Journal of Molecular Biology
Prp8 in a Reduced Spliceosome Lacks a Conserved Toggle that Correlates with Splicing Complexity across Diverse Taxa
Graphical Abstract
Introduction
Eukaryotes contain a split-gene structure in which coding exon sequences are interrupted by non-coding intron sequences. Intron excision and exon ligation take place through two sequential transesterifications catalyzed by the spliceosome (reviewed in Ref. [1]). This large RNA–protein assembly consists of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs), each containing a unique snRNA and associated proteins, that assemble on the pre-mRNA substrate. In the first step of splicing, a conserved adenosine within the intron, selected by virtue of base pairing between the U2 snRNA and intron branch sequence, performs a nucleophilic displacement at the 5′ splice site yielding the free 5′ exon and lariat intermediate. In the second step, attack of the 5′ exon at the 3′ splice site produces the ligated exons and lariat intron as products.
Spliceosome assembly is a multi-step process that proceeds, following snRNP biogenesis, through discrete complexes and involves significant structural and conformational rearrangements [2], [3]. Recruitment of the U4/U6•U5 tri-snRNP to a complex containing pre-mRNA associated with the U1 and U2 snRNPs involves the displacement of the former at the 5′ splice site by the U6 snRNA to yield the B complex. Unwinding of a U4/U6 snRNA duplex within the B complex, mediated by the Brr2 helicase, allows formation of a U6 internal stem loop and U2/U6 snRNA structure to produce the pre-catalytic activated spliceosome referred to as Bact. Together these U6 and U2/U6 snRNA structures comprise the active site of the spliceosome that catalyzes the two splicing transesterifications. Rearrangement leads to the B* complex, which catalyzes the first step and results in the C complex. Further remodeling yields the C* complex where the second step occurs followed by a transition to the post-catalytic P complex [4], [5], [6].
The highly conserved U5 snRNP protein Prp8 (61% identity between Saccharomyces cerevisiae and humans [7]) plays key roles in the regulation of spliceosome assembly and transitions through the two catalytic steps while remaining intimately associated with the spliceosomal core [8], [9], [10]. Prp8 contains reverse transcriptase-like, endonuclease-like, and RNase H-like (RH) domains [11] believed to be derived from an ancestral group II intron maturase [12], [13] as well as a C-terminal Jab-MPN domain that regulates the activity of Brr2 [14], [15], [16].
A large number of mutant prp8 alleles related to spliceosome activation and the two transesterification steps have been characterized in yeast (reviewed in Ref. [17]). A clustering of these alleles led us and others to structurally and functionally characterize the domain of Prp8 corresponding to the RH fold ([18], [19], [20]; Fig. 1A, B). Sequence analysis was unable to predict the RH fold due to a 17-amino-acid insertion between the first and second β-strands of the RH domain. Crystallographically, we observed two conformations of the RH domain: in the closed conformation, the insertion corresponds to an anti-parallel β-hairpin; while in the second, open conformation, the hairpin is disrupted to yield a loop translated ~ 45° back with respect to the closed structure ([21]; Fig. 1A). A hydrated magnesium ion observed in the second conformation, bound in a site conserved from RNase H, possibly acts to stabilize the open structure.
X-ray structural analyses of Prp8 RH domain mutants affecting either step of splicing, or U4/U6 snRNA unwinding, as characterized in yeast and yeast splicing extracts, revealed a stabilization or destabilization of one of the two RH domain conformations [21]. This transition between two conformations corresponded to favoring the first step of splicing (at the expense of the second) or the second step of splicing (at the expense of the first), as shown in both in vitro and yeast reporter splicing assays, suggesting a switch mechanism to regulate splicing. Recently, it has been proposed that the two conformations of the RH domain represent a toggle mechanism related to proofreading, where the closed conformation favors accurate but inefficient splicing and the open conformation favors inaccurate but efficient splicing [22]. This toggle may relate to different conformations of the U2 snRNA, involving stem IIc (catalytically active) and the alternate, mutually exclusive stem IIa (catalytically inactive; [23]). Deletion of the 17 amino acids comprising the RH domain hairpin is lethal in S. cerevisiae [20], [22].
The best-characterized spliceosomes are those from humans and budding yeast. Although these spliceosomes are highly conserved, there are significant differences between them. In order to more fully understand the function of the RH domain of Prp8, we have examined it structurally in an organism distantly related to budding yeast and humans and also carried out a comparative bioinformatic analysis across diverse eukaryotic taxa. As a starting point, we chose Prp8 from the red alga Cyanidioschyzon merolae due to evidence of a considerably less complex spliceosome in this organism compared to either humans or yeast [24]. The genome of C. merolae contains only 27 introns in a genome with 4803 genes [25]. A computational and biochemical analysis revealed the presence of a greatly reduced spliceosome containing only 40 core proteins and completely lacking the U1 snRNA and its associated proteins [24]. Here we report our x-ray structural analysis of the RH domain of C. merolae Prp8 (CmPrp8) showing that while the RNase H fold is maintained, the highly conserved β-hairpin is absent. We have extended this analysis across diverse taxa showing that the presence or absence of the hairpin corresponds with intron number in agreement with the observation that among red algal genomes spliceosome complexity is associated with a greater number of introns [26]. We also show that the loss of this hairpin corresponds with the predicted absence of factors that associate with the RH hairpin that act at multiple steps of spliceosome assembly or progression supporting its role as a regulatory feature in complex spliceosomes.
Section snippets
Identification of the Prp8 RH domain of C. merolae and comparison across taxa
Protein components of the C. merolae (CM) splicing machinery were previously identified by searching the National Center for Biotechnology Information database using sequences of two or more species to retrieve the C. merolae homolog [24]. This analysis confirmed the existence of a CM Prp8 ortholog. In examining the alignment of CM and human Prp8 sequences, we noted a gap of 13 amino acids corresponding to the β-hairpin insertion in the human and yeast RH domains. To determine if this loss was
Discussion
Recent studies have provided detailed static representations of multiple steps in spliceosome assembly, activation, and catalysis (reviewed in Ref. [46]). Comparison of these spliceosomes on a structural level will yield insights into the mechanism of splicing. However, given the high conservation of the spliceosomal machinery, sequence comparison across species is also a useful approach to dissecting spliceosome function in light of these structures. These comparisons should inform our
Identification, cloning, and expression of the Prp8 RH domain from C. merolae
A cDNA corresponding to amino acids 1848–2065 of C. merolae Prp8 was cloned into the pMAL expression vector, which was used to transform Escherichia coli. Expression of the maltose binding protein-tagged protein in LB medium supplemented with 2 g/L dextrose was followed by centrifugation, resuspension of the cell pellet in lysis buffer [50 mM Tris (pH 8), 500 mM NaCl, 5 mM BME] and lysis by sonication. The cleared lysate was run over an amylose column, and protein was eluted in the lysis buffer
Acknowledgments
This work was supported by operating grants to A.M.M. from the Natural Sciences and Engineering Research Council of Canada and from the Canadian Institutes of Health Research as well as Natural Sciences and Engineering Research Council of Canada operating grants to S.D.R. and N.M.F. E.L.G. was supported by a doctoral fellowship from Alberta Innovates Health Solutions.
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