Unpaired loop and bulge regions can be unstructured or form tertiary struc tural modules, both of which can be readily recognized by RBPs. In Olaparib contrast, double stranded RNAs, in general, do not provide good platforms for RBP binding struc tured RNA regions captured by gPAR CLIP generally had low CLS values, likely resulting from crosslinking and or RNase T1 cleavage inefficiency. In structured regions, 4 thiouridines are more likely to be locked in U Inhibitors,Modulators,Libraries A or U G pairing, preventing crosslinking to proteins. In addition, structured regions are less accessi ble to RNase attack during sequencing fragment prepara tion, resulting in under representation in gPAR CLIP libraries. Nevertheless, despite their low crosslinking effi ciencies, Ts in double stranded, paired RNA regions show extremely high conservation compared to Ts with no crosslinking evidence.

These data indicate that RNAs with high secondary structure are evolutionarily con served and can serve as functional, secondary structure motifs recognized by select RBPs. RBP binding sites functioning as cis regulatory ele ments are expected to be under purifying selection. We identified a substantial fraction of conserved ele ments in UTRs overlapping RBP crosslinking Inhibitors,Modulators,Libraries sites. This represents an underestimation because RBPs and RNAs that are not expressed under our experimental conditions or that fail to crosslink will not be captured. Although crosslinking sites in general are more highly conserved than non crosslinking sites Inhibitors,Modulators,Libraries in UTRs, many sites are not well conserved and might represent species specific cis regulatory elements that allow adaptation to different environments and stressors.

A preference of RBP binding to 3 UTRs observed in this study and others is consistent with the function and evolution of 3 UTRs as major sites for post transcrip tional regulation. Unlike protein coding regions, 3 UTRs do not directly engage ribosomes during translation and therefore provide accessible platforms for RBP binding and RNP assembly. One Inhibitors,Modulators,Libraries important aspect of gene regula tion is combinatorial Inhibitors,Modulators,Libraries control, which allows a single gene to be controlled by more than one regulator. In our study, 23% of all nucleotides in annotated 3 UTRs were located within RPB crosslinking sites, corresponding to an average of 1 crosslinking site, on average 23 nucleotides long, in every 100 nucleotides.

For a median sized yeast 3 UTR that is 166 nucleotides long, there are, on average, 2 RBP crosslinking sites, suggesting that most yeast genes are subject to combinatorial post transcriptional regula tion. Since S. cerevisiae lacks post transcriptional regula tion by the highly conserved and pervasive microRNA regulatory pathway, combinatorial regulation download catalog by RBPs may play a more prominent role than in organisms with small RNA mediated post transcriptional gene regulation.