RPL30 regulation of splicing reveals distinct roles for Cbp80 in U1 and U2 snRNP cotranscriptional recruitment

Abstract

Pre-mRNA splicing is catalyzed by the spliceosome, and its control is essential for correct gene expression. While splicing repressors typically interfere with transcript recognition by spliceosomal components, the yeast protein L30 blocks spliceosomal rearrangements required for the engagement of U2 snRNP (small ribonucleoprotein particle) to its own transcript RPL30. Using a mutation in the RPL30 binding site that disrupts this repression, we have taken a genetic approach to reveal that regulation of splicing is restored in this mutant by deletion of the cap-binding complex (CBC) component Cbp80. Indeed, our data indicate that Cbp80 plays distinct roles in the recognition of the intron by U1 and U2 snRNP. It promotes the initial 5' splice site recognition by U1 and, independently, facilitates U2 recruitment, depending on sequences located in the vicinity of the 5' splice site. These results reveal a novel function for CBC in splicing and imply that these molecular events can be the target of a splicing regulator.

FIGURE 1.

L30 binds, but does not repress, the RPL30 5A C9U transcript. (A) Schematic representation of the RPL30 kink-turn bound by L30, wt, and mutant versions, as indicated (SS: splice site, the AUG is at the end of the first exon). Numbers refer to the start of transcription. (B) Effect of mutations in RPL30 on L30 binding. RPL30 transcripts (0.5 pmol, nt 1–123) were incubated with buffer (lanes 1,7,13,19) or with increasing amounts of MBP:L30 (50, 100, 200, 500, and 1000 ng; lanes 2–6, 8–12, 14–18, and 20–24, respectively). Transcripts were wt (lanes 1–6), C9U (lanes 7–12), 5A (lanes 13–18), and 5A-C9U (lanes 19–24). Reactions were analyzed in a 6% acrylamide gel. (C) Both C9U and 5A C9U transcripts fail to accumulate pre-mRNA under conditions of L30 excess. W303 (lanes 1,2) or yJV25 (producing excess L30, lanes 3–6) cells were transformed with pLCUP plasmids (schematized on the bottom) bearing the indicated mutations (top). RNA was extracted, subjected to Northern analysis, and probed with LCUP sequences (D) snRNP coimmunoprecipitation with L30. RPL30 transcripts (nt 1–347) were incubated under splicing conditions with MBP:L30. Reactions were immunoprecipitated with anti-MBP, and pelleted RNA was subjected to Northern analysis to detect RPL30, U1 and U2 snRNA, as indicated. Lane 1, RPL30 +12; lane 2, RPL30 5A C9U; lane 3, RPL30; lane 4, no transcript added; lane 5, no antibody added; lane 6, 1% of the input.

FIGURE 2.

Screen for synthetic enhancers of L30 repression of splicing. (A) Reporter plasmids based on the fusion between RPL30 exon 1 and intron with the CUP1 ORF. LCUPIF transcripts (left) produce Cup1 protein only when unspliced, while LCUP RNAs (right) need to be spliced to encode the protein. (B) Phenotype, identified as growth in medium-containing copper, of cells with constitutive excess of L30 (yJV25) and transformed with either pLCUPIF (upper panel) or pLCUP 5A (bottom panel). Under repression conditions (“+” rows), pLCUPIF confers copper resistance while pLCUP-5A does not. When L30 repression is abolished by the C9U mutation (“−” rows), pLCUP confers tolerance while pLCUPIF does not. Serial one-fifth dilutions were spotted in each case. (C) Screen strategy to select mutations that restore inhibition of splicing by L30 on a C9U transcript. Corresponding Northern analyses are shown in panels D and E. Strain yJV25 with the plasmid pLCUPIF-C9U (Cu-sensitive) was UV-irradiated and SLR mutants were selected on plates containing 0.3 mM copper. Colonies showing LCUPIF C9U pre-mRNA accumulation (panel D) were cured of the plasmid and transformed with pLCUP 5A C9U, rendering them Cu sensitive again because of increased repression, unless the slr mutation is complemented or suppressed. Thus, cells were transformed with a YCp50-based wt genomic library and the transformants selected on 0.7 mM copper, and pCBP80 was identified (panel E). (D) Northern analysis of RNA from SLR mutant cells transformed with pLCUPIF C9U (lanes 3–8). As controls, RNA extracted from yJV25 cells harboring pLCUPIF wt (lane 1) or C9U (lane 2) were loaded in the same gel. Precursor (p) and mature (m) LCUPIF transcripts are indicated. (E) Northern analysis of RNA extracted from SLR4, SLR5, and SLR7 (lanes 3–8) and yJV25 cells (lanes 1,2), transformed with pLCUP 5A C9U alone (odd lanes), or plus pCBP80 (even lanes). Precursor (p) and mature (m) LCUP transcripts are indicated. In D and E U3 was used as loading control.

FIGURE 3.

Deletion of CBP80 is synthetic with L30 repression of splicing. (A) Decreased levels of Cbp80-TAP in SLR5 mutants. Cbp80 protein was TAP-tagged at the C terminus in yJV25 (wt, lane 1), SLR5 (lane 2), and cbp20Δ (Y02074). Extracts were subjected to Western analyses, as indicated. Tubulin was used as loading control. (B) Deletion of CPB80 produces the same phenotype as that of SLR5. One-fifth serial dilutions of wt (yJV25, top), cbp80Δ (yJV35, middle), and SLR5 (bottom) cells transformed with pLCUP 5A C9T were spotted on copper-containing media, as indicated. Copper sensitivity indicates splicing repression of the LCUP 5A C9U transcript (C). Deletion of CBP80 leads to repression of LCUP 5A C9U splicing by L30. Northern analysis of RNA from wt (yJV25, lane 1), SLR5 (lane 2), and cbp80Δ (lane 3) cells. Positions of precursor (p) and mature (m) LGFP 5A C9U are indicated on the right. (D) Splicing efficiency and repression by L30 of the RPL30 intron in wt and cbp80Δ cells, with or without excess L30. Northern analysis of RNA from either wt (even lanes) or cbp80Δ cells (odd lanes) transformed with the pLGFP reporter plasmid, as indicated at the top. Samples in the bottom panel are from cells under excess L30 (pMB73). Positions of precursor (p) and mature (m) LGFP are indicated on the left. pLGFP contains the GFP ORF instead of Cup1 in pLCUP (Vilardell and Warner 1997). pMB73 encodes L30 without the autoregulatory loop (Macías et al. 2008).

FIGURE 4.

In vitro regulation of splicing of RPL30 5A C9U by L30 in the absence of Cpb80. Synthetic RPL30 transcripts, indicated at the top, were incubated under splicing conditions with wt (A) and cbp80Δ (B) extracts, supplemented with MBP:L30 or MBP:Cbp80, as indicated. Upon completion of the reaction, RNA was extracted and analyzed by semiquantitative RT-PCR. Bands corresponding to substrate and spliced RNA are shown. Amounts of RNA, extracts, and recombinant protein were equivalent in all reactions.

FIGURE 5.

Cotranscriptional U1 and U2 recruitments on the RPL30-LacZ transcript in wt and cbp80Δ cells. Horizontal axes show the distance in nucleotides from the start codon. Vertical axes indicate the signal relative to that of the promoter (first primer pair, or PP1). The black bar indicates intron position. The ChIP profiles correspond to wt (panels C and E) or cbp80Δ cells (panels D and F), under normal conditions (black lines) or under L30 excess (gray lines). (A) Scheme showing the positions of the PCR primers used for the ChIP analyses of the RPL30-LacZ intron, relative to the translation start. (B) ChIP against L30 (L30-TAP). In both cases there is L30 excess (pMB73, see Materials and Methods). Black line, wt cells; gray, cbp80Δ cells. In the following panels, ChIP profiles of the indicated proteins are shown, performed on RPL30-LacZ. (C,D) ChIP against U1 snRNP (Snu71-HTB). (E,F) ChIP against U2 snRNP (Lea1-HTB).

FIGURE 6.

Effect of mutations in the RPL30 intron and Cbp80 on cotranscriptional recruitment of U1 and U2 on the RPL30-LacZ gene. Horizontal axes show the distance in nucleotides from the start codon. Vertical axes indicate the signal relative to that of the promoter (first primer pair, or PP1). The black bar indicates intron position. The ChIP profiles correspond to wt (black lines) or cbp80Δ cells (gray lines). (A) Panels indicate different intronic 5′ ends, with a schematic representation of the possible base-pairing with U1 snRNA. GUCAGUAU panels are based on data from Figure 5. (B–D) ChIP profiles of U1 snRNP (Snu71-HTB). (E–G) ChIP profiles of U2 snRNP (Lea1-HTB). (H,I) In vitro splicing of the RPL30 intron, either wt or with reduced affinity for U1 snRNA, in wt or cbp80Δ extracts. Splicing reactions were set up and analyzed as in Figure 4, using wt extracts (H) or extracts from cbp80Δ cells (I), supplemented with MBP:L30 or MBP:Cbp80, as indicated. In the UC transcript, intron positions 6 and 7 (AU) have been mutated to UC, which cannot base-pair to U1. Bands corresponding to substrate and spliced RNA are shown. Amounts of RNA, extracts, and recombinant protein were equivalent in all reactions (see Materials and Methods).