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 disnRNP 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: pre-mRNA splicing; RNA helicase structure and function; remodeling of RNA-protein complexes; spliceosome catalytic activation; X-ray crystallography] Supplemental material is available for this article.Received September 14, 2015; revised version accepted November 13, 2015.Splicing entails the removal of noncoding sequences (introns) from primary transcripts and the concomitant ligation of neighboring coding regions (exons). It is mediated by a highly dynamic, multimegadalton RNA protein (RNP) molecular machine, the spliceosome, which consists of five small nuclear RNPs (snRNPs; U1, U2, U4, U5, and U6 in the case of the major spliceosome) and numerous non-snRNPs (Wahl et al. 2009). For each round of splicing, a spliceosome is assembled de novo on a substrate by the stepwise recruitment of snRNPs and nonsnRNPs. After assembly of a precatalytic complex, the spliceosome is catalytically activated and carries out the two consecutive steps of a splicing reaction before it is disassembled and its subunits are recycled. Each assembly, activation, catalysis, and disassembly step involves profound rearrangements of the spliceosomal RNP interaction networks, mediated predominantly by eight conserved superfamily 2 (SF2) NTPases/RNA helicases (Staley and Guthrie 1998). The most extensive rearrangements occur during spliceosome activation. In the precatalytic spliceosome, U4 and U6 snRNPs form a disnRNP by base-pairing of their snRNAs and are associated with U5 snRNP via protein-protein interactions. During spliceosome activation, the U5 snRNP-specific Brr2 helicase unwinds the U4/U6 ...

Functions and regulation of the Brr2 RNA helicase during splicing

“…Within this NTR, a helical "plug" domain and a PWI-like domain are interspersed among extended, intrinsically disordered regions. 54,55 In crystal structures of yeast Brr2 in complex with a C-terminal Jab1/ MPN-like (Jab1) domain of the Prp8 protein, the NTR runs along one entire flank of the helicase cassettes and extensively contacts both NC and CC. 55,56 Following »100 disordered N-terminal residues, the plug domain wedges between the HB and the RecA2 domains of the NC.…”

“…Chemical crosslinking and mass spectrometry indicated that in solution the PWI-like domain is further displaced toward and contacts the CC. 55 Immediately preceding the NC, an irregularly structured "N-terminal cassette clamp" (NC-clamp) tightly wraps around the NC and connects its RecA and WH domains.…”

“…Recent structural and functional studies showed that Brr2 activity is also regulated by its long NTR 55 and that this region is required for efficient splicing. 44,55 In vitro experiments assessing Brr2 activity upon stepwise NTR truncations revealed that the NTR down-regulates Brr2.…”

“…44,55 In vitro experiments assessing Brr2 activity upon stepwise NTR truncations revealed that the NTR down-regulates Brr2. 55 NTR-based auto-inhibition of Brr2 is implemented by at least 2 mechanisms.…”

“…44,55 In vitro experiments assessing Brr2 activity upon stepwise NTR truncations revealed that the NTR down-regulates Brr2. 55 NTR-based auto-inhibition of Brr2 is implemented by at least 2 mechanisms. First, comparison of structures of full-length Brr2 in complex with the Prp8 Jab1 domain 55,56 with the structure of Brr2 bound to U4/U6 disnRNA in the framework of a yeast tri-snRNP 12 indicated that the plug domain of the NTR upon folding back onto the helicase cassettes competes with accommodation of stem I of the substrate, which Brr2 has to unwind during spliceosome activation.…”