Brr2解旋U4/U6 SnRNAs的调控机制

前体mRNA(precursor messager RNA,pre-mRNA)剪接是去除内含子和将外显子彼此连接形成成熟mRNA的过程。剪接过程在一个呈动态变化的大核糖核蛋白(ribonucleoprotein, RNP)复合体,即剪接体催化作用下完成。DExD/H-box RNA解旋酶在剪接体组装、激活及解聚过程中都发挥着重要作用。Brr2(bad response to refrigeration 2)这种DExD/H-box RNA解旋酶是构成U5稳定的亚单位。Brr2含有两个串联解旋酶盒结构,在剪接体激活中负责U4/U6的解旋,还参与剪接体催化及解聚过程,因此Brr2在剪接过程中必需具备严格的调控机制。在剪接过程中,Prp8的C端包含两个连续的RNase H域和Jab1/MPN域,能够正负调控Brr2活性。Snu114在调节Brr2活性中具有非常重要的作用。此外,Brr2通过C端解旋酶盒(C-terminal cassette, CC)与N末端域(N-terminal region)进行分子内的自我活性调节。本文综述了近年来在Brr2的分子间和分子内活性调节机制的研究进展,这些不同的调节机制协同作用才确保真核生物pre-mRNA可变剪接的保真性。     

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.     

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.     

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.     

Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae

Mapping of functional protein interactions will help in understanding conformational rearrangements that occur within large complexes like spliceosomes. Because the U5 snRNP plays a central role in pre-mRNA splicing, we undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5 snRNP-associated proteins. DExH-box protein Brr2p interacted specifically with five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p, second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is required for splicing at low temperatures. Co-immunoprecipitation experiments confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p to the spliceosome. We provide evidence that the prp8-1 allele disrupts an interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex unwinding through another interaction with Brr2p. The interactions of Brr2p with a wide range of proteins suggest a particular function for the C-terminal half, bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p promotes other RNA rearrangements, acting synergistically with other spliceosomal proteins, including the structurally related Prp2p and Prp16p. Overall, these protein interaction studies shed light on how splicing factors regulate the order of events in the large spliceosome complex.     

Base pairing with U6atac snRNA is required for 5' splice site activation of U12-dependent introns in vivo.

The minor U12-dependent class of eukaryotic nuclear pre-mRNA introns is spliced by a distinct spliceosomal mechanism that requires the function of U11, U12, U5, U4atac, and U6atac snRNAs. Previous work has shown that U11 snRNA plays a role similar to U1 snRNA in the major class spliceosome by base pairing to the conserved 5'' splice site sequence. Here we show that U6atac snRNA also base pairs to the 5'' splice site in a manner analogous to that of U6 snRNA in the major class spliceosome. We show that splicing defective mutants of the 5'' splice site can be activated for splicing in vivo by the coexpression of compensatory U6atac snRNA mutants. In some cases, maximal restoration of splicing required the coexpression of compensatory U11 snRNA mutants. The allelic specificity of mutant phenotype suppression is consistent with Watson-Crick base pairing between the pre-mRNA and the snRNAs. These results provide support for a model of the RNA-RNA interactions at the core of the U12-dependent spliceosome that is strikingly similar to that of the major class U2-dependent spliceosome.     

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 Å 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 splicing.     

A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome.

Prior to the chemical steps of mRNA splicing, the extensive base-pairing interaction between the U4 and U6 spliceosomal snRNAs is disrupted. Here, we use a mutational analysis in yeast to demonstrate a conserved base-pairing interaction between the U6 and U2 snRNAs that is mutually exclusive with the U4-U6 interaction. In this novel pairing, conserved sequences in U6 interact with a sequence in U2 that is immediately upstream of the branch point recognition region. Remarkably, the residues in U6 that can be consequently juxtaposed with the intron substrate include those that have been proposed previously to be catalytic. Both the first and second steps of splicing are inhibited when this base-paired structure is mutated. These observations, together with the high conservation of the U2-U6 structure, lead us to propose that it might be a component of the spliceosomal active site.     

Splicing of a divergent subclass of AT-AC introns requires the major spliceosomal snRNAs.

AT-AC introns constitute a minor class of eukaryotic pre-mRNA introns, characterized by 5''-AT and AC-3'' boundaries, in contrast to the 5''-GT and AG-3'' boundaries of the much more prevalent conventional introns. In addition to the AT-AC borders, most known AT-AC introns have highly conserved 5'' splice site and branch site sequence elements of 7-8 nt. Intron 6 of the nucleolar P120 gene and intron 2 of the SCN4A voltage-gated skeletal muscle sodium channel are AT-AC introns that have been shown recently to be processed via a unique splicing pathway involving several minor U snRNAs. Interestingly, intron 21 of the same SCN4A gene and the corresponding intron 25 of the SCN5A cardiac muscle sodium channel gene also have 5''-AT and AC-3'' boundaries, but they have divergent 5'' splice site and presumptive branch site sequences. Here, we report the accurate in vitro processing of these two divergent AT-AC introns and show that they belong to a functionally distinct subclass of AT-AC introns. Splicing of these introns does not require U12, U4atac, and U6atac snRNAs, but instead requires the major spliceosomal snRNAs U1, U2, U4, U5, and U6. Previous studies showed that G --> A mutation at the first position and G --> C mutation at the last position of a conventional yeast or mammalian GT-AG intron suppress each other in vivo, suggesting that the first and last bases participate in an essential non-Watson-Crick interaction. Our results show that such introns, hereafter termed AT-AC II introns, occur naturally and are spliced by a mechanism distinct from that responsible for processing of the apparently more common AT-AC I introns.     

Splicing factor Prp8 governs U4/U6 RNA unwinding during activation of the spliceosome

The pre-mRNA 5' splice site is recognized by the ACAGA box of U6 spliceosomal RNA prior to catalysis of splicing. We previously identified a mutant U4 spliceosomal RNA, U4-cs1, that masks the ACAGA box in the U4/U6 complex, thus conferring a cold-sensitive splicing phenotype in vivo. Here, we show that U4-cs1 blocks in vitro splicing in a temperature-dependent, reversible manner. Analysis of splicing complexes that accumulate at low temperature shows that U4-cs1 prevents U4/U6 unwinding, an essential step in spliceosome activation. A novel mutation in the evolutionarily conserved U5 snRNP protein Prp8 suppresses the U4-cs1 growth defect. We propose that wild-type Prp8 triggers unwinding of U4 and U6 RNAs only after structurally correct recognition of the 5' splice site by the U6 ACAGA box and that the mutation (prp8-201) relaxes control of unwinding.     

Metal binding and substrate positioning by evolutionarily invariant U6 sequences in catalytically active protein-free snRNAs

We have previously shown that a base-paired complex formed by two of the spliceosomal RNA components, U6 and U2 small nuclear RNAs (snRNAs), can catalyze a two-step splicing reaction that depended on an evolutionarily invariant region in U6, the ACAGAGA box. Here we further analyze this RNA-catalyzed reaction and show that while the 5′ and 3′ splice site substrates are juxtaposed and positioned near the ACAGAGA sequence in U6, the role of the snRNAs in the reaction is beyond mere juxtaposition of the substrates and likely involves the formation of a sophisticated active site. Interestingly, the snRNA-catalyzed reaction is metal dependent, as is the case with other known splicing RNA enzymes, and terbium(III) cleavage reactions indicate metal binding by the U6/U2 complex within the evolutionarily conserved regions of U6. The above results, combined with the structural similarities between U6 and catalytically critical domains in group II self-splicing introns, suggest that the base-paired complex of U6 and U2 snRNAs is a vestigial ribozyme and a likely descendant of a group II-like self-splicing intron.     

The ribosomal translocase homologue Snu114p is involved in unwinding U4/U6 RNA during activation of the spliceosome

Snu114p is a yeast U5 snRNP protein homologous to the ribosomal elongation factor EF-2. Snu114p exhibits the same domain structure as EF-2, including the G-domain, but with an additional N-terminal domain. To test whether Snu114p in the spliceosome is involved in rearranging RNA secondary structures (by analogy to EF-2 in the ribosome), we created conditionally lethal mutants. Deletion of this N-terminal domain (snu114ΔN) leads to a temperature-sensitive phenotype at 37°C and a pre-mRNA splicing defect in vivo. Heat treatment of snu114ΔN extracts blocked splicing in vitro before the first step. The snu114ΔN still associates with the tri-snRNP, and the stability of this particle is not significantly impaired by thermal inactivation. Heat treatment of snu114ΔN extracts resulted in accumulation of arrested spliceosomes in which the U4 RNA was not efficiently released, and we show that U4 is still base paired with the U6 RNA. This suggests that Snu114p is involved, directly or indirectly, in the U4/U6 unwinding, an essential step towards spliceosome activation.     

Sm and Sm-like proteins belong to a large family: identification of proteins of the U6 as well as the U1, U2, U4 and U5 snRNPs.

Several small nuclear RNAs (snRNAs), including the spliceosomal U1, U2, U4 and U5 snRNAs, are associated with Sm proteins. These eight small proteins form a heteromeric complex that binds to snRNAs and plays a major role in small nuclear ribonucleoprotein (snRNP) biogenesis and transport. These proteins are also a major target for autoantibodies in the human disease systemic lupus erythematosus. By sequence comparison I have shown that all the known Sm proteins share a common structural motif which might explain their immunological cross-reactivity. Database searches using this motif uncovered a large number of Sm-like proteins from plants, animals and fungi. These proteins have been grouped in at least 13 different subfamilies. Genes encoding divergent yeast members were cloned and used to produce tagged fusion proteins. Some of these proteins are canonical Sm proteins as they associate with the yeast U1, U2, U4/U6 and U5 snRNAs. Surprisingly, one Sm-like protein was found to be a component of the U6 snRNP. These findings have implications for the structure of the Sm protein complex, spliceosomal snRNP evolution, snRNA transport and modification as well as the involvement of Sm proteins in systemic lupus erythematosus.     

FBP21’s C-Terminal Domain Remains Dynamic When Wrapped around the c-Sec63 Unit of Brr2 Helicase

Based on our recent finding that FBP21 regulates human Brr2 helicase activity involved in the activation of the spliceosomal B-complex, we investigated the structural and dynamic contribution of FBP21 to the interaction. By using NMR spectroscopy, we could show that the 50 C-terminal residues of FBP21 (FBP21^326–376), which are sufficient to fully form the interaction with the C-terminal Sec63 unit of Brr2 (Brr2^C-Sec63), adopt a random-coil conformation in their unbound state. Upon interaction with Brr2^C-Sec63, 42 residues of FBP21^326–376 cover the large binding site on Brr2^C-Sec63 in an extended conformation. Short charged motifs are steering complex formation, still allowing the bound state to retain dynamics. Based on fragment docking in combination with experimental restraints, we present models of the complex structure. The FBP21^326–376/Brr2^C-Sec63 interaction thus presents an example of an intrinsically disordered protein/ordered-protein interaction in which a large binding site provides high specificity and, in combination with conformational disorder, displays a relatively high affinity.     

Identification of novel snRNA-specific Sm proteins that bind selectively to U2 and U4 snRNAs in Trypanosoma brucei

In eukaryotes the seven Sm core proteins bind to U1, U2, U4, and U5 snRNAs. In Trypanosoma brucei, Sm proteins have been implicated in binding both spliced leader (SL) and U snRNAs. In this study, we examined the function of these Sm proteins using RNAi silencing and protein purification. RNAi silencing of each of the seven Sm genes resulted in accumulation of SL RNA as well as reduction of several U snRNAs. Interestingly, U2 was unaffected by the loss of SmB, and both U2 and U4 snRNAs were unaffected by the loss of SmD3, suggesting that these snRNAs are not bound by the heptameric Sm complex that binds to U1, U5, and SL RNA. RNAi silencing and protein purification showed that U2 and U4 snRNAs were bound by a unique set of Sm proteins that we termed SSm (specific spliceosomal Sm proteins). This is the first study that identifies specific core Sm proteins that bind only to a subset of spliceosomal snRNAs.     

Functional stabilization of an RNA recognition motif by a noncanonical N-terminal expansion

RNA recognition motifs (RRMs) constitute versatile macromolecular interaction platforms. They are found in many components of spliceosomes, in which they mediate RNA and protein interactions by diverse molecular strategies. The human U11/U12-65K protein of the minor spliceosome employs a C-terminal RRM to bind hairpin III of the U12 small nuclear RNA (snRNA). This interaction comprises one side of a molecular bridge between the U11 and U12 small nuclear ribonucleoprotein particles (snRNPs) and is reminiscent of the binding of the N-terminal RRMs in the major spliceosomal U1A and U2B″ proteins to hairpins in their cognate snRNAs. Here we show by mutagenesis and electrophoretic mobility shift assays that the β-sheet surface and a neighboring loop of 65K C-terminal RRM are involved in RNA binding, as previously seen in canonical RRMs like the N-terminal RRMs of the U1A and U2B″ proteins. However, unlike U1A and U2B″, some 30 residues N-terminal of the 65K C-terminal RRM core are additionally required for stable U12 snRNA binding. The crystal structure of the expanded 65K C-terminal RRM revealed that the N-terminal tail adopts an α-helical conformation and wraps around the protein toward the face opposite the RNA-binding platform. Point mutations in this part of the protein had only minor effects on RNA affinity. Removal of the N-terminal extension significantly decreased the thermal stability of the 65K C-terminal RRM. These results demonstrate that the 65K C-terminal RRM is augmented by an N-terminal element that confers stability to the domain, and thereby facilitates stable RNA binding.     

The EF-G-like GTPase Snu114p regulates spliceosome dynamics mediated by Brr2p, a DExD/H box ATPase

Binding of a pre-mRNA substrate triggers spliceosome activation, whereas the release of the mRNA product triggers spliceosome disassembly. The mechanisms that underlie the regulation of these rearrangements remain unclear. We find evidence that the GTPase Snu114p mediates the regulation of spliceosome activation and disassembly. Specifically, both unwinding of U4/U6, required for spliceosome activation, and disassembly of the postsplicing U2/U6.U5.intron complex are repressed by Snu114p bound to GDP and derepressed by Snu114p bound to GTP or nonhydrolyzable GTP analogs. Further, similar to U4/U6 unwinding, spliceosome disassembly requires the DExD/H box ATPase Brr2p. Together, our data define a common mechanism for regulating and executing spliceosome activation and disassembly. Although sequence similarity with EF-G suggests Snu114p functions as a molecular motor, our findings indicate that Snu114p functions as a classic regulatory G protein. We propose that Snu114p serves as a signal-dependent switch that transduces signals to Brr2p to control spliceosome dynamics.