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Dramatically Reduced Spliceosome in Cyanidioschyzon Merolae

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Dramatically Reduced Spliceosome in Cyanidioschyzon Merolae Dramatically reduced spliceosome in PNAS PLUS Cyanidioschyzon merolae Martha R. Starka, Elizabeth A. Dunnb, William S. C. Dunna, Cameron J. Grisdalec, Anthony R. Danielea, Matthew R. G. Halsteada, Naomi M. Fastc, and Stephen D. Radera,b,1 aDepartment of Chemistry, University of Northern British Columbia, Prince George, BC, V2N 4Z9 Canada; and Departments of bBiochemistry and Molecular Biology, and cBotany, University of British Columbia, Vancouver, BC, V6T 1Z4 Canada Edited by Joan A. Steitz, Howard Hughes Medical Institute, Yale University, New Haven, CT, and approved February 9, 2015 (received for review September 1, 2014) The human spliceosome is a large ribonucleoprotein complex that C. merolae is an acidophilic, unicellular red alga that grows at catalyzes pre-mRNA splicing. It consists of five snRNAs and more temperatures of up to 56 °C (6). At 16.5 million base pairs, its than 200 proteins. Because of this complexity, much work has genome is similar in size to that of S. cerevisiae and contains focusedontheSaccharomyces cerevisiae spliceosome, viewed as a comparable number of genes; however only one tenth as many a highly simplified system with fewer than half as many splicing introns were annotated in C. merolae: 26 intron-containing factors as humans. Nevertheless, it has been difficult to ascribe genes, 0.5% of the genome (6). The small number of introns in a mechanistic function to individual splicing factors or even to dis- C. merolae raises the questions of whether the full complexity cern which are critical for catalyzing the splicing reaction. We have of the canonical splicing machinery has been maintained or C merolae identified and characterized the splicing machinery from the red alga whether . also harbors a reduced set of splicing factors. Cyanidioschyzon merolae We have undertaken a comprehensive bioinformatic survey of , which has been reported to harbor only C merolae 26 intron-containing genes. The U2, U4, U5, and U6 snRNAs contain the . splicing machinery, identifying four snRNAs (U2, expected conserved sequences and have the ability to adopt second- U4, U5, and U6) and 69 splicing proteins, 43 of which are pre- ary structures and form intermolecular base-pairing interactions, as dicted to be associated with small nuclear ribonucleoprotein in other organisms. C. merolae has a highly reduced set of 43 identifi- (snRNPs) or part of complexes that associate directly with the spliceosome. Surprisingly, we were not able to identify any can- able core splicing proteins, compared with ∼90 in budding yeast and BIOCHEMISTRY didates for the U1 snRNA or U1-associated proteins, leading us ∼140 in humans. Strikingly, we have been unable to find a U1 snRNA to conclude that C. merolae does not contain a U1 snRNP. The candidate or any predicted U1-associated proteins, suggesting that C merolae C merolae profile of splicing proteins retained in . provides a means splicing in . may occur without the U1 small nuclear ribo- of assessing the contribution of specific proteins to the splicing nucleoprotein particle. In addition, based on mapping the identified reaction, at least insofar as the missing ones clearly are not always proteins onto the known splicing cycle, we propose that there is far essential for splicing. The U2 snRNP is the most complex particle, C merolae less compositional variability during splicing in . than in with 10 proteins present in addition to the Sm proteins. Many U5- other organisms. The observed reduction in splicing factors is consis- associated proteins also are retained, whereas the Prp19/CDC5L tent with the elimination of spliceosomal components that play a pe- complex (NTC), step-specific proteins, and tri-snRNP–specific ripheral or modulatory role in splicing, presumably retaining those proteins are largely predicted to be absent. Indeed, the C. merolae with a more central role in organization and catalysis. spliceosome is notable for the almost complete absence of proteins that join or leave the spliceosome after B complex formation, pre-mRNA splicing | spliceosome core | U1 snRNP | genome reduction | suggesting that the initial core contains all the components required splicing mechanism to carry out the splicing reaction. The novelty of a splicing system re-mRNA splicing occurs by two transesterification reactions Significance Pthat are catalyzed by the spliceosome, a large macromolec- ular assembly of five snRNAs and more than 200 proteins in The spliceosome—the molecular particle responsible for re- humans (1). These components are thought to assemble onto moving interrupting sequences from eukaryotic messenger each new pre-mRNA transcript in an ordered fashion through RNA—is one of the most complex cellular machines. Consisting the recognition and binding of three highly conserved sequences of five snRNAs and over 200 proteins in humans, its numerous ′ ′ in the transcript: the 5 splice site, the branch site, and the 3 changes in composition and shape during splicing have made it splice site (2, 3). Some of these interactions occur via direct difficult to study. We have characterized an algal spliceosome RNA/RNA base pairing between the transcript and snRNAs; for that is much smaller, with only 43 identifiable core proteins, the ′ example, both U1 and U6 snRNAs base pair to the 5 splice site majority of which are essential for viability in other organisms. of the pre-mRNA transcript, and, similarly, U2 snRNA base We propose that this highly reduced spliceosome has retained pairs to the branch site (3). only the most critical splicing factors. Cyanidioschyzon merolae Given the complexity of the human spliceosome, it is of con- therefore provides a powerful system to examine the spliceo- siderable interest to find a more tractable splicing system with some’s catalytic core, enabling future advances in understanding fewer components to study the core processes of splicing (assembly, Saccharomyces cerevisiae the splicing mechanism and spliceosomal organization that are catalysis, and fidelity). The (yeast) challenging in more complex systems. spliceosome has been proposed as a simplified model system, because it contains only about 100 proteins (4). Indeed, substantial Author contributions: M.R.S., N.M.F., and S.D.R. designed research; M.R.S., E.A.D., W.S.C.D., progress in understanding the spliceosome has been made by C.J.G., A.R.D., M.R.G.H., and S.D.R. performed research; C.J.G. contributed new reagents/ studying yeast splicing (3, 5). Nevertheless, the yeast spliceosome analytic tools; M.R.S., E.A.D., W.S.C.D., C.J.G., A.R.D., N.M.F., and S.D.R. analyzed data; and is still a highly complex system in which to investigate the role of M.R.S., E.A.D., C.J.G., N.M.F., and S.D.R. wrote the paper. individual proteins, let alone attempt to develop a completely The authors declare no conflict of interest. defined splicing system for more incisive experiments. The publi- This article is a PNAS Direct Submission. cation of the Cyanidioschyzon merolae genome sequence revealed 1To whom correspondence should be addressed. Email: [email protected]. that it has only 27 introns (6), indicating that it might be a simpler This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. system in which to investigate splicing. 1073/pnas.1416879112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1416879112 PNAS Early Edition | 1of10 Downloaded by guest on September 25, 2021 reduced to ∼40 proteins suggests that C. merolae may be a more a shorter U2 snNRA. U4 has an 18-nt insertion, relative to the tractable system for studying the central processes of pre-mRNA human sequence, starting at nucleotide 77 (Fig. S1). This in- splicing. sertion falls in the central domain of U4, in the middle of the hypothetical U4/U6 stem III, which is evolutionarily conserved, Results but for which there is no experimental support (10, 11). In ad- C. merolae Transcripts Are Spliced. Twenty-seven introns were dition, the 5′ stem-loop of U6 is ∼10 bp longer than that in other identified computationally in the C. merolae genome (figure S2 in species (Fig. S1). Finally, U2 snRNA lacks ∼60 nucleotides from ref. 6). To ascertain whether the intron-containing transcripts are the 3′ end, after the Sm-binding site, a region that generally in fact spliced, we performed RT-PCR on DNase-treated total forms one or more stem-loops in other organisms (12). No RNA. Each primer pair, except for those for gene CMK245C, C. merolae snRNAs have stem-loops predicted downstream of resulted in two PCR products, a longer intron-containing ampli- their Sm site. con and a shorter amplicon corresponding to spliced mRNA (Fig. U4 and U6 snRNAs in other organisms are known to form an 1). The CMK245C transcript appears to be completely spliced. extended base-pairing interaction, allowing them to comigrate Two junctions (CMQ117C and CMO094C) were confirmed by on a nondenaturing gel. To test whether this interaction also sequencing reverse transcriptase products, and the remaining occurs in C. merolae, we compared cold phenol-extracted total introns were observed to splice at their expected junctions based RNA (Fig. 2B, lanes 1 and 3) with the same samples incubated on our sequencing of background mRNAs in the RNA immuno- for 5 min at 70 °C to disrupt base pairing (Fig. 2B, lanes 2 and 4). precipitation sequencing (RIP-seq) Illumina library (see below Heat treatment resulted in the disappearance of the low-mobility and SI Materials and Methods). band detected by both U4 (lane 1) and U6 (lane 3) probes and the appearance of higher-mobility bands detected by U4 (lane 2) Identification of snRNAs. Given that intron-containing transcripts and U6 (lane 4) probes. Control experiments with S. cerevisiae are spliced in C. merolae, we sought to identify components snRNAs (lanes 5–8) demonstrate the analogous interaction.
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