After trimming unbound RNA, RBP protected fragments were ligated to linkers, con verted to cDNAs, Enzastaurin manufacturer and subjected to Illumina high throughput sequencing. We term this global PAR CLIP method gPAR CLIP. There are two caveats associated with our gPAR CLIP protocol. First, during crosslinking, the cells were in nutrient free buffer and incubated on ice, which could trigger changes in RBP binding. Second, we limited our analysis to mRNAs from the top of the sucrose gradient, which mostly consist of non translating mRNAs, so our conclusions apply to non translating mRNAs. To define the dynamic landscape of RBP RNA interac tions, we constructed duplicate Inhibitors,Modulators,Libraries gPAR CLIP and mRNA seq libraries, including incorporation of 4sU, for the wild type Saccharomyces cerevisiae strain cultured in complete media or subjected to glucose or nitrogen Inhibitors,Modulators,Libraries star vation for 2 hours.

An average of 10 million reads were sequenced from each gPAR CLIP library. Of the 72% of reads that mapped uniquely to the genome, over 70% contained one or two T to C conversion events, the sig nature substitution induced by 4sU crosslinking. Inhibitors,Modulators,Libraries From overlapping reads, we derived clusters representing RNA regions crosslinked to pro teins. Crosslinking site read coverage was normalized to mRNA expression levels calculated as reads per million mapped reads per kilobase of transcript. Because our approach captures protein RNA interac tions for potentially all RBPs, we cannot rule out the possibility that some clusters are located proximally to true RBP binding sites. therefore, we refer to our gPAR CLIP read clusters as crosslinking sites.

We empirically assigned a false discovery rate to each crosslinking site by deriving clusters from Inhibitors,Modulators,Libraries mRNA seq reads with one or two T to C mismatches representing sequencing error and comparing the T to C conversion rate of these clusters to those derived from gPAR CLIP reads. Using a 1% FDR thresh old, we reproducibly identified 80,883 crosslinking sites that are, on average, 23 nucleotides long 65,992 in protein coding sequences, 4,508 in 5 UTRs, 8,525 in 3 UTRs, and 818 in introns. Of 6,717 annotated protein coding tran scripts, 6,228 have at least one crosslinking site. Because CDS crosslinking sites exhibited three nucleo tide periodicity, a hallmark of ribosome binding, we separately analyzed CDS, 5 UTR, and 3 UTR crosslinking sites.

We observed high correlation between gPAR CLIP read coverage Inhibitors,Modulators,Libraries of 5 UTRs and CDSs, sup porting a prominent role for 5 UTRs in translational regulation. However, gPAR CLIP read cover age of 3 UTRs correlated poorly with both 5 UTRs selleck chem Nutlin-3a and CDS, suggesting a greater role in post transcriptional regulation. As expected, correla tion between total mRNA seq read coverage across all transcript regions was approximately equal. We also observed very high correla tion of gPAR CLIP and mRNA seq read coverage between replicates over each genic region, reflecting low technical variation between replicates.