RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.

RNA-binding proteins (RBPs) exert a broad range of biological functions. To explore the scope of RBPs across eukaryotic evolution, we determined the in vivo RBP repertoire of the yeast Saccharomyces cerevisiae and identified 678 RBPs from yeast and additionally 729 RBPs from human hepatocytic HuH-7 cells. Combined analyses of these and recently published data sets define the core RBP repertoire conserved from yeast to man. Conserved RBPs harbour defined repetitive motifs within disordered regions, which display striking evolutionary expansion. Only 60% of yeast and 73% of the human RBPs have functions assigned to RNA biology or structural motifs known to convey RNA binding, and many intensively studied proteins surprisingly emerge as RBPs (termed ‘enigmRBPs'), including almost all glycolytic enzymes, pointing to emerging connections between gene regulation and metabolism. Analyses of the mitochondrial hydroxysteroid dehydrogenase (HSD17B10) uncover the RNA-binding specificity of an enigmRBP.

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.

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.

(2011) In vivo and in vitro analysis of 6S RNA-templated short transcripts in Bacillussubtilis, RNA Biology, 8:5,[839][840][841][842][843][844][845][846][847][848][849]

Bacillus subtilis 6S-1 RNA binds to the housekeeping RNA polymerase (r A -RNAP) and directs transcription of short 'product' RNAs (pRNAs). Here, we demonstrate that once newly synthesized pRNAs form a sufficiently stable duplex with 6S-1 RNA, a structural rearrangement is induced in cis, which involves base-pairing between sequences in the 5 0 -portion of the central bulge and nucleotides that become available as a result of pRNA invasion. The rearrangement decreases 6S-1 RNA affinity for r A -RNAP. Among the pRNA length variants synthesized by r A -RNAP (up to B14 nt), only the longer ones, such as 12-14-mers, form a duplex with 6S-1 RNA that is sufficiently long-lived to induce the rearrangement. Yet, an LNA (locked nucleic acid) 8-mer can induce the same rearrangement due to conferring increased duplex stability. We propose that an interplay of rate constants for polymerization (k pol ), for pRNA:6S-1 RNA hybrid duplex dissociation (k off ) and for the rearrangement (k conf ) determines whether pRNAs dissociate or rearrange 6S-1 structure to trigger 6S-1 RNA release from r A -RNAP. A bioinformatic screen suggests that essentially all bacterial 6S RNAs have the potential to undergo a pRNA-induced structural rearrangement.

Evolutionary conserved mitochondrial nucleases are involved in programmed cell death and normal cell proliferation in lower and higher eukaryotes. The endo/exonuclease Nuc1p, also termed ‘yeast Endonuclease G (EndoG)’, is a member of this class of enzymes that differs from mammalian homologs by the presence of a 5′–3′ exonuclease activity in addition to its broad spectrum endonuclease activity. However, this exonuclease activity is thought to be essential for a function of the yeast enzyme in DNA recombination and repair. Here we show that higher eukaryotes in addition to EndoG contain its paralog ‘EXOG’, a novel EndoG-like mitochondrial endo/exonuclease. We find that during metazoan evolution duplication of an ancestral nuclease gene obviously generated the paralogous EndoG- and EXOG-protein subfamilies in higher eukaryotes, thereby maintaining the full endo/exonuclease activity found in mitochondria of lower eukaryotes. We demonstrate that human EXOG is a dimeric mitochondrial enzyme that displays 5′–3′ exonuclease activity and further differs from EndoG in substrate specificity. We hypothesize that in higher eukaryotes the complementary enzymatic activities of EndoG and EXOG probably together account for both, the lethal and vital functions of conserved mitochondrial endo/exonucleases.

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