The tumor suppressor Cdc73 functionally associates with CPSF and CstF 3' mRNA processing factors - PubMed (original) (raw)
The tumor suppressor Cdc73 functionally associates with CPSF and CstF 3' mRNA processing factors
Orit Rozenblatt-Rosen et al. Proc Natl Acad Sci U S A. 2009.
Abstract
The CDC73 tumor suppressor gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. Its product, the Cdc73 protein, is a component of the RNA polymerase II and chromatin-associated human Paf1 complex (Paf1C). Here, we show that Cdc73 physically associates with the cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF) complexes that are required for the maturation of mRNA 3' ends in the cell nucleus. Immunodepletion experiments indicate that the Cdc73-CPSF-CstF complex is necessary for 3' mRNA processing in vitro. Microarray analysis of CDC73 siRNA-treated cells revealed INTS6, a gene encoding a subunit of the Integrator complex, as an in vivo Cdc73 target. Cdc73 depletion by siRNA resulted in decreased INTS6 mRNA abundance, and decreased association of CPSF and CstF subunits with the INTS6 locus. Our results suggest that Cdc73 facilitates association of 3' mRNA processing factors with actively-transcribed chromatin and support the importance of links between tumor suppression and mRNA maturation.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Cdc73 associates with CPSF and CstF subunits. (A) Purification of Cdc73 complexes by immunoprecipitation with anti-Cdc73 antibodies, Ab648 (Left) and Ab649 (Right), from 293T cell lysates, with (+BP) or without (−BP) blocking peptide control, shown after gel electrophoresis and Coomassie blue staining. * indicates Ig heavy chain, and ** indicates angiomotin, which cross-reacts with Ab649 (4). Note that the left gel was used to identify the Paf1C components [Reproduced with permission from ref. (Copyright 2005, American Society for Microbiology)]. (B) Immunoprecipitation (IP) with anti-Cdc73 Ab648 or Ab649 from HeLa nuclear extracts and immunoblotting with the indicated antibodies. Lysates were used directly (−RNase) or treated with RNase A (+RNase). Negative controls included immunoprecipitation with normal rabbit serum (IgG) and the respective blocking peptides (+BP). HeLa nuclear extracts were loaded to indicate the position of the endogenous protein (Input). (C) HeLa nuclear extracts were immunoprecipitated with anti-CPSF-30 or anti-CstF-64 antibodies. Immunoprecipitates were resolved and then immunoblotted with either anti-Cdc73 Ab 1 (Upper) or the respective anti-CPSF or anti-CstF antibody (Lower). Normal rabbit IgG and the appropriate blocking peptides (BP) were used as controls.
Fig. 2.
The Cdc73 complex is associated with 3′ mRNA processing activity. (A) Nuclear extracts (NE) were immunodepleted with the indicated amounts of anti-Cdc73 Ab648 as shown. Depleted NEs were resolved by gel electrophoresis and immunoblotted with the indicated antibodies. (B) Input NE (lanes 1 and 4), NE after immunodepletion with 10 μL of anti-Cdc73 antibody (lanes 2 and 5), or bead-immunodepleted NE (lanes 3 and 6) were used in an in vitro cleavage assay using labeled adenoviral L3 (Ad L3; lanes 1–3) or human ApoCI (lanes 4–6) substrates. Arrows indicate the precursor mRNA (pre-mRNA), the 5′ cleavage products (5′ CP), and the 3′ cleavage products (ApoCI 3′ CP, and Ad L3 3′ CP). (C) A schematic of the pG3CMVL3 DNA template and the position of the polyadenylation site is shown. Cotranscriptional cleavage and polyadenylation were performed on this DNA template with either bead-depleted or Cdc73-depleted HeLa nuclear extract. RNA products were then separated into nonpolyadenylated (poly(A)−) and polyadenylated (poly(A)+) fractions by oligo(dT) selection, and analyzed on a denaturing gel. (D) At the top is a schematic of the pCMVAdML DNA template. The expected sizes of the pre-mRNA and the processed/spliced mRNA are 680 and 560 bp, respectively. Eight microliters or 10 μL of bead-depleted (Left) or Cdc73-depleted (Right) nuclear extract was used to transcribe and splice the DNA template. RNA products were analyzed on denaturing gel.
Fig. 3.
Cdc73 regulates the expression of Ints6. (A) Mean fold change for the genes whose expression was most significantly decreased upon knockdown of Cdc73, as assayed by 3 independent probe sets on Affymetrix U133A 2.0 arrays. (B) Expression of CDC73, INTS6, BANP, and LIFR was measured by quantitative real-time RT-PCR in HeLa cells transfected with siRNAs directed to either luciferase (LUC), lamin (LMNA), or CDC73 (_CDC73_-1, _CDC73_-2). Transcript levels were normalized to the LUC control. Error bars indicate SD for 3 independent experiments. (C) HeLa cells were transfected with the indicated siRNAs, and cell lysates were subjected to immunoblot analysis (IB) with the indicated antibodies. (D) (Upper) A scheme representing the INTS6 transcript and predicted poly(A) sites located 3,780 bp [p(A) site 1], 6,335 bp [p(A) site 2], and 7,431 bp [p(A) site 3] from the transcriptional start site. Expression of INTS6 transcript was measured by using quantitative real-time RT-PCR with the depicted CDS, Ext1 and Ext2 primers in HeLa cells transfected with the indicated siRNAs. (Lower) Level of extended transcript was calculated by normalizing Ext1 or Ext2 transcript levels to the CDS transcript level. Error bars indicate SEM for 3 independent experiments.
Fig. 4.
Cdc73 facilitates recruitment of CPSF and CstF subunits to the INTS6 locus. (A) Schematic of the INTS6 locus indicating primer sets used to quantify ChIPs. The arrow indicates the transcriptional start site and the gray box indicates the beginning of the coding sequence. (B) ChIPs were performed using the indicated antibodies or without antibody (−Ab) to determine the background level, in HeLa cells treated with LUC or _CDC73_-1 siRNAs. Precipitated chromatin was used for quantitative real-time PCR amplification with the primer INTS6 5′ UTR primer set. Error bars indicate SEM for 4 independent experiments.
References
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- Shattuck TM, et al. Somatic and germ-line mutations of the HRPT2 gene in sporadic parathyroid carcinoma. N Engl J Med. 2003;349:1722–1729. - PubMed
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