RNA processing occurs co-transcriptionally through the dynamic recruitment of RNA processing factors to RNA polymerase II (RNAPII). However, transcriptome-wide identification of protein-RNA interactions specifically assembled on transcribing RNAPII is challenging. Here, we develop the targeted RNA immunoprecipitation sequencing (tRIP-seq) method that detects protein-RNA interaction sites in thousands of cells. The high sensitivity of tRIP-seq enables identification of protein-RNA interactions at functional subcellular levels. Application of tRIP-seq to the FUS-RNA complex in the RNAPII machinery reveals that FUS binds upstream of alternative polyadenylation (APA) sites of nascent RNA bound to RNAPII, which retards RNAPII and suppresses the recognition of the polyadenylation signal by CPSF. Further tRIP-seq analyses demonstrate that the repression of APA is achieved by a complex composed of FUS and U1 snRNP on RNAPII, but not by either one alone. Moreover, our analysis reveals that FUS mutations in familial amyotrophic lateral sclerosis (ALS) that impair the FUS-U1 snRNP interaction aberrantly activate the APA sites. tRIP-seq provides new insights into the regulatory mechanism of cotranscriptional RNA processing by RNA processing factors.

Although large exons cannot be readily recognized by the spliceosome, many are evolutionarily conserved and constitutively spliced for inclusion in the processed transcript. Furthermore, whether large exons may be enriched in a certain subset of proteins, or mediate specific functions, has remained unclear. Here, we identify a set of nearly 3,000 SRSF3‐dependent large constitutive exons (S3‐LCEs) in human and mouse cells. These exons are enriched for cytidine‐rich sequence motifs, which bind and recruit the splicing factors hnRNP K and SRSF3. We find that hnRNP K suppresses S3‐LCE splicing, an effect that is mitigated by SRSF3 to thus achieve constitutive splicing of S3‐LCEs. S3‐LCEs are enriched in genes for components of transcription machineries, including mediator and BAF complexes, and frequently contain intrinsically disordered regions (IDRs). In a subset of analyzed S3‐LCE‐containing transcription factors, SRSF3 depletion leads to deletion of the IDRs due to S3‐LCE exon skipping, thereby disrupting phase‐separated assemblies of these factors. Cytidine enrichment in large exons introduces proline/serine codon bias in intrinsically disordered regions and appears to have been evolutionarily acquired in vertebrates. We propose that layered splicing regulation by hnRNP K and SRSF3 ensures proper phase‐separation of these S3‐LCE‐containing transcription factors in vertebrates.

During mRNA transcription, diverse RNA-binding proteins (RBPs) are recruited to RNA polymerase II (RNAP II) transcription machinery. These RBPs bind to distinct sites of nascent RNA to co-transcriptionally operate mRNA processing. Recent studies have revealed a close relationship between transcription and co-transcriptional RNA processing, where one affects the other’s activity, indicating an essential role of protein–RNA interactions for the fine-tuning of mRNA production. Owing to their limited amount in cells, the detection of protein–RNA interactions specifically assembled on the transcribing RNAP II machinery still remains challenging. Currently, cross-linking and immunoprecipitation (CLIP) has become a standard method to detect in vivo protein–RNA interactions, although it requires a large amount of input materials. Several improved methods, such as infrared-CLIP (irCLIP), enhanced CLIP (eCLIP), and target RNA immunoprecipitation (tRIP), have shown remarkable enhancements in the detection efficiency. Furthermore, the utilization of an RNA editing mechanism or proximity labeling strategy has achieved the detection of faint protein–RNA interactions in cells without depending on crosslinking. This review aims to explore various methods being developed to detect endogenous protein–RNA interaction sites and discusses how they may be applied to the analysis of co-transcriptional RNA processing.

Mammalian internal exons are usually between 50 and 200 bp long. Their average length is ~150 bp, and only 5% of them are larger than 300 bp. Although previous studies showed that splice site recognition becomes less efficient with increasing exon size, the mechanism how large exons are correctly spliced is not well understood. To investigate the role of RNA binding proteins (RBPs) in the splicing of large exons, we analyzed 842 publicly available RNA‐seq datasets from GEO, in which 48 canonical splicing factors were individually knocked down. MISO analysis was performed to detect exons that were specifically included or excluded by knockdown of an RBP, and average lengths of these exons were calculated. Our analysis revealed that SRSF3 is the top RBP that promotes inclusion of the largest exon set (390 exons per analysis, 337 bp on average). Interestingly, most of the SRSF3‐dependent large exons are annotated as constitutive exons in the public database. Consistently, these exons have high splice site strengths. Motif analysis revealed that the SRSF3‐dependent large exons are greatly enriched with C‐rich sequences, which are preferentially recognized by SRSF3. In addition, our analysis also detected the enrichment of C‐stretches, which are potential binding sites for hnRNP K. ENCODE eCLIP shows that HNRNPK extensively binds to the SRSF3‐dependent large exons. To examine whether hnRNP K is involved in the regulation of SRSF3‐dependent splicing of large exons, we knocked down Hnrnpk along with Srsf3‐knockdown using C2C12 myoblasts. RNA‐seq analysis revealed that Srsf3‐silencing alone induced skipping of 374 exons, of which average length is 395 bp. Co‐silencing of Srsf3 and Hnrnpk rescued skipping of 142 exons by Srsf3‐silencing, of which average length is still long, 458 bp. These results indicate that substantial number of large exons require SRSF3 to prevent their skipping by hnRNP K. To investigate the molecules associated with SRSF3 and hnRNP K in the splicing of large exons, we next conducted proteomic approach that defines the native protein complexes in the chromatin fraction. Cells expressing FLAG tagged SRSF3 or hnRNP K were lysed under the physiological salt concentration, and isolated chromatin fractions were immunoprecipitated with At‐FLAG antibody. Mass spectrometry analysis of the immunoprecipitants revealed that SRSF3 is mainly associated with U1 snRNPs, U2AFs, and SF1, and to a lesser extent with hnRNPs, while hnRNP K is preferentially associated with hnRNPs and U1 snRNPs, but not with U2AF or SF1. Thus, SRSF3 and hnRNP K are involved in early but distinct stages of spliceosome assembly. Together, our analysis identified the antagonistic splicing regulation of SRSF3 and HNRNPK on C‐rich large exons.