Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly

Abstract

Base-pairing of U4 and U6 snRNAs during di-snRNP assembly requires large-scale remodeling of RNA structure that is chaperoned by the U6 snRNP protein Prp24. We investigated the mechanism of U4/U6 annealing in vitro using an assay that enables visualization of ribonucleoprotein complexes and faithfully recapitulates known in vivo determinants for the process. We find that annealing, but not U6 RNA binding, is highly dependent on the electropositive character of a 20 Å-wide groove on the surface of Prp24. During annealing, we observe the formation of a stable ternary complex between U4 and U6 RNAs and Prp24, indicating that displacement of Prp24 in vivo requires additional factors. Mutations that stabilize the U6 'telestem' helix increase annealing rates by up to 15-fold, suggesting that telestem formation is rate-limiting for U4/U6 pairing. The Lsm2-8 complex, which binds adjacent to the telestem at the 3' end of U6, provides a comparable rate enhancement. Collectively, these data identify domains of the U6 snRNP that are critical for one of the first steps in assembly of the megaDalton U4/U6.U5 tri-snRNP complex, and lead to a dynamic model for U4/U6 pairing that involves a striking degree of evolved cooperativity between protein and RNA.

Figure 1.

Prp24 binds U6 RNA with high affinity and specificity. (A) Top: primary structure of Prp24. White regions are disordered and were deleted from the crystallization construct as previously described (24). RRM4 is an occluded RRM (oRRM), with terminal α-helices masking its β-sheet face (22,24). Unless indicated otherwise, all assays herein used full-length protein and RNA. Bottom: molecular architecture of the U6 snRNP core. Regions of U6 RNA (black) that form U4/U6 Stem I and Stem II are highlighted in green and purple, respectively. (B) Schematic of U6 RNA annealing to U4 RNA to form U4/U6. (C) Native gel analysis of full-length Prp24 binding to U6 (top) and U4 (bottom) RNAs.

Figure 2.

Prp24 catalyzes annealing of U4 and U6 RNAs, and remains bound to product di-RNA. (A) Two-color gel demonstrating tight binding of Prp24 to Cy3-U6 (lanes 5 and 6) and weaker binding of Prp24 to Cy5-U4 (lanes 2 and 3). Co-localization of Cy3 and Cy5 fluorescence in the presence of U4 and U6 (lanes 7 and 9) shows that the slowest-migrating species (orange) contains both RNAs, and the increased mobility upon treatment with proteinase K (lanes 8 and 10) shows that the di-snRNA retains bound Prp24. Annealing reactions were incubated at 30°C for 90 min prior to loading. (B) Time-dependent formation of U4/U6, using radiolabeled U4 snRNA and unlabeled U6 and Prp24. Control reactions in lanes 1–4 were incubated for 90 min. (C) Quantification of Prp24-dependent annealing from proteinase K treated lanes (10–14) in (B) compared to protein-independent annealing (lane 4 in B).

Figure 3.

The in vitro annealing assay recapitulates in vivo phenotypes of U6 RNA substitutions. (A) Secondary structure of U6 RNA bound to Prp24 (24), with the boxed region of the ISL shown with substitutions at right. (B) Time course of U4/U6 annealing for reactions containing the indicated substitutions in U6.

Figure 4.

The components of the U6 snRNP core efficiently promote annealing. (A) Full-length (residues 1–444) and truncated (residues 34–400) Prp24 catalyze U4/U6 annealing with similar rates. Lane 1 contains U4 RNA with full-length Prp24 and lane 2 contains U4 with truncated Prp24. Lanes 3–18 contain full-length U6 RNA (unlabeled), U4 RNA and the indicated form of Prp24 incubated 0–90 min at 30°C. Samples in lanes 7–10 and 15–18 were treated with proteinase K before electrophoresis. (B) Truncated U6 RNA (30–101) with two stabilizing mutations (U100C/U101C) anneals more rapidly than full length U6 (1–112). Lanes 1 and 2 contain only U4 and full-length U6, while lanes 3 and 4 contain U4 and truncated U6. Lanes 2 and 4 are proteinase K treated. Lanes 5–20 correspond to lanes 3–18 of Panel A, but with full length Prp24 and full length or truncated U6 as indicated. (C) Annealing timecourses for experiments shown in Panels A and B.

Figure 5.

Mutations within the telestem affect annealing rate. (A) Secondary structure of U6 showing the position of telestem-stabilizing (U100C/U101C) or destabilizing (U37C) mutations. Base specific contacts with Prp24 are highlighted in red. (B) Annealing gel comparing full-length wild-type U6 to U6-U100C/U101C (top) or U6-U37C (bottom). A control lane containing the annealing reaction at 90 min that was not proteinase K-treated precedes each time course of annealing reactions treated with proteinase K. (C) Annealing timecourses for reactions containing wild-type, U100C/U101C and U37C variants of full-length U6.

Figure 6.

Stabilization of the U6 telestem results in significant rate enhancement of U4/U6 annealing. (A) Secondary structure of the lower telestem, with tested mutations boxed. (B) U4/U6 annealing rates of mutant U6 RNAs. Mutations predicted to destabilize the telestem (numbers 1–6) or to be isoenergetic with wild type RNA (nos. 7–9) had little effect on annealing rate, while those predicted to be slightly stabilizing (nos. 10–12) or stabilizing (nos. 13–17) significantly increased the annealing rate. (C) Protein-free annealing of U4 and U6 RNAs at 90 min. The percentage of U4 incorporated into U4/U6 is shown. Mutations that enhance the protein-free annealing rate are marked with an asterisk. (D) Electrophoretic mobility of U4/U6•Prp24 species after 90 min of annealing.

Figure 7.

Reduction of net positive charge in the electropositive groove decreases the rate of U4/U6 annealing without affecting U6 RNA-binding. (A) Electrostatic surface of Prp24, contoured from +8 kT/e (blue) to −8 kT/e (red). Positions of mutations within the electropositive groove (blue text) and outside the groove (green text) are indicated. (B) Annealing gel showing a single time point (90 min) for wild-type Prp24 and each of the nine mutants. Samples were divided into native (odd lanes) and proteinase K-treated (even lanes). Substitutions in the electropositive groove (blue text) reduce the amount of U4/U6 after 90 min, while substitutions outside this region (green text) do not. (C) Rate of U4/U6 formation over time for wild-type (black), constructs with an electropositive groove mutation (blue text) and constructs with mutations outside of this region (green text). (D) Wild-type and ‘6mut’ Prp24 are equally active for U6 binding. Labeled U6 (5 nM Cy5-U6) was supplemented with 500 nM unlabeled U6, and the binding of stoichiometric amounts of Prp24 was monitored. (E) Native gel analysis of U6-Prp24 binding (top) and U4-Prp24 binding (bottom) using wild-type and mutant full-length Prp24. ‘6mut’ refers to the presence of K50A/K77A/K78A/R81A/R131A/R134A mutations. (F) Binding curves (simple one site binding model) of wild-type versus mutant protein with full-length U6 and U4.

Figure 8.

The Lsm2–8 ring enhances U4/U6 annealing. (A) Two-color native gel with Cy5-U4 (red) and Cy3-U6 (green). The Lsm2–8 ring alone binds U4 and U6 RNAs (lanes 4 and 8), but exhibits strong cooperative binding with Prp24 only on U6 RNA (cf. lanes 3 and 7). Lsm2–8 enhances Prp24-mediated annealing (cf. lanes 10 and 12; yellow shows colocalized RNAs), but does not catalyze annealing alone (lane 14). Annealing reactions were incubated at 30°C for 90 minutes prior to loading. (B) Time-dependent formation of U4/U6, using radiolabeled U4 snRNA and unlabeled U6, Prp24 (50 nM) and Lsm2–8 (50 nM). (C) Model for the rate enhancement conferred by stabilizing the telestem or by inclusion of the Lsm2–8 ring. The C-terminal decapeptide of Prp24 (the SNFFL box) interacts with Lsm2–8.