Characterization of specific protein-RNA complexes associated with the coupling of polyadenylation and last-intron removal

Abstract

Polyadenylation and splicing are highly coordinated on substrate RNAs capable of coupled polyadenylation and splicing. Individual elements of both splicing and polyadenylation signals are required for the in vitro coupling of the processing reactions. In order to understand more about the coupling mechanism, we examined specific protein-RNA complexes formed on RNA substrates, which undergo coupled splicing and polyadenylation. We hypothesized that formation of a coupling complex would be adversely affected by mutations of either splicing or polyadenylation elements known to be required for coupling. We defined three specific complexes (A(C)', A(C), and B(C)) that form rapidly on a coupled splicing and polyadenylation substrate, well before the appearance of spliced and/or polyadenylated products. The A(C)' complex is formed by 30 s after mixing, the A(C) complex is formed between 1 and 2 min after mixing, and the B(C) complex is formed by 2 to 3 min after mixing. A(C)' is a precursor of A(C), and the A(C)' and/or A(C) complex is a precursor of B(C). Of the three complexes, B(C) appears to be a true coupling complex in that its formation was consistently diminished by mutations or experimental conditions known to disrupt coupling. The characteristics of the A(C)' complex suggest that it is analogous to the spliceosomal A complex, which forms on splicing-only substrates. Formation of the A(C)' complex is dependent on the polypyrimidine tract. The transition from A(C)' to A(C) appears to require an intact 3'-splice site. Formation of the B(C) complex requires both splicing elements and the polyadenylation signal. A unique polyadenylation-specific complex formed rapidly on substrates containing only the polyadenylation signal. This complex, like the A(C)' complex, formed very transiently on the coupled splicing and polyadenylation substrate; we suggest that these two complexes coordinate, resulting in the B(C) complex. We also suggest a model in which the coupling mechanism may act as a dominant checkpoint in which aberrant definition of one exon overrides the normal processing at surrounding wild-type sites.

FIG. 1.

Diagram of the 392-nt splicing and PA RNA substrate (WT Sp/PA), also called MXSVL (23). It is composed of two separable cassettes, one for splicing and one for PA. Each cassette can function on its own in splicing and PA reactions, respectively. When together, they can function in coupled splicing and PA under the appropriate conditions (see text). The splicing cassette is derived from the adenovirus major late region; the 5′-SS (5′), 3′-SS (3′), and the PPT are shown. The PA cassette contains the PA signal from the SV40 late coding region, the SVLPA, SV40 nt 2531 to 2731. The structural features of the SVLPA are shown: these include the USEs, the AAUAAA, the cleavage and PA site (An), and the DSEs (see the text for details). When the substrate is spliced, polyadenylated exons 1 and 2 are joined.

FIG. 2.

(A) Wild-type and mutant Sp/PA substrates used in these studies. At the top is the diagram of the WT Sp/PA substrate detailed in Fig. 1; below are diagrams of various mutant substrates. These include the following: −3′-SS Sp/PA, which cannot splice and is debilitated in coupling, due to a point mutation in the 3′-SS; −AUA Sp/PA, which cannot polyadenylate and is debilitated in coupling, due to mutation of the AAUAAA sequence and linker substitution mutagenesis of the USEs; and −PPT Sp/PA, which cannot splice and is debilitated in coupling, due to mutation of the PPT. The bottom diagram shows three separate LSMs in the DSEs of the SVLPA, DM1, DM2, and DM3. The exact sequences mutated are shown in panel B. DM2 and DM4 debilitate PA and coupling; DM3 has little effect on either process and is included as a positive control. (B) The sequence of the downstream region of the SVLPA starting at AAUAAA and showing the cleavage and PA site (arrow), the first U-rich region (U; double underline), the G-rich region (G; single underline), and the second U-rich region (U′; single underline). The thick underlined areas show the sequence mutated by linker substitution in the three downstream mutations DM2, DM3, and DM4.

FIG. 3.

Time course studies of protein-RNA complex formation on the WT Sp/PA substrate and a nonspecific RNA substrate containing no processing elements. The complexes noted are a nonspecific complex (N), which forms rapidly on either substrate, and three specific complexes (A_C, A_C′, and B_C), which form only on the WT Sp/PA complex. The subscript C (for “coupled”) is used to denote these complexes as forming on a coupled Sp/PA substrate and to differentiate them from spliceosomal complexes, which form on splicing-only substrates.

FIG. 4.

Comparison of the rate of complex formation with the appearance of spliced and polyadenylated products. Reaction mixtures were split in half, with one-half being used for complex formation analysis and the other being analyzed for processing products: polyadenylated but not spliced (S−A+); spliced but not polyadenylated (S+A−); and spliced and polyadenylated (S+A+), which represents the fully processed product. See the text for details.

FIG. 5.

Comparison of splicing and PA complexes with known spliceosomal complexes and correlation of complexes with coupling. The top panel shows time course studies (in minutes) of complex formation on the splicing cassette (WT Sp only) from the WT Sp/PA substrate (See Fig. 1) as well as mutant splicing cassettes with a point mutation in the 3′-SS (−3′ SS) and LSM of the PPT (−PPT). The well-characterized spliceosomal complexes E, H, A, B, and C are noted. The bottom panel shows similar complex formation time courses (in minutes) with the WT Sp/PA substrate and mutant Sp/PA substrates (Fig. 2A) containing a point mutation in the 3′-SS (−3′ SS) and mutation of the PPT (−PPT). The last four lanes show complex formation on the isolated PA cassette from the WT Sp/PA substrate (Fig. 1).

FIG. 6.

Correlation of complexes with coupling: effects of DSE mutations on complex formation. The panel shows time course studies (in minutes) of complex formation on Sp/PA substrates, including the wild-type substrate (WT Sp/PA) and substrates with LSMs in the downstream region of the SVLPA: DM2, DM3, and DM4 (Fig. 1 and 2 and the text).

FIG. 7.

Effects of Mg²⁺ concentration on complex formation on the WT Sp/PA substrate. The time course reactions (in minutes) were identical, except in terms of the magnesium concentrations (1 or 4 mM).

FIG. 8.

Complex formation on the WT Sp/PA substrate in the presence of specific cold competitor (Comp.) RNAs. None, no competitor; Nonspecific, a nonspecific RNA containing no processing elements; Sp. Cas., wild-type splicing cassette; Sp. Cas. −3′SS, splicing cassette with a point mutation in the 3′-SS; Sp. Cas. −PPT, splicing cassette with an LSM in the PPT; PA Cas., PA cassette.