the chaperone HtpG, a new stationary phase tRNA-binding protein. For the first time, the dynamics of the bacterial RBPome have been interrogated, showcasing how this approach can reveal the function of uncharacterised proteins and identify critical RNA-protein interactions for cell growth which could inform new antimicrobial therapies.

Identification of RNA helicases with unwinding activity on angiogenin-processed tRNAs.

Stress-induced tRNA fragmentation upon environmental insult is a conserved cellular process catalysed by endonucleolytic activities targeting mature tRNAs. The resulting tRNA-derived small RNAs (tsRNAs) have been implicated in various biological processes that impact cell-to-cell signalling, cell survival as well as gene expression regulation during embryonic development. However, how endonuclease-targeted tRNAs give rise to individual and potentially biologically active tsRNAs remains poorly understood. Here, we report on the in vivo identification of proteins associated with stress-induced tsRNAs-containing protein complexes, which, together with a 'tracer tRNA' assay, were used to uncover enzymatic activities that can bind and process specific endonuclease-targeted tRNAs in vitro. Among those, we identified conserved ATP-dependent RNA helicases which can robustly separate tRNAs with endonuclease-mediated 'nicks' in their anticodon loops. These findings shed light on the existence of cellular pathways dedicated to producing individual tsRNAs after stress-induced tRNA hydrolysis, which adds to our understanding as to how tRNA fragmentation and the resulting tsRNAs might exert physiological impact.

nf-core/clipseq - a robust Nextflow pipeline for comprehensive CLIP data analysis.

Crosslinking and immunoprecipitation (CLIP) technologies have become a central component of the molecular biologists' toolkit to study protein-RNA interactions and thus to uncover core principles of RNA biology. There has been a proliferation of CLIP-based experimental protocols, as well as computational tools, especially for peak-calling. Consequently, there is an urgent need for a well-documented bioinformatic pipeline that enshrines the principles of robustness, reproducibility, scalability, portability and flexibility while embracing the diversity of experimental and computational CLIP tools. To address this, we present nf-core/clipseq - a robust Nextflow pipeline for quality control and analysis of CLIP sequencing data. It is part of the international nf-core community effort to develop and curate a best-practice, gold-standard set of pipelines for data analysis. The standards enabled by Nextflow and nf-core, including workflow management, version control, continuous integration and containerisation ensure that these key needs are met. Furthermore, multiple tools are implemented ( e.g. for peak-calling), alongside visualisation of quality control metrics to empower the user to make their own informed decisions based on their data. nf-core/clipseq remains under active development, with plans to incorporate newly released tools to ensure that pipeline remains up-to-date and relevant for the community. Engagement with users and developers is encouraged through the nf-core GitHub repository and Slack channel to promote collaboration. It is available at https://nf-co.re/clipseq.

Positional motif analysis reveals the extent of specificity of protein-RNA interactions observed by CLIP.

BACKGROUND: Crosslinking and immunoprecipitation (CLIP) is a method used to identify in vivo RNA-protein binding sites on a transcriptome-wide scale. With the increasing amounts of available data for RNA-binding proteins (RBPs), it is important to understand to what degree the enriched motifs specify the RNA-binding profiles of RBPs in cells. RESULTS: We develop positionally enriched k-mer analysis (PEKA), a computational tool for efficient analysis of enriched motifs from individual CLIP datasets, which minimizes the impact of technical and regional genomic biases by internal data normalization. We cross-validate PEKA with mCross and show that the use of input control for background correction is not required to yield high specificity of enriched motifs. We identify motif classes with common enrichment patterns across eCLIP datasets and across RNA regions, while also observing variations in the specificity and the extent of motif enrichment across eCLIP datasets, between variant CLIP protocols, and between CLIP and in vitro binding data. Thereby, we gain insights into the contributions of technical and regional genomic biases to the enriched motifs, and find how motif enrichment features relate to the domain composition and low-complexity regions of the studied proteins. CONCLUSIONS: Our study provides insights into the overall contributions of regional binding preferences, protein domains, and low-complexity regions to the specificity of protein-RNA interactions, and shows the value of cross-motif and cross-RBP comparison for data interpretation. Our results are presented for exploratory analysis via an online platform in an RBP-centric and motif-centric manner ( https://imaps.goodwright.com/apps/peka/ ).

N6-methyladenosine (m6A) reader Pho92 is recruited co-transcriptionally and couples translation to mRNA decay to promote meiotic fitness in yeast.

N6- methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here, we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3'end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.

Ultraplex: A rapid, flexible, all-in-one fastq demultiplexer.

Background: The first step of virtually all next generation sequencing analysis involves the splitting of the raw sequencing data into separate files using sample-specific barcodes, a process known as "demultiplexing". However, we found that existing software for this purpose was either too inflexible or too computationally intensive for fast, streamlined processing of raw, single end fastq files containing combinatorial barcodes. Results: Here, we introduce a fast and uniquely flexible demultiplexer, named Ultraplex, which splits a raw FASTQ file containing barcodes either at a single end or at both 5' and 3' ends of reads, trims the sequencing adaptors and low-quality bases, and moves unique molecular identifiers (UMIs) into the read header, allowing subsequent removal of PCR duplicates. Ultraplex is able to perform such single or combinatorial demultiplexing on both single- and paired-end sequencing data, and can process an entire Illumina HiSeq lane, consisting of nearly 500 million reads, in less than 20 minutes. Conclusions: Ultraplex greatly reduces computational burden and pipeline complexity for the demultiplexing of complex sequencing libraries, such as those produced by various CLIP and ribosome profiling protocols, and is also very user friendly, enabling streamlined, robust data processing. Ultraplex is available on PyPi and Conda and via Github.

psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation.

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.

RNA modifications detection by comparative Nanopore direct RNA sequencing.

RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. In recent years, a growing number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework that identifies modifications from these data. Our strategy compares an RNA sample of interest against a non-modified control sample, not requiring a training set and allowing the use of replicates. We show that Nanocompore can detect different RNA modifications with position accuracy in vitro, and we apply it to profile m6A in vivo in yeast and human RNAs, as well as in targeted non-coding RNAs. We confirm our results with orthogonal methods and provide novel insights on the co-occurrence of multiple modified residues on individual RNA molecules.

How Do You Identify m6 A Methylation in Transcriptomes at High Resolution? A Comparison of Recent Datasets.

A flurry of methods has been developed in recent years to identify N6-methyladenosine (m6A) sites across transcriptomes at high resolution. This raises the need to understand both the common features and those that are unique to each method. Here, we complement the analyses presented in the original papers by reviewing their various technical aspects and comparing the overlap between m6A-methylated messenger RNAs (mRNAs) identified by each. Specifically, we examine eight different methods that identify m6A sites in human cells with high resolution: two antibody-based crosslinking and immunoprecipitation (CLIP) approaches, two using endoribonuclease MazF, one based on deamination, two using Nanopore direct RNA sequencing, and finally, one based on computational predictions. We contrast the respective datasets and discuss the challenges in interpreting the overlap between them, including a prominent expression bias in detected genes. This overview will help guide researchers in making informed choices about using the available data and assist with the design of future experiments to expand our understanding of m6A and its regulation.

Analysis of RNA-Seq datasets reveals enrichment of tissue-specific splice variants for nuclear envelope proteins.

Laminopathies yield tissue-specific pathologies, yet arise from mutation of ubiquitously-expressed genes. A little investigated hypothesis to explain this is that the mutated proteins or their partners have tissue-specific splice variants. To test this, we analyzed RNA-Seq datasets, finding novel isoforms or isoform tissue-specificity for: Lap2, linked to cardiomyopathy; Nesprin 2, linked to Emery-Dreifuss muscular dystrophy and Lmo7, that regulates the Emery-Dreifuss muscular dystrophy linked emerin gene. Interestingly, the muscle-specific Lmo7 exon is rich in serine phosphorylation motifs, suggesting regulatory function. Muscle-specific splice variants in non-nuclear envelope proteins linked to other muscular dystrophies were also found. Nucleoporins tissue-specific variants were found for Nup54, Nup133, Nup153 and Nup358/RanBP2. RT-PCR confirmed novel Lmo7 and RanBP2 variants and specific knockdown of the Lmo7 variantreduced myogenic index. Nuclear envelope proteins were enriched for tissue-specific splice variants compared to the rest of the genome, suggesting that splice variants contribute to its tissue-specific functions.