Structural elucidation of a PRP8 core domain from the heart of the spliceosome

Ritchie, Dustin B.; Schellenberg, M.J.; Gesner, Emily M.; Raithatha, Sheetal A.; Stuart, David A.; MacMillan, Andrew M.

doi:10.1038/nsmb.1505

Cited by 66 publications

(84 citation statements)

References 32 publications

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“…Prp8 has been cross-linked to the 5Ј splice site and appears to functionally interact with both splice sites during splicing (23,24). Recent crystallographic data show that a region of Prp8 that cross-links to the 5Ј splice site folds into an RNase H domain (25)(26)(27), which is remarkable, because RNase H domains catalyze the cleavage of phosphodiester bonds in RNA by using a 2-metal-ion mechanism (28). However, because the Prp8 RNase H domain structure contains only 2 of the 4 conserved carboxylates required for metal ion coordination, it is not able to coordinate metal ions by itself.…”

mentioning

confidence: 99%

The spliceosome as ribozyme hypothesis takes a second step

Butcher

2009

Proc. Natl. Acad. Sci. U.S.A.

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T he spliceosome is a massive assembly of 5 RNAs and many proteins that, together, catalyze precursor-mRNA (pre-mRNA) splicing. The splicing mechanism involves 2 steps: (i) cleavage of the 5Ј exon-intron phosphodiester bond and formation of a new 2Ј-5Ј bond resulting in an intron ''lariat,'' and (ii) exon ligation (Fig. 1A). This 2-step phosphoryl transfer mechanism is suspiciously identical to the reaction catalyzed by the group II self-splicing introns, which are ribozymes. The precedent for a ribozymecatalyzed splicing mechanism, combined with the fact that the spliceosome has an RNA core that has been highly conserved for Ͼ1 billion years, led researchers to hypothesize that the spliceosome may use an RNA-based catalytic mechanism (1). However, the spliceosome as ribozyme hypothesis has been exceedingly difficult to prove, for 2 major reasons. First, the spliceosome contains many proteins that are essential for splicing (2). Second, the uncatalyzed rate of RNA ligation in vitro is significant when measured over many hours (3). This latter point makes it difficult to establish whether an inefficient reaction is actually caused by a catalytic rate enhancement or occurs spontaneously because of template-driven proximity. This is a particular concern for 1-step phosphoryl transfer reactions. Furthermore, it is possible to serendipitously obtain unrelated activities from RNAs, perhaps because a significant region of RNA conformational space retains some degree of catalytic activity (4). Therefore, it is important to be very cautious when interpreting an inefficient RNA-based reaction. The relevance of using engineered, protein-free RNA systems to study splicing has been recently argued (5, 6). In a recent issue of PNAS, Valadkhan et al. (7) demonstrated that an RNA complex derived from the spliceosome can perform a reaction that resembles splicing (Fig. 1B). This finding is significant because the reaction appears to be identical to the second step of splicing and is Ϸ10-fold more efficient than previous studies that used similar ).An interesting aspect of this new reaction is that the first step seems to be generated via hydrolysis rather than by a 2Ј-5Ј branching reaction (Fig. 1B). This finding is different from spliceosomecatalyzed pre-mRNA splicing (Fig. 1 A), but has precedence among the group II ribozmes (11). The second step of the reaction, however, results in exon ligation and formation of a new 5Ј-3Ј bond, just as in splicing. This reaction also has sequence requirements that closely parallel those of the spliceosome and requires magnesium. These data support, but do not prove, the idea that the reaction is related to pre-mRNA splicing. In future experiments, it will be important to investigate this reaction further for its mechanistic parallels to splicing. Currently, it is not clear how the substrate RNAs are coordinated in the reaction, and whether or not the system faithfully recapitulates the interactions in the spliceosome remains to be determined. Another important question concerns how the cata...

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confidence: 99%

The spliceosome as ribozyme hypothesis takes a second step

Butcher

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Prp8 is the largest and most highly conserved spliceosomal protein 80 and forms a scaffold, on which the catalytic core of the spliceosome is assembled. 39,81 At its C-terminus, Prp8 comprises an RNase H-like (RH) domain [82][83][84] followed by a Jab1 domain, 85,86 both of which can regulate Brr2 function. The RH domain can bind to the single-stranded region of the U4 snRNA neighboring U4/U6 stem I (the U4 central domain), thus hindering Brr2 entry and inhibiting the helicase by substrate competition.…”

Section: Brr2 Regulation Via Trans-acting Factorsmentioning

confidence: 99%

Functions and regulation of the Brr2 RNA helicase during splicing

2016

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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

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“…Intriguingly, recent high resolution structural studies of a fragment of Prp8 close to its C-terminal domain has indicated the presence of a degenerate RNase H-like motif in this protein. [82][83][84][85] While the general topology of the domain is conserved, only one of the three catalytic residues at the active site is present. This residue, an aspartate, is involved in coordinating both catalytic metals in the active site of canonical RNase H domains.…”

Section: Insights Into Spliceosomal Catalysis In the Years To Comementioning

confidence: 99%

“…Mutation of this residue in Prp8 in yeast in one study resulted in no detectable growth defect, while in another it caused protein misfolding and led to a lethal phenotype. 82,83 No metal binding was observed by the region corresponding to the active site of the RNase H domain when the crystals were grown in up to 200 mM MgCl 2 , and it bound RNA with a Kd of 20 to over 200 µM, depending on the RNA species tested. [82][83][84] What made this RNase H-like domain particularly interesting was that the amino acids corresponding to its active site are positioned adjacent to residues in Prp8 that were previously shown to crosslink to the 5' splice site in precatalytic spliceosomes.…”

Section: Insights Into Spliceosomal Catalysis In the Years To Comementioning

confidence: 99%

“…82,83 No metal binding was observed by the region corresponding to the active site of the RNase H domain when the crystals were grown in up to 200 mM MgCl 2 , and it bound RNA with a Kd of 20 to over 200 µM, depending on the RNA species tested. [82][83][84] What made this RNase H-like domain particularly interesting was that the amino acids corresponding to its active site are positioned adjacent to residues in Prp8 that were previously shown to crosslink to the 5' splice site in precatalytic spliceosomes. 86 However, the efficiency of the formation of this crosslink was decreased as the precatalytic spliceosomes progressed to become catalytically active spliceosomal complexes.…”

Section: Insights Into Spliceosomal Catalysis In the Years To Comementioning

confidence: 99%

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