A core complex of CPSF73, CPSF100, and Symplekin may form two different cleavage factors for processing of poly(A) and histone mRNAs - PubMed (original) (raw)

A core complex of CPSF73, CPSF100, and Symplekin may form two different cleavage factors for processing of poly(A) and histone mRNAs

Kelly D Sullivan et al. Mol Cell. 2009.

Abstract

Metazoan histone mRNAs are unique: their pre-mRNAs contain no introns, and the mRNAs are not polyadenylated, ending instead in a conserved stem-loop structure. In Drosophila, canonical poly(A) signals are located downstream of the normal cleavage site of each histone gene and are utilized when histone 3' end formation is inhibited. Here we define a subcomplex of poly(A) factors that are required for histone pre-mRNA processing. We demonstrate that Symplekin, CPSF73, and CPSF100 are present in a stable complex and interact with histone-specific processing factors. We use chromatin immunoprecipitation to show that Symplekin and CPSF73, but not CstF50, cotranscriptionally associate with histone genes. Depletion of SLBP recruits CstF50 to histone genes. Knockdown of CPSF160 or CstF64 downregulates Symplekin but does not affect histone pre-mRNA processing or association of Symplekin with the histone locus. These results suggest that a common core cleavage factor is required for processing of histone and polyadenylated pre-mRNAs.

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Figures

Figure 1. Knockdown of pre-mRNA processing factors results in misprocessed histone mRNA

(A) A schematic of one tandem repeat of the histone gene locus is shown. The numbers indicate the distance between the 5’ or 3’ ends of the mRNAs . (B) Schematic of S1 nuclease protection assay. A 670 nt DNA fragment containing 650 nts of the 3’ end of H2A gene and 20 nts of plasmid sequence at the 5’ end was labeled with α-32P-dCTP on the 3’ end. The probe was hybridized to total cell RNA and digested with S1 nuclease. Properly processed H2A mRNA yields a protected fragment of 340 nt extending to the 3’ end of the mRNA while misprocessed transcripts give fragments ranging in size from 340-600nt. Any read-through protects a 650 nt fragment. Two gels were necessary to accommodate all samples in the same order in Figs. 1C, 1D and 2A, boundaries have been clearly indicated. Relative amounts of properly processed histone pre-mRNA from lanes 3, 4 and 9 were not significantly different from a control treated sample on the same gel. (C) Dmel-2 cells were treated with the indicated dsRNAs and total RNA was prepared. 12.5 μg of RNA was analyzed by S1 nuclease protection assay. Diagrams at left indicate RNA species corresponding to protected fragments. The undigested probe is shown in lane 13. Read-through transcripts (RT) are marked with an arrow. (D) 25 μg of total RNA was fractionated on oligo(dT) cellulose and the purified poly(A)+ RNA was used for S1 nuclease protection assay as in (B). A small amount of the processed histone mRNA nonspecifically binds to the oligo(dT) cellulose.

Figure 2. The core cleavage factor interacts with histone specific processing factors

(A) The indicated proteins were RNAi-depleted and NEs were prepared. 15 μg of NE was resolved by SDS-PAGE and western blots were performed using indicated antibodies. The loading control is a cross-reacting band from the Symplekin blot. (B) Dmel-2 NEs were used in IP experiments with indicated antibodies under high detergent conditions. α-HA and beads alone IPs were included as controls. Inputs represent 2.5% of starting material. (C) IPs were performed as in A except under low detergent conditions. (D) Diagram of core cleavage factor interactions with poly(A) and histone specific RNA processing factors.

Figure 3. RNA processing factors are recruited to histone genes in vivo

(A) Diagram of ChIP assay design. The oligo sets correspond to the coding regions of H2A, H2B, H3 and H4, the promoters of the H2A/H2B and H3/H4 pairs and the H3/H1 intergenic region. Control oligo sets were designed to exon 6 and the proximal poly(A) site of the domino gene. (B) ChIP assays were performed using antibodies to the Rpb3 subunit of RNA polymerase II, CPSF73, Symplekin and CstF50. Columns represent average relative ChIP signal normalized to the maximal value for each antibody. Data are presented as the average of 3 independent experiments ± SD.

Figure 4. Loss of a histone specific processing factor results in recruitment of additional poly(A) factors to histone genes

(A) ChIP assays were performed using lysates prepared from Dmel-2 cells treated with control (blue) or SLBP (red) dsRNAs. Columns represent the average relative ChIP signal normalized to the maximal value for each antibody. Data are presented as the average of 3 independent experiments +SD. Blue bars are untreated cells; red bars are values for SLBP knockdown cells. (B) Dmel-2 cells treated with control dsRNAs (top) or SLBP dsRNAs (bottom) were fixed and probed for CstF50 (green) and Mpm-2 (red), and also stained with DAPI (blue). The number of cells with Mpm-2 foci, and the number of cells with Mpm-2 foci that also contained CstF50 foci were counted. (C) Western blots of Dmel-2 cells treated with PTB dsRNA (control) or dsRNA targeting SLBP and CstF50. (D) Total RNA was prepared from cells treated as in (C) or with dsRNAs against SLBP or CstF50. S1 nuclease protection assays were performed on total RNA (lanes 1-4) or poly(A)+ RNA (lanes 5-8) as in Fig. 1 C, D.

Figure 5. Knockdown of poly(A) specific factors does not affect recruitment of core cleavage factor to histone genes

ChIP assays were performed as in Fig. 3B on (A) CstF64 or (B) CPSF160 knockdown cells. Columns represent the average relative ChIP signal normalized to the maximal value for each antibody. Data are presented as the average of 3 independent experiments +SD.

Figure 6. Model of histone pre-mRNA processing

(A) Model of normal histone 3’ end formation. (B) Additional poly(A) factors are recruited to histone genes to participate in cleavage and polyadenylation in the absence of histone specific RNA processing proteins.

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A core complex of CPSF73, CPSF100, and Symplekin may form two different cleavage factors for processing of poly(A) and histone mRNAs - PubMed (original) (raw)