Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2

Abstract

Brr2 is a DExD/H-box helicase responsible for U4/U6 unwinding during spliceosomal activation. Brr2 contains two helicase-like domains, each of which is followed by a Sec63 domain with unknown function. We determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308. This, together with sequence similarities between Brr2’s helicase-like domains and domains 1–3 of Hel308, led us to hypothesize that Brr2 contains two consecutive Hel308-like modules (Hel308-I and II). Our structural model and mutagenesis data suggest that Brr2 shares a similar helicase mechanism with Hel308. We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo. We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2/Prp8-CTR complex to U4/U6. Our results have important implications for the mechanism and regulation of Brr2’s activity.

Functions and regulation of the Brr2 RNA helicase during splicing

“…The S1087L RP33 exchange in human Brr2 reduces RNA affinity as well as ATPase and helicase activities 51 and RP33-like N1104L and R1107L exchanges in yeast Brr2 are associated with reduced ATPdependent U4/U6 unwinding in tri-snRNP preparations. 60 Mutation of a Brr2 element equivalent to the Hel308 separator loop resulted in reduction or loss of cell viability, 41,52 in line with a similar duplex disruption mechanism as in Hel308. However, in a yeast U4/U6 U5 tri-snRNP structure, 12 Brr2 is loaded on the U4/U6 di-snRNA with the putative separator loop distant from the U4/U6 duplex portion to be unwound and instead with an edge of the RecA2 domain abutting the end of this duplex region (Fig.…”

“…In contrast, in DEAH/RHA helicases an element equivalent to the separator loop (termed "5 0 HP") has been suggested to mainly control access to the single-stranded RNA binding site. 58,59 Comparative modeling 41,51,52 and a recent electron cryomicroscopic (cryo-EM) structure of a yeast U4/U6 U5 trisnRNP 12 showed that Brr2 engages its U4/U6 di-snRNA substrate in a similar manner as Hel308. A single-stranded region of the U4 snRNA is threaded through the central tunnel of the NC between the RNA-binding motifs of the RecA and HB domains, with 3 0 -portions of the RNA extending toward the CC (Fig.…”

“…This idea is in line with previous proposals of Brr2 functioning as a landing pad for multiple other helicases, which might sequentially interact in similar fashion with the C-terminal cassette. 40,41,52,63,64 Based on the observations that a temperature-sensitive yeast Brr2 variant (E909K), encoded by the slt22-1 allele, is synthetically lethal with mutations in U2 or U6 snRNAs that affect the stability or conformation of U2/U6 helix II, and that the ATPase activity of this variant is no longer stimulated by a U2/ U6 duplex, it was proposed that Brr2 might proofread U2/U6 interactions. 45 Furthermore, different cross-linking profiles of wt Brr2 and a variant exhibiting a residue exchange (G858R) in the NC 5 0 HP/separator loop with U6 snRNA 46 together with the latter variant exhibiting a second step defect and showing synthetic lethality with step 2 mutations in Slu7, Prp18 and Prp16 46,49 suggest that Brr2 participates in a transient opening of the catalytic core between the 2 steps of splicing, which is characterized by the intermittent disruption of U6-5SS and U2-U6 interactions.…”

“…2A,B). 41,52 Only the N-terminal helicase cassette (NC) is active in nucleotide hydrolysis and RNA unwinding, 51 and the enzymatic activity of the NC alone is required for splicing in vivo. 27 The C-terminal cassette (CC) is still able to bind ATP, 51 but it contains noncanonical residues in the ATP binding and hydrolysis motifs, which abrogate the ATPase activity.…”

Contribution of SNRNP200 sequence variations to retinitis pigmentosa

“…Further, the Prp8-CTR facilitates the binding of the Brr2/Prp8-CTR complex to U4/U6 in yeast. 41 In this current study, we have identified a potentially pathogenic mutation, p.R1779H, which is located between the second helicase CTR and sec-63 domains. It is the first candidate mutation found in the hel308-II module for RP.…”