Rearrangement of competing U2 RNA helices within the spliceosome promotes multiple steps in splicing

Abstract

Nuclear pre-messenger RNA (pre-mRNA) splicing requires multiple spliceosomal small nuclear RNA (snRNA) and pre-mRNA rearrangements. Here we reveal a new snRNA conformational switch in which successive roles for two competing U2 helices, stem IIa and stem IIc, promote distinct splicing steps. When stem IIa is stabilized by loss of stem IIc, rapid ATP-independent and Cus2p-insensitive prespliceosome formation occurs. In contrast, hyperstabilized stem IIc improves the first splicing step on aberrant branchpoint pre-mRNAs and rescues temperature-sensitive U6-U57C, a U6 mutation that also suppresses first-step splicing defects of branchpoint mutations. A second, later role for stem IIa is revealed by its suppression of a cold-sensitive allele of the second-step splicing factor PRP16. Our data expose a spliceosomal progression cycle of U2 stem IIa formation, disruption by stem IIc, and then reformation of stem IIa before the second catalytic step. We propose that the competing stem IIa and stem IIc helices are key spliceosomal RNA elements that optimize juxtaposition of the proper reactive sites during splicing.

Figure 1.

(A) A 5′ portion of yeast U2 snRNA showing stem IIa or stem IIc forms and various mutations used in this study that promote either form. (B) Secondary structure of interactions between U2 and U6 snRNAs required for first transesterification: Mutation at U6–U57 to A or C is indicated.

Figure 2.

U2 stem IIa formation regulates the rate, Cus2p, and ATP dependence of prespliceosome formation. (A) U2-ΔCC rapidly forms Cus2p and ATP-independent prespliceosomes at identical rates in vitro. Shown is native gel analysis of a 0- to 40-min time course of spliceosome assembly on pre-RP51A in U6-depleted cus2Δ (lanes 1–5), CUS2+ (lanes 6–10), CUS2+ U2-ΔCC (lanes 11–20), or cus2Δ U2-ΔCC (lanes 21–30) splicing extracts in the presence of AMP-PCP (lanes 1–5,16–20,26–30) or ATP (lanes 6–10,11–15,22–25). Samples were taken at 0 min (lanes 1,6,11,16,21,26), 1 min (lanes 2,7,12,17,22,27), 5 min (lanes 3,8,13,18,23,28), 20 min (lanes 4,9,14,19,24,29), and 40 min (lanes 5,10,15,20,25,30) after pre-RP51A addition. (CC1) Commitment complex 1; (CC2) commitment complex 2; (PS) prespliceosomes. (B) HA-Cus2p preferentially coimmunoprecipitates U2 snRNA that favors U2–stem IIc. Extracts prepared from strains containing HA-Cus2p and U2 (lanes 1,2), U2-ΔCC (lanes 3,4), or U2–G53A (lanes 5,6) were incubated with anti-HA antibody 12CA5 prebound to Protein A Sepharose and washed with 50 mM NET buffer. Bound RNA was extracted and used as a template for primer extension with U2 snRNA-specific primer. Lanes are 1/10 total (1,3,5) and coimmunopreciptated fractions (2,4,6). Below is average percentage of U2 bound and standard deviations from three independent coimmunopreciptations.

Figure 3.

Stabilized U2 stem IIa decreases, but hyperstabilized U2 stem IIc increases, the first step of splicing on C256A and A259C branch site mutant ACT1-CUP1 reporter in vivo. (A) ACT1-CUP1 reporter pre-mRNA indicating 5′ splice site and branch site mutants used in this study. (B) Copper growth of strains expressing ACT1-CUP1 reporters (indicated) and either wild-type (lanes 1,2), U2-ΔCC (lanes 3,4), or U2–IIc+ (lanes 5,6). Lanes 1, 3, and 5 show growth on 0.15 mM Cu++, while lanes 2, 4, and 6 indicate the highest copper concentration allowing growth. Boxes indicate strains where growth was observed on higher (black) or lower (white) Cu++ when compared with wild-type U2 snRNA. (C) Primer extension analysis of RNAs from strain DS4D containing wild type (lanes 1–3), C256A (lanes 4–7), A259C (lanes 8–10), A259G, and one of U2 (lanes 1,4,7), U2-ΔCC (lanes 2,5,8), or U2–IIC+ (lanes 3,6,9). Primer complementary to the 3′ exon was used to visualize pre-mRNA, mRNA, and lariat intermediate indicated from top to bottom of gel, respectively. Lane 0 (10) are RNA from isogenic yeast lacking the ACT1-CUP1 reporter. (D) Quantitation of results from three independent experiments, an example of which is presented in B. Dark bars show first-step efficiency calculated as M + L/(P + M + L) and light bars show second-step efficiency calculated as M/(M + L) as per Query and Konarska (2004). Percent splicing efficiency is normalized to the first-step efficiency of wild-type pre-ACT1-CUP1 in wild-type strain set at 100.

Figure 4.

Stem IIc interacts functionally with U6–U57C, while cus2Δ in combination with a Prp5p ATPase mutant increases the first step of splicing on A259C pre-mRNA. (A) Hyperstabilized stem IIc can rescue temperature-sensitive U6–U57C. Growth of fourfold serial dilutions of strain YHM118 carrying temperature-sensitive U6–U57C at restrictive temperature (37°C) is shown with one of U2, U2-ΔCC, or U2–IIc+ snRNAs. (B) A Prp5p ATPase mutant can increase the first step of splicing on A259C ACT1-CUP1 pre-mRNA, and cus2Δ can enhance this. Primer extension analysis of RNAs from DS4D containing wild type (lanes 1–4) or A259C (lanes 5–8) and Prp5p (lanes 1,2,5,6) or Prp5-GNTp (lanes 3,4,7,8) and CUS2+ (lanes 1,3,5,7) or cus2Δ (lanes 2,4,6,8). Lane 0 is control RNA as in Figure 3. Product designations are as in Figure 3. (C) Quantitation of results from three independent experiments, an example of which is presented in A. Calculations of percent efficiency is as in Figure 3.

Figure 5.

Genetic interactions between U2–stem IIa, U6–U57A, and Prp16p, a DExD/H protein involved in first-to-second step transition suggest a role for U2–IIa at this transition. U2-ΔCC and U2-24, but not U2–IIc+, can suppress the cold-sensitive phenotype of prp16-302 at 18°C. This rescue is enhanced when U2-ΔCC and U6–U57A (also a suppressor of prp16-302) (McPheeters 1996) or U2-24 and U6–U57A are coexpressed. Growth of fourfold dilutions of strain YHM187 at restrictive temperature (18°C) is shown with one of U2, U2-ΔCC, or U2-24 as the sole source of U2 snRNA and U6, or U6–U57A as the sole source of U6 snRNA.

Figure 6.

A model showing the roles of U2–stem IIa, U2–stem IIc in steps leading to the first-to-second step transition in pre-mRNA splicing. Formation of U2–stem IIa as the rate-limiting and ATP-determining product (I) leading to prespliceosomes (II) (Fig. 2). This step is catalyzed by ATPase activity of Prp5p and regulated by Cus2p. (III) A role for formed U2–stem IIc, and not U2–stem IIa, during the first step (Fig. 3). (IV) A role for reformed U2–stem IIa between first and second step either as an important scaffold for PRP16 action at catalytic site or a direct PRP16 substrate in disrupting U2–stem IIc (see also Fig. 5).