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Gene expression involves transcription, translation and the turnover of mRNAs and proteins. The degree to which protein abundances scale with mRNA levels and the implications in cases where this dependency breaks down remain an intensely debated topic. Here we review recent mRNA–protein correlation studies in the light of the quantitative parameters of the gene expression pathway, contextual confounders and buffering mechanisms. Although protein and mRNA levels typically show reasonable correlation, we describe how transcriptomics and proteomics provide useful non-redundant readouts. Integrating both types of data can reveal exciting biology and is an essential step in refining our understanding of the principles of gene expression control.Gene expression is typically measured at the level of either mRNAs or proteins. In this Review, Buccitelli and Selbach discuss how large-scale comparative studies are characterizing the degree to which mRNA and protein levels correlate. They discuss technical and biological reasons why mRNA and protein levels may be particularly concordant or disconnected, as well as the different biological information provided by these non-redundant readouts of gene expression.

Liquid–liquid phase separation has been shown to underlie the formation and disassembly of membraneless organelles in cells, but the cellular mechanisms that control this phenomenon are poorly understood. A prominent example of regulated and reversible segregation of liquid phases may occur during mitosis, when membraneless organelles disappear upon nuclear-envelope breakdown and reappear as mitosis is completed. Here we show that the dual-specificity kinase DYRK3 acts as a central dissolvase of several types of membraneless organelle during mitosis. DYRK3 kinase activity is essential to prevent the unmixing of the mitotic cytoplasm into aberrant liquid-like hybrid organelles and the over-nucleation of spindle bodies. Our work supports a mechanism in which the dilution of phase-separating proteins during nuclear-envelope breakdown and the DYRK3-dependent degree of their solubility combine to allow cells to dissolve and condense several membraneless organelles during mitosis. The dual-specificity kinase DYRK3 acts as a central ‘dissolvase’, mediating the phase transitions of several types of membraneless organelles during mitosis.

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium. RNA binding proteins are important regulators of RNA function. Here the authors describe a method for isolation of RNA-protein complexes that does not rely on a specific RNA sequence or motif, and demonstrate the approach by providing the global RNA-bound proteomes of human HEK293 cells and Salmonella Typhimurium.

RiboTaper quantifies the three-nucleotide periodicity in Ribo-seq data to find translated open reading frames (ORFs). The de novo inferred set of ORFs comprehensively defines the cellular proteome across a wide expression range and comprises few additional translated noncoding regions. RNA-sequencing protocols can quantify gene expression regulation from transcription to protein synthesis. Ribosome profiling (Ribo-seq) maps the positions of translating ribosomes over the entire transcriptome. We have developed RiboTaper (available at https://ohlerlab.mdc-berlin.de/software/ ), a rigorous statistical approach that identifies translated regions on the basis of the characteristic three-nucleotide periodicity of Ribo-seq data. We used RiboTaper with deep Ribo-seq data from HEK293 cells to derive an extensive map of translation that covered open reading frame (ORF) annotations for more than 11,000 protein-coding genes. We also found distinct ribosomal signatures for several hundred upstream ORFs and ORFs in annotated noncoding genes (ncORFs). Mass spectrometry data confirmed that RiboTaper achieved excellent coverage of the cellular proteome. Although dozens of novel peptide products were validated in this manner, few of the currently annotated long noncoding RNAs appeared to encode stable polypeptides. RiboTaper is a powerful method for comprehensive de novo identification of actively used ORFs from Ribo-seq data.

Gene expression is a multistep process that involves the transcription, translation and turnover of messenger RNAs and proteins. Although it is one of the most fundamental processes of life, the entire cascade has never been quantified on a genome-wide scale. Here we simultaneously measured absolute mRNA and protein abundance and turnover by parallel metabolic pulse labelling for more than 5,000 genes in mammalian cells. Whereas mRNA and protein levels correlated better than previously thought, corresponding half-lives showed no correlation. Using a quantitative model we have obtained the first genome-scale prediction of synthesis rates of mRNAs and proteins. We find that the cellular abundance of proteins is predominantly controlled at the level of translation. Genes with similar combinations of mRNA and protein stability shared functional properties, indicating that half-lives evolved under energetic and dynamic constraints. Quantitative information about all stages of gene expression provides a rich resource and helps to provide a greater understanding of the underlying design principles. Mammalian gene expression control quantified Gene expression is a multistep process that involves transcription, translation and turnover of messenger RNAs (mRNAs) and proteins, but little is known about how the combined effect of these events shapes gene expression. Schwanhäusser et al . have now quantified gene expression in mammalian cells by simultaneously measuring protein and mRNA abundance and turnover by parallel metabolic pulse labelling for more than 5,000 genes. A model is used to predict synthesis rates of mRNAs and proteins. The cellular abundance of proteins seems to be predominantly controlled at the level of translation.

MaxQuant is a quantitative proteomics software package designed for analyzing large mass spectrometric data sets. It is specifically aimed at high-resolution mass spectrometry (MS) data. Currently, Thermo LTQ-Orbitrap and LTQ-FT-ICR instruments are supported and Mascot is used as a search engine. This protocol explains step by step how to use MaxQuant on stable isotope labeling by amino acids in cell culture (SILAC) data obtained with double or triple labeling. Complex experimental designs, such as time series and drug-response data, are supported. A standard desktop computer is sufficient to fulfill the computational requirements. The workflow has been stress tested with more than 1,000 liquid chromatography/mass spectrometry runs in a single project. In a typical SILAC proteome experiment, hundreds of thousands of peptides and thousands of proteins are automatically and reliably quantified. Additional information for identified proteins, such as Gene Ontology, domain composition and pathway membership, is provided in the output tables ready for further bioinformatics analysis. The software is freely available at the MaxQuant home page.

Telomere maintenance by telomerase activation or alternative lengthening of telomeres (ALT) is a major determinant of poor outcome in neuroblastoma. Here, we screen for ALT in primary and relapsed neuroblastomas ( n  = 760) and characterize its features using multi-omics profiling. ALT-positive tumors are molecularly distinct from other neuroblastoma subtypes and enriched in a population-based clinical sequencing study cohort for relapsed cases. They display reduced ATRX/DAXX complex abundance, due to either ATRX mutations (55%) or low protein expression. The heterochromatic histone mark H3K9me3 recognized by ATRX is enriched at the telomeres of ALT-positive tumors. Notably, we find a high frequency of telomeric repeat loci with a neuroblastoma ALT-specific hotspot on chr1q42.2 and loss of the adjacent chromosomal segment forming a neo-telomere. ALT-positive neuroblastomas proliferate slowly, which is reflected by a protracted clinical course of disease. Nevertheless, children with an ALT-positive neuroblastoma have dismal outcome. Alternative lengthening of telomeres (ALT) is associated with a poor outcome in neuroblastoma. Here, the authors find that ALT is associated with mutated ATRX and/or reduced protein abundance, frequent telomeric repeat loci and heterochromatic telomeric chromatin.

Despite their lack of a defined 3D structure, intrinsically disordered regions (IDRs) of proteins play important biological roles. Many IDRs contain short linear motifs (SLiMs) that mediate protein-protein interactions (PPIs), which can be regulated by post-translational modifications like phosphorylation. 20% of pathogenic missense mutations are found in IDRs, and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms. Here, we employ peptide-based interaction proteomics to investigate 36 disease-associated mutations affecting phosphorylation sites. Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides, often due to disrupted phosphorylation-dependent SLiMs. We focused on a mutation of a serine phosphorylation site in the transcription factor GATAD1, which causes dilated cardiomyopathy. We find that this phosphorylation site mediates interaction with 14-3-3 family proteins. Follow-up experiments reveal the structural basis of this interaction and suggest that 14-3-3 binding affects GATAD1 nucleocytoplasmic transport by masking a nuclear localisation signal. Our results demonstrate that pathogenic mutations of human phosphorylation sites can significantly impact protein-protein interactions, offering insights into potential molecular mechanisms underlying pathogenesis. Here the authors characterise the impact of phosphorylation site mutations in intrinsically disordered regions (IDRs) on protein-protein interactions, highlighting the critical role of phosphorylation of IDRs in health and disease.

Background There is increasing evidence that transcripts or transcript regions annotated as non-coding can harbor functional short open reading frames (sORFs). Loss-of-function experiments have identified essential developmental or physiological roles for a few of the encoded peptides (micropeptides), but genome-wide experimental or computational identification of functional sORFs remains challenging. Results Here, we expand our previously developed method and present results of an integrated computational pipeline for the identification of conserved sORFs in human, mouse, zebrafish, fruit fly, and the nematode C. elegans . Isolating specific conservation signatures indicative of purifying selection on amino acid (rather than nucleotide) sequence, we identify about 2,000 novel small ORFs located in the untranslated regions of canonical mRNAs or on transcripts annotated as non-coding. Predicted sORFs show stronger conservation signatures than those identified in previous studies and are sometimes conserved over large evolutionary distances. The encoded peptides have little homology to known proteins and are enriched in disordered regions and short linear interaction motifs. Published ribosome profiling data indicate translation of more than 100 novel sORFs, and mass spectrometry data provide evidence for more than 70 novel candidates. Conclusions Taken together, we identify hundreds of previously unknown conserved sORFs in major model organisms. Our computational analyses and integration with experimental data show that these sORFs are expressed, often translated, and sometimes widely conserved, in some cases even between vertebrates and invertebrates. We thus provide an integrated resource of putatively functional micropeptides for functional validation in vivo .