Heterogeneous Nuclear Ribonucleoprotein (HnRNP) K Genome-wide Binding Survey Reveals Its Role in Regulating 3′-End RNA Processing and Transcription Termination at the Early Growth Response 1 (EGR1) Gene through XRN2 Exonuclease*

Abstract

The heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a nucleic acid-binding protein that acts as a docking platform integrating signal transduction pathways to nucleic acid-related processes. Given that hnRNPK could be involved in other steps that compose gene expression the definition of its genome-wide occupancy is important to better understand its role in transcription and co-transcriptional processes. Here, we used chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) to analyze the genome-wide hnRNPK-DNA interaction in colon cancer cell line HCT116. 9.1/3.6 and 7.0/3.4 million tags were sequenced/mapped, then 1809 and 642 hnRNPK binding sites were detected in quiescent and 30-min serum-stimulated cells, respectively. The inspection of sequencing tracks revealed inducible hnRNPK recruitment along a number of immediate early gene loci, including EGR1 and ZFP36, with the highest densities present at the transcription termination sites. Strikingly, hnRNPK knockdown with siRNA resulted in increased pre-RNA levels transcribed downstream of the EGR1 polyadenylation (A) site suggesting altered 3'-end pre-RNA degradation. Further ChIP survey of hnRNPK knockdown uncovered decreased recruitment of the 5'-3' exonuclease XRN2 along EGR1 and downstream of the poly(A) signal without altering RNA polymerase II density at these sites. Immunoprecipitation of hnRNPK and XRN2 from intact and RNase A-treated nuclear extracts followed by shotgun mass spectrometry revealed the presence of hnRNPK and XRN2 in the same complexes along with other spliceosome-related proteins. Our data suggest that hnRNPK may play a role in recruitment of XRN2 to gene loci thus regulating coupling 3'-end pre-mRNA processing to transcription termination.