The sole LSm complex in Cyanidioschyzon merolae associates with pre-mRNA splicing and mRNA degradation factors.

Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are found in all three domains of life and are known to promote a variety of RNA processes such as base-pair formation, unwinding, RNA degradation, and RNA stabilization. In eukaryotes, (L)Sm proteins have been studied, inter alia, for their role in pre-mRNA splicing. In many organisms, the LSm proteins form two distinct complexes, one consisting of LSm1-7 that is involved in mRNA degradation in the cytoplasm, and the other consisting of LSm2-8 that binds spliceosomal U6 snRNA in the nucleus. We recently characterized the splicing proteins from the red alga Cyanidioschyzon merolae and found that it has only seven LSm proteins. The identities of CmLSm2-CmLSm7 were unambiguous, but the seventh protein was similar to LSm1 and LSm8. Here, we use in vitro binding measurements, microscopy, and affinity purification-mass spectrometry to demonstrate a canonical splicing function for the C. merolae LSm complex and experimentally validate our bioinformatic predictions of a reduced spliceosome in this organism. Copurification of Pat1 and its associated mRNA degradation proteins with the LSm proteins, along with evidence of a cytoplasmic fraction of CmLSm complexes, argues that this complex is involved in both splicing and cytoplasmic mRNA degradation. Intriguingly, the Pat1 complex also copurifies with all four snRNAs, suggesting the possibility of a spliceosome-associated pre-mRNA degradation complex in the nucleus.

Dramatically reduced spliceosome in Cyanidioschyzon merolae.

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.

Splicing diversity revealed by reduced spliceosomes in C. merolae and other organisms.

Pre-mRNA splicing has been considered one of the hallmarks of eukaryotes, yet its diversity is astonishing: the number of substrate introns for splicing ranges from hundreds of thousands in humans to a mere handful in certain parasites. The catalytic machinery that carries out splicing, the spliceosome, is similarly diverse, with over 300 associated proteins in humans to a few tens in other organisms. In this Point of View, we discuss recent work characterizing the reduced spliceosome of the acidophilic red alga Cyanidioschyzon merolae, which further highlights the diversity of splicing in that it does not possess the U1 snRNP that is characteristically responsible for 5' splice site recognition. Comparisons to other organisms with reduced spliceosomes, such as microsporidia, trypanosomes, and Giardia, help to identify the most highly conserved splicing factors, pointing to the essential core of this complex machine. These observations argue for increased exploration of important biochemical processes through study of a wider ranger of organisms.

In vitro reconstitution of yeast splicing with U4 snRNA reveals multiple roles for the 3' stem-loop.

U4 small nuclear RNA (snRNA) plays a fundamental role in the process of premessenger RNA splicing, yet many questions remain regarding the location, interactions, and roles of its functional domains. To address some of these questions, we developed the first in vitro reconstitution system for yeast U4 small nuclear ribonucleoproteins (snRNPs). We used this system to examine the functional domains of U4 by measuring reconstitution of splicing, U4/U6 base-pairing, and triple-snRNP formation. In contrast to previous work in human extracts and Xenopus oocytes, we found that the 3' stem-loop of U4 is necessary for efficient base-pairing with U6. In particular, the loop is sensitive to changes in both length and sequence. Intriguingly, a number of mutations that we tested resulted in more stable interactions with U6 than wild-type U4. Nevertheless, each of these mutants was impaired in its ability to support splicing, indicating that these regions of U4 have functions subsequent to base pair formation with U6. Our data suggest that one such function is likely to be in tri-snRNP formation, when U5 joins the U4/U6 di-snRNP. We have identified two regions, the upper stem of the 3' stem-loop and the central domain, that promote tri-snRNP formation. In addition, the loop of the 3' stem-loop promotes di-snRNP formation, while the central domain and the 3'-terminal domain appear to antagonize di-snRNP formation.

Identification of Alternative Polyadenylation in Cyanidioschyzon merolae Through Long-Read Sequencing of mRNA.

Alternative polyadenylation (APA) is widespread among metazoans and has been shown to have important impacts on mRNA stability and protein expression. Beyond a handful of well-studied organisms, however, its existence and consequences have not been well investigated. We therefore turned to the deep-branching red alga, Cyanidioschyzon merolae, to study the biology of polyadenylation in an organism highly diverged from humans and yeast. C. merolae is an acidothermophilic alga that lives in volcanic hot springs. It has a highly reduced genome (16.5 Mbp) and has lost all but 27 of its introns and much of its splicing machinery, suggesting that it has been under substantial pressure to simplify its RNA processing pathways. We used long-read sequencing to assess the key features of C. merolae mRNAs, including splicing status and polyadenylation cleavage site (PAS) usage. Splicing appears to be less efficient in C. merolae compared with yeast, flies, and mammalian cells. A high proportion of transcripts (63%) have at least two distinct PAS's, and 34% appear to utilize three or more sites. The apparent polyadenylation signal UAAA is used in more than 90% of cases, in cells grown in both rich media or limiting nitrogen. Our documentation of APA for the first time in this non-model organism highlights its conservation and likely biological importance of this regulatory step in gene expression.

Efficient splinted ligation of synthetic RNA using RNA ligase.

RNA ligation allows the creation of large RNA molecules from smaller pieces. This can be useful in a number of contexts: to generate molecules that are larger than can be directly synthesized; to incorporate site-specific changes or RNA modifications within a large RNA in order to facilitate functional and structural studies; to isotopically label segments of large RNAs for NMR structural studies; and to construct libraries of mutant RNAs in which one region is extensively mutagenized or modified. The impediment to widespread use of RNA ligation is the low and variable efficiency of standard ligation strategies, which frequently preclude joining more than two pieces of RNA together.We describe a method using RNA ligase (Rligation), rather than DNA ligase (Dligation), in a splint-mediated ligation reaction that joins RNA molecules with high efficiency. RNA ligase recognizes single-stranded RNA ends, which are held in proximity to one another by the splint. Monitoring the reaction is easily accomplished by denaturing gel electrophoresis and ethidium bromide staining. Using this technique, it is possible to generate a wide range of modified RNAs from synthetic oligoribonucleotides.

Complementation of U4 snRNA in S. cerevisiae splicing extracts for biochemical studies of snRNP assembly and function.

Pre-messenger RNA splicing is a surprisingly complex and dynamic process, the details of which remain largely unknown. One important method for studying splicing involves the replacement of endogenous splicing components with their synthetic counterparts. This enables changes in protein or nucleic acid sequence to be tested for functional effects, as well as the introduction of chemical moieties such as cross-linking groups and fluorescent dyes. To introduce the modified component, the endogenous one must be removed and a method found to reconstitute the active splicing machinery. In extracts prepared from S. cerevisiae, reconstitution has been accomplished with the small, nuclear RNAs U6, U2, and U5.We describe a comparable method to reconstitute active U4 small, nuclear RNA (snRNA) into a splicing extract. In order to remove the endogenous U4 it is necessary to target it for oligo-directed RNase H degradation while active splicing is under way, i.e., in the presence of a splicing transcript and ATP. This allows complete degradation of endogenous U4 and subsequent replacement with an exogenous version. In contrast to the procedures described for depletion of U6, U2, or U5 snRNAs, depletion of U4 requires concurrent active splicing. The ability to reconstitute U4 in yeast extract allows a variety of structural and functional studies to be carried out.

An RNA ligase-mediated method for the efficient creation of large, synthetic RNAs.

RNA ligation has been a powerful tool for incorporation of cross-linkers and nonnatural nucleotides into internal positions of RNA molecules. The most widely used method for template-directed RNA ligation uses DNA ligase and a DNA splint. While this method has been used successfully for many years, it suffers from a number of drawbacks, principally, slow and inefficient product formation and slow product release, resulting in a requirement for large quantities of enzyme. We describe an alternative technique catalyzed by T4 RNA ligase instead of DNA ligase. Using a splint design that allows the ligation junction to mimic the natural substrate of RNA ligase, we demonstrate several ligation reactions that appear to go nearly to completion. Furthermore, the reactions generally go to completion within 30 min. We present data evaluating the relative importance of various parameters in this reaction. Finally, we show the utility of this method by generating a 128-nucleotide pre-mRNA from three synthetic oligoribonucleotides. The ability to ligate synthetic or in vitro transcribed RNA with high efficiency has the potential to open up areas of RNA biology to new functional and biophysical investigation. In particular, we anticipate that site-specific incorporation of fluorescent dyes into large RNA molecules will yield a wealth of new information on RNA structure and function.