Common Design Principles in the Spliceosomal RNA Helicase Brr2 and in the Hel308 DNA Helicase

Abstract

Brr2 is a unique DExD/H box protein required for catalytic activation and disassembly of the spliceosome. It contains two tandem helicase cassettes that both comprise dual RecA-like domains and a noncanonical Sec63 unit. The latter may bestow the enzyme with unique properties. We have determined crystal structures of the C-terminal Sec63 unit of yeast Brr2, revealing three domains, two of which resemble functional modules of a DNA helicase, Hel308, despite lacking significant sequence similarity. This structural similarity together with sequence conservation between the enzymes throughout the RecA-like domains and a winged helix domain allowed us to devise a structural model of the N-terminal active cassette of Brr2. We consolidated the model by rational mutagenesis combined with splicing and U4/U6 di-snRNA unwinding assays, highlighting how the RecA-like domains and the Sec63 unit form a functional entity that appears suitable for unidirectional and processive RNA duplex unwinding during spliceosome activation and disassembly.

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.…”

“…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.…”

“…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.…”

“…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.…”