A noncanonical PWI domain in the N-terminal helicase-associated region of the spliceosomal Brr2 protein

Abstract

The spliceosomal RNA helicase Brr2 is required for the assembly of a catalytically active spliceosome on a messenger RNA precursor. Brr2 exhibits an unusual organization with tandem helicase units, each comprising dual RecA-like domains and a Sec63 homology unit, preceded by a more than 400-residue N-terminal helicase-associated region. Whereas recent crystal structures have provided insights into the molecular architecture and regulation of the Brr2 helicase region, little is known about the structural organization and function of its N-terminal part. Here, a near-atomic resolution crystal structure of a PWI-like domain that resides in the N-terminal region of Chaetomium thermophilum Brr2 is presented. CD spectroscopic studies suggested that this domain is conserved in the yeast and human Brr2 orthologues. Although canonical PWI domains act as low-specificity nucleic acid-binding domains, no significant affinity of the unusual PWI domain of Brr2 for a broad spectrum of DNAs and RNAs was detected in band-shift assays. Consistently, the C. thermophilum Brr2 PWI-like domain, in the conformation seen in the present crystal structure, lacks an expanded positively charged surface patch as observed in at least one canonical, nucleic acid-binding PWI domain. Instead, in a comprehensive yeast two-hybrid screen against human spliceosomal proteins, fragments of the N-terminal region of human Brr2 were found to interact with several other spliceosomal proteins. At least one of these interactions, with the Prp19 complex protein SPF27, depended on the presence of the PWI-like domain. The results suggest that the N-terminal region of Brr2 serves as a versatile protein-protein interaction platform in the spliceosome and that some interactions require or are reinforced by the PWI-like domain.

Functions and regulation of the Brr2 RNA helicase during splicing

“…Consistent with Brr2 held in an inhibited state, human tri-snRNP preparations are stable in the presence of ATP, 94 indicating that the human tri-snRNP structure represents a resting state of the tri-snRNP before delivery to the spliceosome. Furthermore, in full agreement with the suggested role as a multi-protein interaction device and as an anchor to the tri-snRNP, 54,55 the Brr2 NTR interacts with several other proteins in the human tri-snRNP, with the plug contacting Prp6 and the RH domain of Prp8 and the PWI domain interacting with Snu114 and Sad1 (Fig. 4B).…”

“…44,55 Shortening of the NTR led to unconventional, ATP-dependent, Brr2-mediated tri-snRNP disruption into U4/U6 di-snRNP and U5 snRNP, 55 and removal of the first 120 residues of the NTR in yeast Brr2 elicited an increased loss of U5 and U6 snRNAs during spliceosome activation. 44 Similar to the CC, Y2H 54 and pulldown studies 44 suggested that the NTR interacts with a large number of splicing factors implicated in different steps of splicing, consistent with the NTR supporting recruitment or anchoring of Brr2 to U5 snRNP and the tri-snRNP. 55 The diverse protein interactions of the NTR might help to reinforce, fine-tune or release Brr2 auto-inhibition via the NTR.…”

“…47 This organization would leave the Brr2 NTR available to engage in contacts with and possibly regulate the activity of Prp16. 54 However, the limited local resolution in the corresponding region of the Prp16-containing C complex structure does not unequivocally reveal the state of the NTR or the Jab1 domain.…”

“…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.…”

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

“…Residues 258-273, which we term the "intercassette (IC) clamp," adopt an extended conformation with a central 90°bend and laterally tape the helicase cassettes together. Residues 284-393 form a PWI domain (Absmeier et al 2015) that is connected to neighboring elements by flexible linkers and is held above the IC clamp by crystal packing contacts. Finally, residues 420-474 embrace the N-terminal cassette, resembling an "N-terminal cassette (NC) clamp."…”