CAPTURE of the human U2 snRNA genes expands the repertoire of associated factors.

In order to identify factors involved in transcription of human snRNA genes and 3' end processing of the transcripts, we have carried out CRISPR affinity purification in situ of regulatory elements (CAPTURE), which is deadCas9-mediated pull-down, of the tandemly-repeated U2 snRNA genes in human cells. CAPTURE enriched many factors expected to be associated with human snRNA genes including pol II, NELF, CDK9, SPT5 and several subunits of the Integrator Complex. SPT6, Mediator subunits 16, 20 and 23, TAF15, CDK7, CDK9, Cyclin K, and the SPT16 subunit of FACT were also enriched. Several polyadenylation factors, including CPSF1, CPSF6 and CstF1-3 were also enriched by U2 gene CAPTURE. These add to the polyadenylation factors CstF2, Pcf11 and Ssu72 which we previously showed are associated with the human U1 and U2 genes. Analysis of genome-wide studies and ChIP-qPCR confirms the association of SPT6 with the U2 genes. In addition, SPT6 knockdown causes loss of subunit 3 of the Integrator complex from the U2 genes and transcriptional readthrough, indicating a functional role in snRNA gene expression. CAPTURE has therefore expanded the repertoire of transcription and RNA processing factors associated with these genes. Overall design: ChIP-seq of CDK12 in human HEK293 cells