The PRoteomics IDEntifications (PRIDE) database (https://www.ebi.ac.uk/pride/) is the world’s largest data repository of mass spectrometry-based proteomics data, and is one of the founding members of the global ProteomeXchange (PX) consortium. In this manuscript, we summarize the developments in PRIDE resources and related tools since the previous update manuscript was published in Nucleic Acids Research in 2016. In the last 3 years, public data sharing through PRIDE (as part of PX) has definitely become the norm in the field. In parallel, data re-use of public proteomics data has increased enormously, with multiple applications. We first describe the new architecture of PRIDE Archive, the archival component of PRIDE. PRIDE Archive and the related data submission framework have been further developed to support the increase in submitted data volumes and additional data types. A new scalable and fault tolerant storage backend, Application Programming Interface and web interface have been implemented, as a part of an ongoing process. Additionally, we emphasize the improved support for quantitative proteomics data through the mzTab format. At last, we outline key statistics on the current data contents and volume of downloads, and how PRIDE data are starting to be disseminated to added-value resources including Ensembl, UniProt and Expression Atlas.

Our comprehensive analysis of alternative splicing across 32 The Cancer Genome Atlas cancer types from 8,705 patients detects alternative splicing events and tumor variants by reanalyzing RNA and whole-exome sequencing data. Tumors have up to 30% more alternative splicing events than normal samples. Association analysis of somatic variants with alternative splicing events confirmed known trans associations with variants in SF3B1 and U2AF1 and identified additional trans-acting variants (e.g., TADA1, PPP2R1A). Many tumors have thousands of alternative splicing events not detectable in normal samples; on average, we identified ≈930 exon-exon junctions ("neojunctions") in tumors not typically found in GTEx normals. From Clinical Proteomic Tumor Analysis Consortium data available for breast and ovarian tumor samples, we confirmed ≈1.7 neojunction- and ≈0.6 single nucleotide variant-derived peptides per tumor sample that are also predicted major histocompatibility complex-I binders ("putative neoantigens").

The processing of Arabidopsis thaliana microRNAs (miRNAs) from longer primary transcripts (pri-miRNAs) requires the activity of several proteins, including DICER-LIKE1 (DCL1), the double-stranded RNA-binding protein HYPONASTIC LEAVES1 (HYL1), and the zinc finger protein SERRATE (SE). It has been noted before that the morphological appearance of weak se mutants is reminiscent of plants with mutations in ABH1/CBP80 and CBP20, which encode the two subunits of the nuclear cap-binding complex. We report that, like SE, the cap-binding complex is necessary for proper processing of pri-miRNAs. Inactivation of either ABH1/CBP80 or CBP20 results in decreased levels of mature miRNAs accompanied by apparent stabilization of pri-miRNAs. Whole-genome tiling array analyses reveal that se, abh1/cbp80, and cbp20 mutants also share similar splicing defects, leading to the accumulation of many partially spliced transcripts. This is unlikely to be an indirect consequence of improper miRNA processing or other mRNA turnover pathways, because introns retained in se, abh1/cbp80, and cbp20 mutants are not affected by mutations in other genes required for miRNA processing or for nonsense-mediated mRNA decay. Taken together, our results uncover dual roles in splicing and miRNA processing that distinguish SE and the cap-binding complex from specialized miRNA processing factors such as DCL1 and HYL1. P osttranscriptional gene regulation by microRNAs (miRNAs) is essential for the development and function of multicellular eukaryotes (1). In plants, miRNAs are processed from primary transcripts that contain partially complementary foldbacks of variable lengths (pri-miRNAs). Pri-miRNAs are similar to messenger RNAs (mRNAs) in being transcribed by DNA-dependent RNA polymerase II (pol II) and carrying a seven-methyl guanosine (m 7 G) cap at the 5Ј end and a polyadenosine (polyA) tail at the 3Ј end (1). Pri-miRNAs are processed to yield 20-to 22-nt-long mature miRNAs by an RNAseIII-like domain containing protein called DICER-LIKE1 (DCL1) (2-4). DCL1 interacts with the double-stranded (ds)RNA-binding protein HYPONASTIC LEAVES1 (HYL1) and the zinc finger protein SERRATE (SE) to ensure proper processing of pri-miRNAs (5-13). In common with dcl1 mutants, pri-miRNA levels are increased in plants that lack HYL1 or SE activity, whereas the amounts of mature miRNAs are reduced (5-9). All three proteins are found in nuclear processing centers, called D-bodies or SmD3/SmB nuclear bodies (12, 13).Mutants deficient in miRNA biogenesis suffer from a large range of morphological defects. Plants with null alleles of DCL1 or SE die as embryos, and even moderate reduction of DCL1 activity leads to a broad spectrum of developmental abnormalities (8,14,15). The weak se-1 allele causes only mild defects, including an alteration of phyllotaxis and the name-sake serrated leaves (16,17). It has been noted before that the se-1 phenotype is reminiscent of that of another mutant with impaired RNA metabolism, ABA hypersensitive 1 (abh1). Both mutants respond more strongly t...

rnA-protein complexes play pivotal roles in many central biological processes. Although methods based on highthroughput sequencing have advanced our ability to identify the specific rnAs bound by a particular protein, there is a need for precise and systematic ways to identify rnA interaction sites on proteins. We have developed an experimental and computational workflow combining photo-induced crosslinking, high-resolution mass spectrometry and automated analysis of the resulting mass spectra for the identification of cross-linked peptides, cross-linking sites and the cross-linked rnA oligonucleotide moieties of such rnA-binding proteins. the workflow can be applied to any rnA-protein complex of interest or to whole proteomes. We applied the approach to human and yeast mrnA-protein complexes in vitro and in vivo, demonstrating its powerful utility by identifying 257 cross-linking sites on 124 distinct rnA-binding proteins. the open-source software pipeline developed for this purpose, rnP xl , is available as part of the openms project.RNA molecules bind to proteins to form ribonucleoprotein complexes (RNPs). These are indispensable for the synthesis, stability, transport and activity of mRNAs 1 and noncoding RNAs 2,3 . RNA-binding proteins (RBPs) assume numerous functions in RNPs. RBPs can modulate or stabilize RNA structures, thereby making RNA catalytically active, for example, during pre-mRNA splicing 4 . RNA can also guide a catalytically active RBP to its destination; examples of this are microRNA-or long noncoding RNA-mediated translational control and epigenetic modulation 5,6 . RBPs are also involved in splicing and can recruit or repel other proteins, induce hydrolysis of RNA or protect RNA from degradation.

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