Intrinsically Disordered Protein Ntr2 Modulates the Spliceosomal RNA Helicase Brr2.

Precursor messenger RNA splicing is mediated by the spliceosome, a large and dynamic molecular machine composed of five small nuclear RNAs and numerous proteins. Many spliceosomal proteins are predicted to be intrinsically disordered or contain large disordered regions, but experimental validation of these predictions is scarce, and the precise functions of these proteins are often unclear. Here, we show via circular dichroism spectroscopy, dynamic light scattering, and NMR spectroscopy that the yeast spliceosomal disassembly factor Ntr2 is largely intrinsically disordered. Peptide SPOT analyses, analytical size-exclusion chromatography, and surface plasmon resonance measurements revealed that Ntr2 uses an N-terminal region to bind the C-terminal helicase unit of the Brr2 RNA helicase, an enzyme involved in spliceosome activation and implicated in splicing catalysis and spliceosome disassembly. NMR analyses suggested that Ntr2 does not adopt a tertiary structure and likely remains disordered upon complex formation. RNA binding and unwinding studies showed that Ntr2 downregulates Brr2 helicase activity in vitro by modulating the fraction of helicase molecules productively bound to the RNA substrate. Our data clarify the nature of a physical link between Brr2 and Ntr2, and point to the possibility of a functional Ntr2-Brr2 interplay during splicing.

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.

ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts.

Multicomponent biological systems perform a wide variety of functions and are crucially important for a broad range of critical health and disease states. A multitude of applications in contemporary molecular and synthetic biology rely on efficient, robust and flexible methods to assemble multicomponent DNA circuits as a prerequisite to recapitulate such biological systems in vitro and in vivo. Numerous functionalities need to be combined to allow for the controlled realization of information encoded in a defined DNA circuit. Much of biological function in cells is catalyzed by multiprotein machines typically made up of many subunits. Provision of these multiprotein complexes in the test-tube is a vital prerequisite to study their structure and function, to understand biology and to develop intervention strategies to correct malfunction in disease states. ACEMBL is a technology concept that specifically addresses the requirements of multicomponent DNA assembly into multigene constructs, for gene delivery and the production of multiprotein complexes in high-throughput. ACEMBL is applicable to prokaryotic and eukaryotic expression hosts, to accelerate basic and applied research and development. The ACEMBL concept, reagents, protocols and its potential are reviewed in this contribution.

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

The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.

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.