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. 2013 Aug;19(8):1054-63.
doi: 10.1261/rna.037069.112. Epub 2013 Jun 21.

The Nuclear Cap-Binding Complex Interacts With the U4/U6·U5 tri-snRNP and Promotes Spliceosome Assembly in Mammalian Cells

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Free PMC article

The Nuclear Cap-Binding Complex Interacts With the U4/U6·U5 tri-snRNP and Promotes Spliceosome Assembly in Mammalian Cells

Marta Pabis et al. RNA. .
Free PMC article

Abstract

The nuclear cap-binding complex (CBC) binds to the 7-methyl guanosine cap present on every RNA polymerase II transcript. CBC has been implicated in many aspects of RNA biogenesis; in addition to roles in miRNA biogenesis, nonsense-mediated decay, 3'-end formation, and snRNA export from the nucleus, CBC promotes pre-mRNA splicing. An unresolved question is how CBC participates in splicing. To investigate CBC's role in splicing, we used mass spectrometry to identify proteins that copurify with mammalian CBC. Numerous components of spliceosomal snRNPs were specifically detected. Among these, three U4/U6·U5 snRNP proteins (hBrr2, hPrp4, and hPrp31) copurified with CBC in an RNA-independent fashion, suggesting that a significant fraction of CBC forms a complex with the U4/U6·U5 snRNP and that the activity of CBC might be associated with snRNP recruitment to pre-mRNA. To test this possibility, CBC was depleted from HeLa cells by RNAi. Chromatin immunoprecipitation and live-cell imaging assays revealed decreased cotranscriptional accumulation of U4/U6·U5 snRNPs on active transcription units, consistent with a requirement for CBC in cotranscriptional spliceosome assembly. Surprisingly, recruitment of U1 and U2 snRNPs was also affected, indicating that RNA-mediated interactions between CBC and snRNPs contribute to splicing. On the other hand, CBC depletion did not impair snRNP biogenesis, ruling out the possibility that decreased snRNP recruitment was due to changes in nuclear snRNP concentration. Taken together, the data support a model whereby CBC promotes pre-mRNA splicing through a network of interactions with and among spliceosomal snRNPs during cotranscriptional spliceosome assembly.

Keywords: cap-binding complex; m7G cap; pre-mRNA splicing; snRNA biogenesis; snRNP.

Figures

FIGURE 1.
FIGURE 1.
Validation of splicing factor binding to CBC. (Upper panel) Extracts from transgenic HeLa cells harboring GFP-tagged splicing factors, indicated left of the panel, were incubated with or without RNase A and subjected to immunoprecipitation with α-GFP. 0.6% of input (Inp) and 33% of the immunoprecipitate were analyzed by Western blot, using α-CBP20 and α-CBP80. Nonspecific background was assessed by immunoprecipitation with nonimmune IgG (IgG). See Supplemental Figure 1, D and E, for additional data and evidence that RNase treatment disrupted snRNPs. (Lower panel) Extracts from the same transgenic HeLa cells treated with and without α-amanitin to inhibit Pol II transcription, as indicated. All experiments were performed two to four times each, and representative gels are shown.
FIGURE 2.
FIGURE 2.
Efficient CBC knockdown does not affect snRNP levels. HeLa cells transduced with retroviral vectors expressing CBP80 shRNA or without shRNA (Control) were assayed on day 6. (A) Assessment of CBC depletion through semiquantitative Western blotting (left panel) of CBP80, CBP20, and GAPDH after CBP80 knockdown (KD). Decreasing amounts of the same lysate were loaded. Comparison of changes in CBP80 and CBP20 RNA and protein levels (right panel). Band intensities were measured as integrated densities from Western blots and normalized to GAPDH. RNA levels were determined by RT-qPCR and normalized to 18S rRNA. N ≥ 8 different knockdowns. Error bars are the SEM. The changes are statistically significant (P < 0.005), as determined by the Student’s t-test. (B) Immunoprecipitation of snRNPs with α-TMG (K121), α-Sm proteins (Y12), and α-SART3. RNA was extracted, resolved on a 10% urea gel, and detected by Northern blot. A longer exposure of α-TMG IP lanes is shown.
FIGURE 3.
FIGURE 3.
CBC depletion reduces cotranscriptional spliceosome assembly and splicing. HeLa cells transduced with control and α-CBP80 shRNA retroviral vectors were subjected to RNA isolation, ChRIP, or ChIP on day 6. (A) Schematic representation of FOS pre-mRNA and mRNA, showing the location of qPCR amplicons detecting RNA with either spliced or unspliced intron 1 (blue) and intron 3 (red). Total RNA (left panel) was reverse-transcribed with an oligo(dT) primer and the relative abundance of spliced to unspliced poly(A)+ RNA assessed by qPCR. Nascent RNA (right panel) was isolated by ChRIP from an α-AcH4 IP and reverse-transcribed, using a primer in intron 2 (for intron 1 splicing) or in exon 4 (for intron 3 splicing). qPCR was conducted to assess the relative abundance of spliced to unspliced RNA. n ≥ 4; error bars are the SEM. (B) Schematic representation of the FOS gene. (Gray lines) The location of qPCR amplicons used in ChIP. The following antibodies were used: α-CBP80, α-Pol II-CTD (4H8), α-U2AF-65 (MC3), α-U5-116K, and α-GFP in case of transgenic cell lines expressing U1-70K-GFP or SF3A1-GFP. All values were calculated as percent input, normalized to nonimmune ChIP, and presented as fold enrichment over an intergenic region. In Pol II ChIP, normalization to intergenic was omitted due to absence of background. N ≥ 3; error bars are the SEM. Significant differences between knockdown and control determined by the Student’s t-test: (*) P < 0.05, (**) P < 0.01.
FIGURE 4.
FIGURE 4.
CBC depletion reduces snRNP interactions with active transcription sites. (A) Diagram of the E3 construct stably integrated into U2OS cells. Expression is driven by a minimal CMV promoter (P) under control of the tetracycline response element and induced in the presence of doxycycline (dox) by the transactivator rtTA. The gene contains three β-globin exons (E1–E3) and a CFP coding sequence, 18 MS2 repeats, a polyadenylation cleavage signal, and a transcription terminator (T). (B) Imaging of nascent RNA at the E3 transcription unit in fixed cells by RNA FISH (Cy3-labeled probe to the MS2 sequence repeats; red) together with the stably integrated U1 snRNP (U1-70K-GFP; green), U2AF65-GFP, or U5 snRNP proteins (Prp8-GFP). Scale bars, 5 μm. (C) FRAP curves show recovery of U1-70K-GFP, U2AF65-GFP, and Prp8-GFP fluorescence at bleached spots placed in nucleoplasm, speckles, or at the E3 transcription site visualized by MS2BP-mCherry. Three independent experiments were performed; in each 10–20 cells were tested. Error bars represent the SEM. For U1-70K-GFP and Prp8-GFP, the differences between recovery curves in control and CBC knockdown cells are statistically significant, as established using the Mann–Whitney test.

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