title: 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.
authors.affiliation: Department of Genetics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, 02-781 Warsaw, Poland. mikula.michal@gmail.com
authors.forename: Michal
authors.initials: M
authors.lastname: Mikula
authors.forename: Karol
authors.initials: K
authors.lastname: Bomsztyk
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Goryca
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Chojnowski
authors.forename: Jerzy
authors.initials: J
authors.lastname: Ostrowski
mesh_terms: D020413:3' Untranslated Regions / Q000502:physiology*; D045744:Cell Line, Tumor; D051766:Early Growth Response Protein 1 / Q000096:biosynthesis* / Q000235:genetics; D005095:Exoribonucleases / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D056426:Genetic Loci / Q000502:physiology; D055106:Genome-Wide Association Study; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D006801:Humans; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D020871:RNA Stability / Q000502:physiology*; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D061806:Transcription Termination, Genetic / Q000502:physiology*; D051816:Tristetraprolin / Q000096:biosynthesis / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D012322:RNA Precursors; D012261:Ribonucleoproteins; D051816:Tristetraprolin; C497552:ZFP36 protein, human; C072074:HNRNPK protein, human; D011061:Poly A; D005095:Exoribonucleases; C120903:XRN2 protein, human
keywords: Chromatin Immunoprecipitation (ChiP); Gene Expression; Mass Spectrometry (MS); RNA Polymerase II; Transcription Termination; XRN2; hnRNPK