Genome-wide Mapping Of Polypyrimidine Binding Proteins’ Binding Sites Reveals Their Diverse Roles In Pre-mRNA Splicing

It’s believed that U2AF65 (U2 snRNP Auxiliary Factor 65 kDd) and PTB (Polypyrimidine binding protein) bind polypyrimidine tract (PPT). In regulating cassette exon splicing, PTB represses exon inclusion in most cases, however, PTB also stimulate some exons’inclusion. Little is known about the mechanism of U2AF65 repressing splicing, the prevailing model is U2AF65 compete out U2AF65 in binding PPT then disrupt spliceosome assembly. U2AF65 is an essential splicing factor, which recognizes 3’splice site (3’ss). During the assembly of spliceosomal E complex, U2AF65 binds the polypyrimidine tract (PPT) sequence upstream of 3’splice site, and its strongly associated partner U2AF35 binds the 3’splice site AG dinucleotide immediately downstream of PPT. The tight heterodimer formed by U2AF65 and U2AF35 is called U2AF which functions cooperatively in recognizing 3’ss and helps U2 snRNP bind to the branch point. U2AF65 has been traditionally considered as a general polypyrimidine tract binding protein, recognizing and binding the PPT region of every intron. However, this theory started to be challenged recently for several reasons. First, in several cases, some other protein factors can replace U2AF65 during in vitro splicing experiments. Second, U2AF65 depletion did not lead to a global splicing deficiency. And it apparently involves in regulation of certain alternative splicing events. A few lines of evidence have shown that may take part in mRNA export and polyadenylation.We completed PTB CILP-seq,4,807,389 PTB RNA tags were identified.58.4% of them locate on introns, which is 17-fold more than that on exon, which is consistent with PTB’s function in pre-mRNA splicing. We statistically combine nearby RNA tags to 64,314 binding peaks, and further merge them into 51,394 binding clusters.Two high-throughput methods have been applied in this study to clarify the diverse roles of U2AF65 in RNA processing including splicing and polyadenylation, as well as in mRNA transport and metabolism. CLIP-seq (Cross-Linking Immuno-Precipitation-high throughput rna Sequencing) methodology was used to obtain the genomewide binding sites of U2AF65 in living Hela cells. RNAseq was then performed using the U2AF65-expressed and U2AF65-silenced Hela cells to obtain both the expression and splicing profiles of these cells. Genome-wide analysis of the association of U2AF65 binding and the altered splicing pattern and mRNA level of the bound gene in response to U2AF65 silencing shall shed light on the global roles of this well-known splicing factors in pre-mRNA processing and mRNA metabolism.CLIP-seq has recovered 7,871,053 of U2AF65-bound tag sequences that mapped onto human genome. Genomic distribution shows that 82% of tags mapped to annotated genes, while 18% to antisense and un-annotated intergenic regions. This result is consistent with the fact that known genes are much more actively transcribed than the intergenic regions, and the mature mRNA is the major component of transcriptome, as revealed by the recent high-throughput sequencing study. A total of 60,973 reliable U2AF65-binding peaks were identified from the tags in the known genes. U2AF65 peaks do not contain any RNA motif with specific sequence, however, are significantly enriched in the uracil residue(U) that is also enriched in polypyrimidine tracts。Consistently, the binding peaks are highly enriched in the exon-intron junction (5% of total peaks),which is 25-fold higher than the polypyrimidine tract binding protein PTB that binds CU-rich RNA motifs with a bias to U. Furthermore, U2AF65 is significantly enriched in the polypyrimidine tract regions of constitutive 3’splice sites, while PTB is strongly excluded from the PPT region。Therefore, for the first time, the genome-wide in vivo data prove the theory that U2AF65 and PTB competes to bind the PPT regions, and U2AF65 clearly is a winner at constitutive 3’splice sites。Moreover, binding sites of these two polypyrimidine tract binding proteins are highly diverge from each other, further implicating that these proteins may be functionally mutual-exclusive. About 10% of the U2AF65 peaks are located in coding sequences of known genes, which is about one-fold higher than those of PTB. This is consistent with the reported role of U2AF65 in mRNA transport.Most of the U2AF65 peaks (85%) are intronic, with a majority away from the 3’splice site. Surprisingly, close to 2% of U2AF65 peaks are in the 5’splice site exon-intron junction. All these binding features suggest that U2AF65 has new roles in pre-mRNA splicing. We found that U2AF65 sites are strongly associated with a number of alternative splicing events, with the top three events being intron retention, alternative 3’splice site and exon skipped, sequentially. We have tested near 20 alternative splicing events associated with U2AF65 binding, and found most of them are altered upon siRNA silencing of U2AF65 expression. This indicates that the intronic binding of U2AF6 can regulates alternative splicing. We preliminarily concluded U2AF65 enhances an alternative exon’s inclusion when binding its immediate upstream, while repress its inclusion when binding its immediate downstream.This study has accomplished the high-throughput sequencing of the transcriptomes of U2AF65-expressed and-depleted Hela cells. About 15 millions of 2x75 nt pair-end sequence have been mapped to the human genome. Further analysis is ongoing, which is anticipated to reveal the novel mechanism of U2AF65 in regulating constitutive and alternative splicing, as well as the new biological function associated with its RNA binding activity. At the end, we anticipate new insights toward to the role of U2AF65 in splicing of many genes related to oncogenesis.