Functions and regulation of the Brr2 RNA helicase during splicing

Abstract

Pre-mRNA splicing entails the stepwise assembly of an inactive spliceosome, its catalytic activation, splicing catalysis and spliceosome disassembly. Transitions in this reaction cycle are accompanied by compositional and conformational rearrangements of the underlying RNA-protein interaction networks, which are driven and controlled by 8 conserved superfamily 2 RNA helicases. The Ski2-like helicase, Brr2, provides the key remodeling activity during spliceosome activation and is additionally implicated in the catalytic and disassembly phases of splicing, indicating that Brr2 needs to be tightly regulated during splicing. Recent structural and functional analyses have begun to unravel how Brr2 regulation is established via multiple layers of intra-and inter-molecular mechanisms. Brr2 has an unusual structure, including a long N-terminal region and a catalytically inactive C-terminal helicase cassette, which can auto-inhibit and auto-activate the enzyme, respectively. Both elements are essential, also serve as protein-protein interaction devices and the N-terminal region is required for stable Brr2 association with the tri-snRNP, tri-snRNP stability and retention of U5 and U6 snRNAs during spliceosome activation in vivo. Furthermore, a C-terminal region of the Prp8 protein, comprising consecutive RNase H-like and Jab1/MPN-like domains, can both up-and downregulate Brr2 activity. Biochemical studies revealed an intricate cross-talk among the various cis-and transregulatory mechanisms. Comparison of isolated Brr2 to electron cryo-microscopic structures of yeast and human U4/U6 U5 tri-snRNPs and spliceosomes indicates how some of the regulatory elements exert their functions during splicing. The various modulatory mechanisms acting on Brr2 might be exploited to enhance splicing fidelity and to regulate alternative splicing. KEYWORDSPre-mRNA splicing; regulation of pre-mRNA splicing; remodeling of RNAprotein complexes; RNA helicase structure, function and regulation; spliceosome catalytic activation

RNA and Proteins: Mutual Respect

“…Rearrangements of pre-mRNA and snRNAs to prepare and position them for catalysis are mainly accomplished by protein helicases 62 . There are eight such type SF2 helicases that associate with the spliceosome along the reaction pathway 63, 64 . These ATP-dependent RNA helicases are not sequence specific; they can unwind any RNA duplex.…”

“…Rather, their specific targets appear to be defined by where and when they associate with the spliceosome. The Brr2 helicase is particularly critical in the transformation of pre-spliceosome intermediates 64– 67 . Brr2 is unusual: it has two helicase domains (only one is active) and a long (450-amino-acid) N-terminal domain 64, 65, 68, 69 .…”

“…The goal of Brr2 in the tri-snRNP is to maintain stasis. As biochemistry experiments of Brr2 show 64 , there is a plug domain at the N-terminus of Brr2’s long N-terminal region (NTR). This plug folds back over the entrance of the helicase to block access of the U4/U6 snRNA duplex to the active site of Brr2.…”

Identification of proteins associated with splicing factors Ntr1, Ntr2, Brr2 and Gpl1 in the fission yeast Schizosaccharomyces pombe

“…Nrl1 also suppresses homologous recombination-dependent R-loop formation and targets transcripts with cryptic introns to form heterochromatin domains at developmental genes and retrotransposons in the fission yeast Schizosaccharomyces pombe [3,4]. In this Extra View, we provide an analysis of the spliceosome complex and validate the specificity of interactions between Nrl1 and spliceosome by purifying TAP-tagged splicing factors Ntr1, Ntr2, Brr2 and SPAC20H4.06c, a G-patch domain-containing protein orthologous to human GPATCH1 protein, which we named Gpl1 (GPATCH1 like 1) [5,6]. We also discuss insights into the protein interaction network of splicing factors including a possible functional interaction between G-patch domain-containing protein Gpl1 and putative RNA helicase SPAC20H4.09.…”

Positive and negative intra-molecular modulation in a dual-cassette RNA helicase

“…Thus, regulation of the activities of the spliceosomal helicases may have a major impact on gene expression and regulation, in particular in higher eukaryotes where alternative splicing is pervasive (26). A number of specific regulatory principles have been delineated for Brr2 (27). For example, besides the two helicase cassettes, Brr2 comprises a large N-terminal region of about 400 residues that can fold back onto the helicase cassettes and autoinhibit the enzyme by substrate competition and conformational clamping (28).…”