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

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

Interplay of cis and trans regulatory mechanisms in the spliceosomal RNA helicase Brr2

RNA helicase Brr2 is implicated in multiple phases of pre-mRNA splicing and thus requires tight regulation. Brr2 can be auto-inhibited via a large N-terminal region folding back onto its helicase core and auto-activated by a catalytically inactive C-terminal helicase cassette. Furthermore, it can be regulated in trans by the Jab1 domain of the Prp8 protein, which can inhibit Brr2 by intermittently inserting a C-terminal tail in the enzyme's RNA-binding tunnel or activate the helicase after removal of this tail. Presently it is unclear, whether these regulatory mechanisms functionally interact and to which extent they are evolutionarily conserved. Here, we report crystal structures of Saccharomyces cerevisiae and Chaetomium thermophilum Brr2-Jab1 complexes, demonstrating that Jab1-based inhibition of Brr2 presumably takes effect in all eukaryotes but is implemented via organism-specific molecular contacts. Moreover, the structures show that Brr2 auto-inhibition can act in concert with Jab1-mediated inhibition, and suggest that the N-terminal region influences how the Jab1 C-terminal tail interacts at the RNA-binding tunnel. Systematic RNA binding and unwinding studies revealed that the N-terminal region and the Jab1 C-terminal tail specifically interfere with accommodation of double-stranded and single-stranded regions of an RNA substrate, respectively, mutually reinforcing each other. Additionally, such analyses show that regulation based on the N-terminal region requires the presence of the inactive C-terminal helicase cassette. Together, our results outline an intricate system of regulatory mechanisms, which control Brr2 activities during snRNP assembly and splicing

Long range allostery mediates cooperative adenine nucleotide binding by the Ski2 like RNA helicase Brr2

Brr2 is an essential Ski2 like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N terminal helicase cassette and a structurally similar but enzymatically inactive C terminal helicase cassette connected by a linker region. Both cassettes contain a nucleotide binding pocket, but it is unclear whether nucleotide binding in these two pockets is related. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and determine whether occupancy of one nucleotide binding site may influence nucleotide binding at the other cassette. Our results show that Brr2 exhibits high specificity for adenine nucleotides, with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C terminal cassette is more than two orders of magnitude higher than that of the active N terminal cassette, as determined by slow nucleotide release. Mutations at the intercassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Moreover, we found that abrogation of nucleotide binding at the C terminal cassette reduces nucleotide binding at the N terminal cassette 70 away. Molecular dynamics simulations identified structural communication lines that likely mediate these long range allosteric effects, predominantly across the intercassette interface. Together, our results reveal intricate networks of intramolecular interactions in the complex Brr2 RNA helicase, which fine tune its nucleotide affinities and which could be exploited to regulate enzymatic activity during splicin

Molecular Mechanism Underlying Inhibition of Intrinsic ATPase Activity in a Ski2 like RNA Helicase

RNA dependent NTPases can act as RNA RNA protein remodeling enzymes and typically exhibit low NTPase activity in the absence of RNA RNA protein substrates. How futile intrinsic NTP hydrolysis is prevented is frequently not known. The ATPase RNA helicase Brr2 belongs to the Ski2 like family of nucleic acid dependent NTPases and is an integral component of the spliceosome. Comprehensive nucleotide binding and hydrolysis studies are not available for a member of the Ski2 like family. We present crystal structures of Chaetomium thermophilum Brr2 in the apo, ADP bound, and ATP amp; 947;S bound states, revealing nucleotide induced conformational changes and a hitherto unknown ATP amp; 947;S binding mode. Our results in conjunction with Brr2 structures in other molecular contexts reveal multiple molecular mechanisms that contribute to the inhibition of intrinsic ATPase activity, including an N terminal region that restrains the RecA like domains in an open conformation and exclusion of an attacking water molecule, and suggest how RNA substrate binding can lead to ATPase stimulatio

Functions and regulation of the Brr2 RNA helicase during splicing

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 down-regulate Brr2 activity. Biochemical studies revealed an intricate cross-talk among the various cis- and trans-regulatory 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

The interaction of DNA repair factors ASCC2 and ASCC3 is affected by somatic cancer mutations

The ASCC3 subunit of the activating signal co integrator complex is a dual cassette Ski2 like nucleic acid helicase that provides single stranded DNA for alkylation damage repair by the amp; 945; ketoglutarate dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3 AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2 ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2 ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2 ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principle