Dramatically reduced spliceosome in Cyanidioschyzon merolae

Abstract

The human spliceosome is a large ribonucleoprotein complex that catalyzes pre-mRNA splicing. It consists of five snRNAs and more than 200 proteins. Because of this complexity, much work has focused on the Saccharomyces cerevisiae spliceosome, viewed as a highly simplified system with fewer than half as many splicing factors as humans. Nevertheless, it has been difficult to ascribe a mechanistic function to individual splicing factors or even to discern which are critical for catalyzing the splicing reaction. We have identified and characterized the splicing machinery from the red alga Cyanidioschyzon merolae, which has been reported to harbor only 26 intron-containing genes. The U2, U4, U5, and U6 snRNAs contain expected conserved sequences and have the ability to adopt secondary structures and form intermolecular base-pairing interactions, as in other organisms. C. merolae has a highly reduced set of 43 identifiable core splicing proteins, compared with ∼90 in budding yeast and ∼140 in humans. Strikingly, we have been unable to find a U1 snRNA candidate or any predicted U1-associated proteins, suggesting that splicing in C. merolae may occur without the U1 small nuclear ribonucleoprotein particle. In addition, based on mapping the identified proteins onto the known splicing cycle, we propose that there is far less compositional variability during splicing in C. merolae than in other organisms. The observed reduction in splicing factors is consistent with the elimination of spliceosomal components that play a peripheral or modulatory role in splicing, presumably retaining those with a more central role in organization and catalysis.pre-mRNA splicing | spliceosome core | U1 snRNP | genome reduction | splicing mechanism P re-mRNA splicing occurs by two transesterification reactions that are catalyzed by the spliceosome, a large macromolecular assembly of five snRNAs and more than 200 proteins in humans (1). These components are thought to assemble onto each new pre-mRNA transcript in an ordered fashion through the recognition and binding of three highly conserved sequences in the transcript: the 5′ splice site, the branch site, and the 3′ splice site (2, 3). Some of these interactions occur via direct RNA/RNA base pairing between the transcript and snRNAs; for example, both U1 and U6 snRNAs base pair to the 5′ splice site of the pre-mRNA transcript, and, similarly, U2 snRNA base pairs to the branch site (3).Given the complexity of the human spliceosome, it is of considerable interest to find a more tractable splicing system with fewer components to study the core processes of splicing (assembly, catalysis, and fidelity). The Saccharomyces cerevisiae (yeast) spliceosome has been proposed as a simplified model system, because it contains only about 100 proteins (4). Indeed, substantial progress in understanding the spliceosome has been made by studying yeast splicing (3,5). Nevertheless, the yeast spliceosome is still a highly complex system in which to investigate the role of individual proteins, let alone attempt ...

Conserved structure of Snu13 from the highly reduced spliceosome of Cyanidioschyzon merolae

“…16 Our catalog of splicing factors in C. merolae revealed no Prp4p/60K homolog. 5 Thus, we propose that Prp3 (90K in humans) is the most likely candidate for a protein interacting with the hydrophobic pocket of Snu13. Structurally, this interaction would mean that Prp3 bridges Stem II of the U4/U6 complex and the 5 0 stem loop of U4 via Snu13.…”

“…The 57% identity between C. merolae and yeast orthologs is one of the highest levels of sequence conservation of any C. merolae splicing protein. 5 Structures of Snu13 homologs have been reported alone and bound to fragments of U4 snRNA. 13,16 To determine whether similarity in primary sequence was reflected at the structural level, we solved the 2.35 Å resolution X-ray structure of CmSnu13 [ Fig.…”

“…3,4 We recently reported a computational and biochemical assessment of the C. merolae splicing factor complement that revealed a substantial reduction in core splicing proteins to only 40. 5 Notably, there was no evidence for the U1 snRNA or any of its associated proteins in this organism. C. merolae therefore provides a minimal model for studying premRNA splicing, and a source of thermostable splicing proteins for crystallographic studies.…”

Eisosome Ultrastructure and Evolution in Fungi, Microalgae, and Lichens

“…The extensively studied extremophilic red alga Cyanidioschizon merolae (48)(49)(50), isolated from hot springs near Naples, Italy, lacks a cell wall and carries no eisosomes in its plasma membrane. A recently isolated red alga, Cyanidioschizon YNP 1A, obtained from hot springs in Yellowstone National Park, WY, has 18S rRNA and RubisCO large subunit gene sequences nearly identical to those of C. merolae (51), and both species have very similar cellular morphologies (see Fig.…”

Eukaryotes first: how could that be?

“…B 370: 20140322 stepwise acquisition of as many as 200 proteins, so many that the spliceosome is often said to resemble the ribosome in the complexity of its structure and assembly. There is variation of course: Saccharomyces cerevisiae has half as many splicing proteins as humans and the thermoacidophilic red alga Cyanidioschyzon merolae has only half that many again-43 identifiable 'core splicing proteins' [67] and apparently no U1 associated proteins (or snRNA). There is a consensus now that LECA was intron-rich, and Irimia and Roy [68] recently concluded that 'by the time of LECA, a very complex, modern-looking spliceosome, composed of at least 78 proteins and all the snRNAs, had already been established'.…”