Proteins begin to interact with nascent RNAs as soon as transcription is initiated. The protein complement decorating an RNA molecule changes dynamically in space and time, orchestrating RNA processing and function in the nucleus and cytoplasm 1 . Ribonucleoprotein (RNP) complexes are key to every step of RNA processing and function, and understanding the roles that RNA-binding proteins (RBPs) play requires methods that identify the set of RNAs that they bind in cells during specific developmental stages, activities or disease states.Numerous methods can characterize the RNA interactions that coordinate RNP assembly. These approaches can be protein-centric, describing the compendium of RNA sites bound by a specific RBP, or RNA-centric, identifying the RNA-bound proteome. The most common protein-centric strategies are based on the immunopurification of an RBP and its associated RNAs, and can be broadly categorized as RNA immunoprecipitation (RIP) or cross-linking and immunoprecipitation (CLIP) approaches. RIP approaches purify the RNAprotein complexes under native conditions 2,3 or using formaldehyde cross-linking 4 . CLIP techniques are more widely used and rely on the irradiation of cells by UV light, which causes proteins in the immediate vicinity of the irradiated bases to irreversibly cross-link to the RNA by a covalent bond 5 (Fig. 1). The covalent cross-links allow stringent purification of the RNA-protein complexes, which is followed by a series of steps to determine the interactions of a specific protein across the transcriptome. CLIP uses a limited RNase treatment of cross-linked RNPs to isolate RNA fragments occupied by the RBP and sequencing of these fragments can identify RBP binding sites, which allows inference of RBP function through determining the location of binding sites relative to, for example, other RBP binding sites or cis-acting elements (Box 1).The development of high-throughput sequencing of RNA isolated by CLIP (HITS-CLIP) has enabled a transcriptome-wide view of RNA binding sites 6 . CLIP techniques have been further developed to identify cross-link sites with nucleotide resolution, either through analysis of mutations in reads (photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP)) 7 or by capturing cDNAs that terminate at the cross-linked peptide during reverse transcription (individual-nucleotide resolution CLIP (iCLIP)) 8 . The development of dedicated bioinformatics workflows has allowed the determination of binding sites and consensus motifs to better understand post-transcriptional regulation 9 .This Primer focuses on experimental and computational aspects of CLIP methods that have been broadly adopted and have generated widely used data sets. We also cover the identification of RBP binding sites by tagging RBPs with enzymes that naturally act on RNA, where the resulting RNA modifications can be identified by high-throughput sequencing 10 , as well as the use of

Highlights d Retapamulin arrests bacterial ribosomes specifically at translation start sites d Retapamulin-assisted Ribo-seq reveals known and cryptic translation start sites d Translation from start sites located within the ORFs may generate functional proteins d Start-stop sites found within some genes may help to regulate gene expression

The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center.ribosome | antibiotics | protein synthesis | nascent peptide | oxazolidinones

Translation efficiency can be affected by mRNA stability and secondary structures, including G-quadruplex structures (G4s). The highly conserved DEAH-box helicase DHX36/RHAU resolves G4s on DNA and RNA in vitro, however a systems-wide analysis of DHX36 targets and function is lacking. We map globally DHX36 binding to RNA in human cell lines and find it preferentially interacting with G-rich and G4-forming sequences on more than 4500 mRNAs. While DHX36 knockout (KO) results in a significant increase in target mRNA abundance, ribosome occupancy and protein output from these targets decrease, suggesting that they were rendered translationally incompetent. Considering that DHX36 targets, harboring G4s, preferentially localize in stress granules, and that DHX36 KO results in increased SG formation and protein kinase R (PKR/EIF2AK2) phosphorylation, we speculate that DHX36 is involved in resolution of rG4 induced cellular stress.