The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

Abstract

The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.

Keywords: RNA helicase structure and function; X-ray crystallography; pre-mRNA splicing; remodeling of RNA–protein complexes; spliceosome catalytic activation.

Figure 1.

Structure of a FL Brr2–Jab1 complex. (A) Simulated annealing composite omit map covering the NTRs (magenta), N-terminal cassette (NC; gray), C-terminal cassette (CC; brown), and Jab1 (gold) in the FL Brr2–Jab1 crystal structure contoured at the 1.0 σ level. Molecular models are shown as ribbons. (B) Scheme of the NTR organization. Numbers above the scheme provide the domain borders, and angled arrows and numbers below the scheme indicate the starting positions of the NTR truncation variants of yeast Brr2. (C) Orthogonal views of the FL Brr2–Jab1 complex structure showing a ribbon of the NTR on the surface of the helicase cassettes and Jab1. The NTR is colored blue to red from the N terminus to the C terminus, the N-terminal cassette is colored dark gray, the C-terminal cassette is beige, and Jab1 is light gray. (D) Close-up views of the plug and IC clamp/PWI region of the NTR (magenta) showing cross-links identified in isolated Brr2. (Solid lines) Zero-length cross-links observed with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM); (dashed lines) cross-links observed with bis(sulfosuccinimidyl) suberate (BS3). The image is rotated 10° to the left about the vertical axis (top panel) and 45° to the top about the horizontal axis (bottom panel) compared with the top panel in B.

Figure 2.

Physiological effects of Brr2 NTR truncations. (A) Yeast growth assay comparing strains that produce the indicated Brr2 variants (FL, T1, T2, and T3) as the sole type of Brr2 protein. The T4 strain was not viable. (B) Western blot analysis monitoring the types and amounts of Brr2 variants relative to the U5 protein Snu114 produced in extracts of the strains shown in A. (C) Yeast growth assay comparing strains that produce the indicated Brr2 variants (FL, T1, T2, T3, and T4) in a FL Brr2 background. (D) Accumulation of intron-containing transcripts of the indicated genes (ACT1, RPL17B, and TEF4) relative to the amounts of intronless THD1 transcripts in yeast strains producing the indicated Brr2 variants (FL, T1, T2, and T3) as the sole type of Brr2 protein. Values and error bars represent means ± standard error of the mean (SEM) of two technical duplicates of at least four independent experiments. Significance (Student's unpaired t-test) is indicated by asterisks. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (E) Western blots of odd-numbered glycerol gradient fractions of extracts from yeast strains producing the indicated Brr2 variants (FL, T1, T2, and T3). (Left panels) Without pretreatment (− ATP). (Right panels) After preincubation with ATP (+ ATP). Proteins are identified at the right. (F) Northern blots of even-numbered glycerol gradient fractions. snRNPs contained in the fractions are indicated below the gels, and snRNAs are identified at the right. (G) Quantification of the data in F. The left panel shows the amounts of U4 plus U6 snRNAs in U4/U6 di-snRNP fractions relative to U4 plus U6 snRNAs in combined di-snRNP and tri-snRNP fractions (before and after the addition of ATP). The right panel shows the amounts of U5 snRNA in U5 snRNP fractions relative to U5 snRNA in combined U5 snRNP and tri-snRNP fractions (before and after the addition of ATP). (H) Solution hybridization analysis (probing for U4 snRNA) of RNAs extracted from the fractions in F and separated by nondenaturing PAGE. Migration positions of the U4/U6 duplex (syn. U4/U6; obtained by annealing of in vitro transcribed RNAs) and single-stranded U4 snRNA are shown at the right. Boil time was chosen so that part of U4/U6 remained associated.

Figure 3.

Effects of NTR truncations on yeast, human, and C. thermophilum Brr2 activities. (A) Apparent K_d values of the indicated Brr2 variants binding to U4/U6 di-snRNAs (organisms are indicated at the left, and protein variants are indicated below the graphs). Values represent means ± SEM of at least two independent experiments. Apparent K_d values were obtained by fitting quantified data from electrophoretic gel mobility shift assays (EMSA) titrations to a single exponential Hill function {fraction bound = A[protein]ⁿ/([protein]ⁿ + K_dⁿ), where A is the fitted maximum of RNA bound, and n is the Hill coefficient} (Ryder et al. 2008). (B) ATPase activities of the indicated Brr2 variants determined by thin-layer chromatography (organisms are indicated at the left, and protein variants are indicated below the graphs). (−) Intrinsic ATPase activities; (+) U4/U6-stimulated ATPase activities. Values represent means ± SEM of at least three independent experiments. (C) Quantification of U4/U6-unwinding time courses using the indicated Brr2 variants (organisms are indicated at the left, and protein variants are indicated below the graphs). Radioactive bands on gels monitoring the unwinding reactions were quantified by densitometry and fit to a first-order reaction [fraction unwound = A{1 − exp(−k_u t)}, where A is the amplitude of the reaction, k_u is the apparent first-order rate constant of unwinding, and t is time]. Data points and error bars represent means ± SEM of at least three independent experiments. (D–F) Close-up views of the structure of the FL Brr2–Jab1 complex illustrating the effects of NTR elements removed from the various truncations. (D) Orthogonal views illustrating steric hindrance of U4/U6 di-snRNA binding by the plug. (E) Conformational clamping of N-terminal and C-terminal cassettes by the IC clamp and the PWI domain. (F) Conformational clamping of the N-terminal cassette by the NC clamp. Views in the left panel of D and in E and F are the same as in the top of Figure 1B.

Figure 4.

Model for the function of the Brr2 NTR. The NTR (magenta) serves to stably anchor Brr2 to the tri-snRNP and autoinhibits Brr2 in isolation and in the tri-snRNP, possibly avoiding engagement of off-target RNAs. Upon formation of a precatalytic spliceosome by association of the tri-snRNP with the A complex, conformational changes are required for Brr2 to engage U4 snRNA. To achieve productive Brr2-mediated tri-snRNP disruption (separation of U4/U6 and release of U4), parts of the Brr2 NTR need to fasten portions of the tri-snRNP that have to remain associated. After U4/U6 unwinding, the NTR might rebind the helicase region of Brr2 and thus shut off the enzyme again.