Substrate-assisted mechanism of RNP disruption by the spliceosomal Brr2 RNA helicase

Abstract

The Brr2 RNA helicase disrupts the U4/U6 di-small nuclear RNAprotein complex (di-snRNP) during spliceosome activation via ATPdriven translocation on the U4 snRNA strand. However, it is unclear how bound proteins influence U4/U6 unwinding, which regions of the U4/U6 duplex the helicase actively unwinds, and whether U4/U6 components are released as individual molecules or as subcomplexes. Here, we set up a recombinant Brr2-mediated U4/U6 disnRNP disruption system, showing that sequential addition of the U4/U6 proteins small nuclear ribonucleoprotein-associated protein 1 (Snu13), pre-mRNA processing factor 31 (Prp31), and Prp3 to U4/U6 di-snRNA leads to a stepwise decrease of Brr2-mediated U4/U6 unwinding, but that unwinding is largely restored by a Brr2 cofactor, the C-terminal Jab1/MPN domain of the Prp8 protein.Brr2-mediated U4/U6 unwinding was strongly inhibited by mutations in U4/U6 di-snRNAs that diminish the ability of U6 snRNA to adopt an alternative conformation but leave the number and kind of U4/U6 base pairs unchanged. Irrespective of the presence of the cofactor, the helicase segregated a Prp3-Prp31-Snu13-U4/U6 RNP into an intact Prp31-Snu13-U4 snRNA particle, free Prp3, and free U6 snRNA. Together, these observations suggest that Brr2 translocates only a limited distance on the U4 snRNA strand and does not actively release RNA-bound proteins. Unwinding is then completed by the partially displaced U6 snRNA adopting an alternative conformation, which leads to dismantling of the Prp3-binding site on U4/U6 di-snRNA but leaves the Prp31-and Snu13-binding sites on U4 snRNA unaffected. In this fashion, Brr2 can activate the spliceosome by stripping U6 snRNA of all precatalytic binding partners, while minimizing logistic requirements for U4/U6 di-snRNP reassembly after splicing.pre-mRNA splicing | RNA helicase | RNP remodeling | small nuclear ribonucleoprotein particle | spliceosome catalytic activation R NA helicases form a large family of nucleotide triphosphatedependent enzymes, many of which can unwind RNA duplexes in vitro (1, 2). However, in vivo, these enzymes typically act on RNA-protein complexes (RNPs) and can also exhibit other activities, including RNA annealing (3, 4), RNA clamping (5), displacement of RNA-bound proteins (6), or displacement of RNAbound RNPs (7). Often, the precise functions of RNA helicases are unclear because their in vivo substrates are mostly unknown. Consequently, the RNP remodeling activities of RNA helicases have typically been studied using model RNPs that these helicases would not encounter naturally (6, 7).Eight superfamily (SF) 2 RNA helicases are required for premRNA splicing by the spliceosome (8). During every splicing reaction, these enzymes drive and control multiple compositional and conformational RNP remodeling events that accompany the initial assembly of an inactive spliceosome, its catalytic activation, its splicing catalysis, and its disassembly (8, 9). The most profound helicase-mediated rearrangements occur during spliceosome activation. In the precatalyt...

Functions and regulation of the Brr2 RNA helicase during splicing

“…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. 29 In full agreement with this structural analysis, RNA binding studies showed that the NTR has no effect on singlestranded RNA binding by Brr2, but inhibits binding of substrates that additionally include a duplex resembling U4/U6 stem I. 56 Second, the NC-clamp connects the RecA-like and WH domains of the active NC and locks the RecA1 and the RecA2 domains in a conformation not conducive to ATP hydrolysis.…”

“…On the other hand, isolated Brr2 is a comparatively weak helicase, 25,40,41 and its U4/U6 di-snRNA substrate is stabilized by extensive base pairing and bound proteins, [12][13][14]29,42,43 suggesting that the helicase may also depend on specific activation to efficiently unwind the U4/U6 duplex at the right time.…”

“…53,88 Recent studies suggested that Jab1-mediated stimulation of Brr2 might be required at the stage of spliceosome catalytic activation, during which Brr2 needs to unwind U4/U6 di-snRNA decorated with U4/U6 proteins; addition of the U4/U6 di-snRNP proteins Snu13, Prp31 and Prp3 led to a stepwise decrease in unwinding activity, which could be efficiently restored upon addition of Jab1 DC . 29 On the molecular level, Jab1-mediated Brr2 activation may in part be due to bridging of the N-terminal HB and RecA2 domains by the proximal portion of the Jab1 tail (Fig. 2B), via which the U4 snRNA strand might be more efficiently entrapped at the NC.…”

“…20 The most dramatic rearrangements occur during spliceosome activation, where the Prp28 helicase aids in the displacement of U1 snRNA from the 5SS, 8,23,24 followed by Brr2 unwinding the U4 and U6 snRNAs [25][26][27] and leading to displacement of U4 snRNA and U4/U6-bound proteins. 28,29 These processes allow U6 to engage in alternative interactions with the 5SS and U2 snRNA, as well as to form a catalytically important internal stem-loop, thereby building up the spliceosome's active site. [30][31][32][33] Here we review results from structural and functional studies, which have begun to shed light on an intricate network of regulatory principles controlling the Brr2 RNA helicase, and discuss how Brr2 regulation may influence splicing fidelity and alternative splicing.…”

A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex

“…Indeed, it has already been shown that deletion of U6 nt 1–54, which would prevent telestem formation, results in a decrease in Brr2 unwinding rate (46). More recently, it has been postulated that a competing structure in U6 actively participates in releasing of U4/U6 di-snRNP proteins and U4 unwinding in a model system (47). This competing structure may initially be the telestem, followed by the ISL.…”