The inactive C-terminal cassette of the dual-cassette RNA helicase BRR2 both stimulates and inhibits the activity of the N-terminal helicase unit

Abstract

The RNA helicase bad response to refrigeration 2 homolog (BRR2) is required for the activation of the spliceosome before the first catalytic step of RNA splicing. BRR2 represents a distinct subgroup of Ski2-like nucleic acid helicases whose members comprise tandem helicase cassettes. Only the N-terminal cassette of BRR2 is an active ATPase and can unwind substrate RNAs. The C-terminal cassette represents a pseudoenzyme that can stimulate RNA-related activities of the N-terminal cassette. However, the molecular mechanisms by which the C-terminal cassette modulates the activities of the N-terminal unit remain elusive. Here, we show that N- and C-terminal cassettes adopt vastly different relative orientations in a crystal structure of BRR2 in complex with an activating domain of the spliceosomal Prp8 protein at 2.4 Å resolution compared with the crystal structure of BRR2 alone. Likewise, inspection of BRR2 structures within spliceosomal complexes revealed that the cassettes occupy different relative positions and engage in different intercassette contacts during different splicing stages. Engineered disulfide bridges that locked the cassettes in two different relative orientations had opposite effects on the RNA-unwinding activity of the N-terminal cassette, with one configuration enhancing and the other configuration inhibiting RNA unwinding compared with the unconstrained protein. Moreover, we found that differences in relative positioning of the cassettes strongly influence RNA-stimulated ATP hydrolysis by the N-terminal cassette. Our results indicate that the inactive C-terminal cassette of BRR2 can both positively and negatively affect the activity of the N-terminal helicase unit from a distance.

Keywords: ATPase; RNA helicase; RNA splicing; allosteric regulation; dual-cassette Ski2-like helicase; intramolecular regulation; nucleic acid-dependent nucleotide triphosphatase (NTPase); pre-mRNA splicing; protein conformation; small nuclear ribonucleoprotein U5 subunit 200 (SNRNP200); superfamily 2 helicase.

Figure 1.

Structures of hBRR2^T1 and introduction of disulfide bridges. A, domain structure of hBRR2^T1. N-ext., N-terminal extension; RecA, RecA-like domains; WH, winged-helix; HB, helical bundle; HLH, helix-loop-helix; IG, immunoglobulin-like domains; L, intercassette linker; Sec63, Sec63 homology units. B, structure of the hBRR2^T1-hJab1^ΔCtail complex. hBRR2^T1 domains are colored as in A; hJab1^ΔCtail is shown as gold. C, structure of the hBRR2^T1 (34). hBRR2^T1 domains are colored as in A. D, zoom into the intercassette interface in the hBRR2^T1-hJab1^ΔCtail complex (region boxed in B). In this and the following figure panels, interacting residues are shown as sticks. Carbon is shown as the respective protein region. Blue, nitrogen; red, oxygen. Dashed lines, hydrogen bonds or salt bridges. Residues mutated to cysteines to introduce a disulfide bridge are labeled in magenta. Rotation symbols represent the view relative to B and C. E, 2F_o − F_c electron density (gray mesh; 1σ level) and F_o − F_c “omit” electron density (green mesh; 3σ level) around the engineered disulfide bridge in hBRR2^{T1-D534C/N1866C} (hBRR2^T1-SS-rot+). hBRR2^{T1-D534C/N1866C} was crystallized in complex with hJab1^ΔCtail. Mutated residues are labeled in magenta. F, zoom into the intercassette interface in isolated hBRR2^T1 (region boxed in C). G, 2F_o − F_c electron density (gray mesh; 1σ level) and F_o − F_c “omit” electron density (green mesh; 3σ level) around the engineered disulfide bridge in hBRR2^{T1-M641C/A1582C} (hBRR2^T1-SS-linear). hBRR2^{T1-M641C/A1582C} was crystallized in isolation. Mutated residues and a neighboring cysteine are labeled in magenta.

Figure 2.

Structural comparisons. Crystal structure of hBRR2^T1 (green) (34) superimposed on the hBRR2^T1-hJab1^ΔCtail structure (blue) and on the hBRR2 subunits (gray) in cryo-EM structures of the U4/U6·U5 tri-snRNP (39), spliceosomal pre-B (40), B (38), B^act (42), C (43), C* (41), and P complexes (44). Alignments were based on NC residues 1–1288. Yellow “mode” vectors indicate structural differences as distances after alignment (displacements of common Cα positions of the compared structures to the isolated hBRR2^T1 reference structure). rot+, apparent rotation of CC in the hBRR2^T1 structure to the CC in the hBRR2^T1-hJab1^ΔCtail structure defined as positive; rot−, opposite apparent rotation sense for the CC seen in all structures of spliceosomal complexes, most prominently in the B complex.

Figure 3.

Analysis of the hBRR2^T1 bearing engineered disulfide bridges. A, DSF-derived melting temperatures for hBRR2^T1 (gray), hBRR2^T1-SS-linear (green), and hBRR2^T1-SS-rot+ (blue) with and without the addition of the reducing agent, DTT, in size-exclusion chromatography buffer. Values represent means ± S.D. (error bars) of at least three independent experiments. ***, p ≤ 0.001. B, DSF-derived melting temperatures for hBRR2^T1 (gray), hBRR2^T1-SS-linear (green), and hBRR2^T1-SS-rot+ (blue) with and without the addition of the reducing agent, DTT, in 40 mm TRIS-HCl, pH 7.5, 50 mm NaCl, 0.5 mm MgCl₂. Values represent means ± S.D. of at least three independent experiments. *, p ≤ 0.05; **, p ≤ 0.01. C, mass-to-charge ratios (m/z) of disulfide-bridged peptides identified in LC-MS analyses. D, extracted ion chromatograms for disulfide bridge–containing peptides of hBRR2^T1-SS-linear present in the nonreduced/oxidized samples and missing in the reduced samples. One exemplary MS1 spectrum of the triply charged bridged peptide is shown. E, extracted ion chromatograms for disulfide bridge–containing peptides of hBRR2^T1-SS-rot+ present in the nonreduced/oxidized samples and missing in the reduced samples. One exemplary MS1 spectrum of the quadruply charged bridged peptide is shown. a.u., arbitrary units.

Figure 4.

Helicase activities. A–D, unwinding of U4*/U6 duplexes by the hBRR2^T1 variants indicated on the left of the gels under the conditions listed on top of the gels. A, enzyme alone, untreated. B, enzyme alone, treated with reducing agent. C, enzyme in complex with hJab1^ΔCtail. D, enzyme with the addition of ySnu13 to the RNA. Top, exemplary gels monitoring the unwinding reactions. U4*, radioactively labeled U4 snRNA. Bottom, quantification of the data at the top. Black, hBRR2^T1; green, hBRR2^T1-SS-linear; blue, hBRR2^T1-SS-rot+. Data points represent means ± S.D. (error bars) of at least three independent experiments.

Figure 5.

ATPase- and RNA-binding activities. A, RNA-stimulated ATPase rates of the indicated hBRR2^T1 variants (hBRR2^T1 (black), hBRR2^T1-SS-linear (green), and hBRR2^T1-SS-rot+ (blue)), analyzed via a radioactive, TLC-based ATPase assay. Data points represent means ± S.D. (error bars) of at least three independent experiments. **, p ≤ 0.01. B, fluorescence polarization–based analysis of RNA-binding activities of the indicated hBRR2^T1 variants (hBRR2^T1 (black), hBRR2^T1-SS-linear (green), and hBRR2^T1-SS-rot+ (blue)). The indicated K_d values were determined by fitting the data to a Hill equation, fraction bound = Ac_protⁿ/(c_protⁿ + K_dⁿ), in which A is the maximum of bound RNA, c_prot is the protein concentration, K_d is the dissociation constant, and n is the Hill coefficient. Values represent means ± S.E.M. (error bars) of at least three independent experiments. C, representative electrophoretic gel mobility shift assays monitoring binding of the indicated hBRR2^T1 variants (hBRR2^T1 (black), hBRR2^T1-SS-linear (green), and hBRR2^T1-SS-rot+ (blue)) to U4*/U6 di-snRNAs. U4*, radioactively labeled U4 snRNA. D, quantification of the data shown in C. The indicated K_d values were determined by fitting the quantified data to a Hill equation, fraction bound = Ac_protⁿ/(c_protⁿ + K_dⁿ), in which A is the maximum of bound RNA, c_prot is the protein concentration, K_d is the dissociation constant, and n is the Hill coefficient. Data points represent means ± S.E.M. of at least three independent experiments.