A Broad Set of Chromatin Factors Influences Splicing

Abstract

Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing.

Fig 1. Purification of spliceosome complexes associated with the U2 snRNP.

(A) Detection by western blot of indicated splicing factors in nuclear extracts from HeLa S3 cells either WT (NE) or stably transduced with FV₅-U2-B (NE_B”). (B) Comparison of the kinetic and quality of spliceosome assembly on a radiolabeled AdML pre-mRNA reporter in the presence of either ATP (lanes 1–3) or ATP-γS (lanes 4–8). Spliceosome complexes A and B were resolved from unspecific RNP (H complex) on a native gel. (C) Spliceosome complexes were purified by Sephacryl-S500 gel filtration from an in vitro-splicing reaction composed of NE_B” and radiolabeled AdML reporter incubated for 45 min in the presence of ATP-γS. Fractions with spliceosome and H complexes selected for anti-FLAG immunoprecipitation are indicated. Degraded RNAs and free proteins appear in fractions 68 to 80. (D) Spliceosome complexes anchored to the U2 snRNP. NE_B”, gel-filtration fractions with either spliceosome (Splic.; lane 2) or H complex (H; lane 3), and the product of the anti-FLAG immunoprecipitation (lanes 4 and 5) were analyzed by western blot using the indicated antibodies. See also Table 1 and S1D and S1E Fig.

Fig 2. A high-throughput siRNA screen for chromatin factors that affect splicing.

(A) Diagram of the bi-cistronic v4-v5–ren and int-ren minigene reporters used to follow splicing by luminescence. Rectangles represent exons, stem-loop structures represent Internal Ribosome Entry Sites (IRES). Splicing regulation leading to a functional Renilla is indicated in Green, while those producing to a non-functional Renilla are marked in Red (B) Outline of the procedure used to screen a library of siRNAs targeting 375 chromatin factors. Each gene was targeted with an average of 3 individual siRNAs, tested in duplicates. (C) List of chromatin factors modulating splicing of the v4-v5-ren reporter in 293 EcR cells. The 63 hits of the siRNA screen are grouped in five categories according to the “String” database (string-db.org). Associated post-translational modifications (PTMs) are indicated to the left of the gene names: Acetylation (Ac), Methylation (Me), Phosphorylation (Ph). Each gene is designated by its name and its NCBI identification number (NCBI Gene Id.). (D and E) Schematic display of BRM-sin3A-HDAC and huCHRAC complexes highlighting the subunits identified by proteomic or by the siRNA screen.

Fig 3. Chromatin factors affect the regulation of endogenous splicing.

SiRNAs targeting 14 chromatin factors and identified as affecting splicing of v4-v5-ren in 293-ECR cells were transfected into HeLa cells as well as siRNA targeting controls. (A) Radiolabeled RT-PCR was then used to examine splicing of exons v4-v5 from endogenous CD44 and exons 2 and 3 from Sam68. The chromatin factors identified by mass spectrometry are highlighted with asterisks. (B) Graphs displaying the percentage of inclusion for six alternative exons previously described as sensitive to U2 snRNP activity [18]. Exon inclusion was detected by radiolabeled RT-PCR (S3D–S3F Fig) using total RNA of HeLa cells depleted for the indicated chromatin factors and controls. Graphs display effects obtained from triplicate experiments; asterisks indicate p-val<0.05. The stippled line shows the percentage of inclusion for untreated and siGlo-transfected cells, while the exons amplified by RT-PCR are drawn on the top of each graph.

Fig 4. Chromatin affects the efficiency of intron removal in vitro.

(A) Diagram of the three different sources of Ftz reporter RNA used to study in vitro the impact of chromatin on splicing. (i) A capped pre-mRNA that was independently transcribed with the T7 RNA polymerase before being added to HeLa nuclear extract; (ii) pre-mRNA was transcribed in HeLa nuclear extract from a naked, or (iii) a chromatinized DNA template. Regardless of the source, the pre-mRNAs are identical, with two exons and one intron originating from the Drosophila Ftz pre-mRNA. (B) Analysis of the products of in vitro splicing of the pre-synthesized RNA reporter (lanes 1–3), of a transcription/splicing reaction transcribing the RNA reporter from a naked DNA template (lanes 4–5), or from a chromatinized template (lanes 6–7). Transcription/splicing was assayed in the absence (-) or in the presence (+) of Gal4-VP16. For each condition, the RNA was resolved on a 6% denaturing polyacrylamide gel; the relative abundance of spliced mRNA indicated at the bottom of each lane was calculated as follows: % splicing = [spliced/(unspliced+spliced)]. The asterisk indicates the labeling of U6 snRNA by a terminal uridylyl transferase present in HeLa nuclear extract [15,46]. (C) The influence of NaB and CoA on in vitro transcription/splicing was evaluated in reactions assembled with naked or chromatinized DNA template. The presence (+) or absence (-) of Gal4-VP16, NaB, CoA and the time of incubation are indicated above the gel. The transcription level was calculated as the sum of unspliced and spliced RNA, and lane 4 was arbitrarily set at 100%. The splicing efficiency was calculated as in Fig 4B. (D) The experimental procedure applied in Fig 4E is displayed with a chronogram. (E) Chromatin affects the quality of pre-mRNP released by RNAPII transcription. The transcription/splicing reactions of naked or nucleosomal template were performed for 45 min (lanes 3 and 7) or 120 min (lanes 1, 2, 4, 5, 6, 8). α-amanitin was added to the reactions after 45 min of incubation (lanes 4 and 8) and the reactions were extended for additional 75 min. The presence (+) or absence (-) of Gal4-VP16 is indicated. The percentage of splicing (% splicing) was calculated as in (B) for lanes 2 and 6; while the percentage of post-transcriptional splicing in lanes 4 and 8 was calculated as followed: % PT splicing = [(spliced₁₂₀-spliced₄₅)/(unspliced+(spliced₁₂₀-spliced₄₅))].