A RECQ5-RNA polymerase II association identified by targeted proteomic analysis of human chromatin

Abstract

Although the active forms of factors involved in DNA-related processes such as DNA replication, repair, and transcription are associated with chromatin, proteins are rarely purified from this source. Here, we describe a protocol for the isolation of chromatin-associated factors and use it to identify proteins interacting with human RNA polymerase II (RNAPII). Our data establish RECQ5 helicase as a bona fide RNAPII-associated protein. The RECQ5-RNAPII interaction is direct and is mediated by the RPB1 subunit of RNAPII, and RECQ5 appears to be the only member of the human RECQ family of helicases that associates with RNAPII. These data suggest an unexpected role for RECQ5 helicase at the interface of transcription and genomic stability.

Fig. 1.

Mass spectrometric analysis of purified human RNAPII complexes. (A) Outline of the purification procedure. (B and C) Equal amounts of the M2 chromatography eluates from control (Mock), nucleoplasm (B), and chromatin fractions (C) were fractionated on a 4–12% bis-Tris gradient protein gel and analyzed by silver staining. Asterisks indicate background protein bands that also are present in the control purification. The mock purification looked similar whether originating from nucleoplasm or chromatin. For MS analysis, a duplicate gel was analyzed by SYPRO Ruby staining, and protein bands were excised for mass spectrometric analysis. Identified proteins are labeled to the right of the respective protein bands.

Fig. 2.

RECQ5 is a bona fide RNAPII-associated protein in human chromatin. (A) Western blotting showing the coimmunoprecipitation of RNAPII with RECQ5. Native chromatin prepared from HEK293 cells stably expressing FLAG-RECQ5 was solubilized and used for immunoprecipitation either with anti-FLAG antibody or nonspecific mouse IgGs. RNAPII was detected with anti-Rpb1 (4H8) antibody. (B) Immunopurification of FLAG-RECQ5 from stable HEK293 cells. Native chromatin was prepared from FLAG-RECQ5 expressing stable cells as described in Fig. 1A. The M2 eluate was resolved by 4–12% bis-Tris gradient PAGE, stained by SYPRO Ruby, and analyzed with a PhosphorImager. Proteins identified by mass spectrometry are labeled to the right of the respective protein bands. Asterisks indicate four C-terminal degradation products of the FLAG-RECQ5 protein. The lane on the left represents the mock purification.

Fig. 3.

Direct interaction between RNAPII and RECQ5 via the C-terminal region of RECQ5 and the RPB1 subunit of RNAPII. (A) Ni-NTA pull-down of RNAPII using purified, His-tagged RECQ5, RECQ5_1–542, and RECQ1. His-tagged proteins attached to Ni-NTA beads were incubated with purified RNAPII. After washing, the beads were boiled, and bound proteins were detected by SDS/PAGE, followed by Western blotting using 4H8 (RPB1, Upper) and anti-His antibodies (Lower). (B) Far Western blot analysis of RECQ5 interaction with individual RPB subunits of RNAPII. Purified RNAPII (from a RECQ5-FLAG purification) was separated by denaturing SDS/PAGE and transferred onto nitrocellulose membrane. Membranes were either incubated with recombinant His-RECQ5 (Right) or His-RECQ5_1–542 (Left), and membrane-bound, His-tagged proteins were then detected by anti-His antibody. Asterisks indicate contaminating mouse IgG heavy and light chains derived from the polymerase purification procedure that were recognized directly by secondary anti-mouse IgG antibody. The origin of the band detected better with the truncated RECQ5 probe (labeled by a double asterisk) is unknown.

Fig. 4.

RNAPII interacts with RECQ5, but not with other helicases in the RECQ5 family. FLAG-RECQ complexes were immunoprecipitated from 293T cells by using FLAG M2-agarose beads. The presence of RNAPII, RAD51, and FLAG-RECQ in each of the RECQ complexes was determined by SDS/PAGE, followed by Western blotting with mouse 4H8 mAb (RPB1, Top), rabbit anti-RAD51 (Middle), and rabbit anti-FLAG antibody (Bottom).