Interaction of the U1 snRNP with nonconserved intronic sequences affects 5' splice site selection

Abstract

Intron definition and splice site selection occur at an early stage during assembly of the spliceosome, the complex mediating pre-mRNA splicing. Association of U1 snRNP with the pre-mRNA is required for these early steps. We report here that the yeast U1 snRNP-specific protein Nam8p is a component of the commitment complexes, the first stable complexes assembled on pre-mRNA. In vitro and in vivo, Nam8p becomes indispensable for efficient 5' splice site recognition when this process is impaired as a result of the presence of noncanonical 5' splice sites or the absence of a cap structure. Nam8p stabilizes commitment complexes in the latter conditions. Consistent with this, Nam8p interacts with the pre-mRNA downstream of the 5' splice site, in a region of nonconserved sequence. Substitutions in this region affect splicing efficiency and alternative splice site choice in a Nam8p-dependent manner. Therefore, Nam8p is involved in a novel mechanism by which a snRNP component can affect splice site choice and regulate intron removal through its interaction with a nonconserved sequence. This supports a model where early 5' splice recognition results from a network of interactions established by the splicing machinery with various regions of the pre-mRNA.

Figure 1

Nam8p affects specifically 5′ splice site recognition. Splicing of reporters carrying a wild-type or mutant RP51A intron interrupting the β-galactosidase-coding sequence was analyzed in wild-type (solid bars) and Δnam8 (open bars) strains. Mutations are underlined.

Figure 2

Nam8p stabilizes the commitment complexes assembled on uncapped pre-mRNA. Commitment complexes were assembled on labeled GpppG-capped (lanes 1–4) or uncapped (lanes 5–8) substrates. An excess of cold pre-mRNA was added and the incubation was resumed for 40 min. (Lanes 1,2 and 5,6) Wild-type extract; (lanes 3,4 and 7,8) Δnam8 extract.

Figure 3

Nam8p specifically cross-links to pre-mRNA. Nam8p–protA-containing extract (lanes 1, 4–6, 10,11) and wild-type extract (lanes 2, 7–9, 12–13) were used to assemble commitment complexes on pre-mRNA, UV cross-linked, treated with RNase T1 and immunoprecipitated. Pellets were analyzed by SDS-PAGE. (Lanes 1–3) No immunoprecipitation; (lanes 4–9) immunoprecipitation with IgG-coupled beads; (lanes 10–13) immunoprecipitation with rabbit anti-Nam8p antibodies and Sepharose beads coupled to Staphyloccocus aureus protein A protein. The pre-mRNAs used as substrates were WT-B, ΔBP; and 5′ SSmut + ΔBP (see text). The band visible below Nam8p–protA in lanes 4–9 is nonspecifically precipitated, corresponds to the unidentified major cross-linked product seen in lanes 1,2, and comigrates in this gel conditions with the untagged version of Nam8p. In lane 3, extract buffer rather than extract was used to demonstrate that the products are not generated by intramolecular RNA cross-linking.

Figure 4

(A) Cross-linking of the Nam8 protein to site-specific-labeled pre-mRNAs. WT-B pre-mRNA was labeled in position −1, +5, and +13, respectively, and used in cross-linking reactions with Nam8p–protA. (Lane 1) Internally labeled pre-mRNA used as a positive control; (lanes 2–4) pre-mRNAs labeled in positions +5, +13, and −1, respectively. X is an unidentified cross-linked product. (B) Nam8p cross-links to an oligonucleotide spanning the 5′ splice site and following residues. RNA oligonucleotides protected at their 5′ end by two deoxynucleotides were used to cross-link Nam8p–protA. (Lane 1) oligonucleotide A overlapping the 5′ splice site and upstream exon sequence; (lane 2) oligonucleotide B spanning the 5′ splice site and following residues; (lane 3) oligonucleotide spanning the branchpoint region; (lane 4) oligonucleotide B; (lane 5) mutant oligonucleotide B carrying a substitution in the 5′ splice site sequence (GUAUaU).

Figure 5

Nam8p interaction with the sequence located downstream of the 5′ splice site affects splicing and splice site choice. (A) Representation of the reporters used in the experiments shown in B and C. An arrowhead indicates the 5′ splice site. (B) Plasmids carrying A, G, U, and H sequences were transformed in the wild-type (black bars) and Δnam8 (white bars) strains. Splicing efficiency, measured as mRNA versus pre-mRNA ratio (M/P; Pikielny and Rosbash 1985), was determined by primer extension and quantification with a PhosphorImager. All the measurements were normalized using the endogenous RP51A signal. (C) Primer extension analysis of the RNA produced by the different constructs in wild-type (WT) and nam8-disrupted (Δ) strains. Owing to multiple transcription initiation sites of the inducible GAL-CYC1 promoter, pre-mRNA and mRNAs appear as multiple bands. Numbers below each lane represent the splicing efficiency of the upstream site versus the downstream site quantified with a PhosphorImager. Samples 1–4 and 5–10 were run in different gels. (D) Nam8p–pre-mRNA interaction is modulated by the sequence following the 5′ splice site. (Top) Oligonucleotides BG and BU used to compete with oligonucleotide B for the cross-linking with Nam8p–protA in yeast extracts. (Bottom) Labeled oligonucleotide B was premixed with oligonucleotides BU (lanes 1–3) or BG (lanes 4–6) in molar ratios of 1:1 (lanes 1,4), 1:3 (lanes 2,5), and 1:10 (lanes 3,6). Subsequently, the different oligonucleotide mixtures were used in cross-linking reactions.

Figure 6

Model for the recognition of the 5′ splice site. The 5′ splice recognition results from a network of interactions established by the splicing machinery with various regions of the pre-mRNA namely CBC–cap structure, U1 snRNA–5′ splice site, and Nam8p region of nonconserved sequence in the intron. Therefore, this network of interactions is based on recognition of conserved (5′ splice site and cap) and nonconserved (downstream intronic region bound by Nam8p) elements in the pre-mRNA. Some interactions are essential, such as U1 snRNA–5′ splice site base pairing (Rosbash and Séraphin 1991). Other interactions contribute positively to stability of the complex but are not absolutely needed. For a pre-mRNA with canonical 5′ splice site, the presence of CBC or Nam8p is not required. CBC stabilizes the complex but is not absolutely necessary as the complexes assembled on uncapped RNA, although present in reduced levels, are functional (Colot et al. 1996; Lewis et al. 1996a). Commitment complexes assembled without Nam8p are stable (Fig. 2) and splicing of pre-mRNAs with canonical 5′ splice site occurs with normal efficiency in the Δnam8 strain (Fig. 1). However, Nam8p becomes indispensable for efficient splicing when the U1 snRNA–pre-mRNA pairing is weakened or with commitment complexes assembled on uncapped pre-mRNA.