The fission yeast methyl phosphate capping enzyme Bmc1 guides 2'-O-methylation of the U6 snRNA.

Splicing requires the tight coordination of dynamic spliceosomal RNAs and proteins. U6 is the only spliceosomal RNA transcribed by RNA Polymerase III and undergoes an extensive maturation process. In humans and fission yeast, this includes addition of a 5' γ-monomethyl phosphate cap by members of the Bin3/MePCE family as well as snoRNA guided 2'-O-methylation. Previously, we have shown that the Bin3/MePCE homolog Bmc1 is recruited to the S. pombe telomerase holoenzyme by the LARP7 family protein Pof8, where it acts in a catalytic-independent manner to protect the telomerase RNA and facilitate holoenzyme assembly. Here, we show that Bmc1 and Pof8 are required for the formation of a distinct U6 snRNP that promotes 2'-O-methylation of U6, and identify a non-canonical snoRNA that guides this methylation. We also show that the 5' γ-monomethyl phosphate capping activity of Bmc1 is not required for its role in promoting snoRNA guided 2'-O-methylation, and that this role relies on different regions of Pof8 from those required for Pof8 function in telomerase. Our results are consistent with a novel role for Bmc1/MePCE family members in stimulating 2'-O-methylation and a more general role for Bmc1 and Pof8 in guiding noncoding RNP assembly beyond the telomerase RNP.

Spliceosome assembly and regulation: insights from analysis of highly reduced spliceosomes.

Premessenger RNA splicing is catalyzed by the spliceosome, a multimegadalton RNA-protein complex that assembles in a highly regulated process on each intronic substrate. Most studies of splicing and spliceosomes have been carried out in human or S. cerevisiae model systems. There exists, however, a large diversity of spliceosomes, particularly in organisms with reduced genomes, that suggests a means of analyzing the essential elements of spliceosome assembly and regulation. In this review, we characterize changes in spliceosome composition across phyla, describing those that are most frequently observed and highlighting an analysis of the reduced spliceosome of the red alga Cyanidioschyzon merolae We used homology modeling to predict what effect splicing protein loss would have on the spliceosome, based on currently available cryo-EM structures. We observe strongly correlated loss of proteins that function in the same process, for example, in interacting with the U1 snRNP (which is absent in C. merolae), regulation of Brr2, or coupling transcription and splicing. Based on our observations, we predict splicing in C. merolae to be inefficient, inaccurate, and post-transcriptional, consistent with the apparent trend toward its elimination in this lineage. This work highlights the striking flexibility of the splicing pathway and the spliceosome when viewed in the context of eukaryotic diversity.

The evolution of pre-mRNA splicing and its machinery revealed by reduced extremophilic red algae.

The Cyanidiales are a group of mostly thermophilic and acidophilic red algae that thrive near volcanic vents. Despite their phylogenetic relationship, the reduced genomes of Cyanidioschyzon merolae and Galdieria sulphuraria are strikingly different with respect to pre-mRNA splicing, a ubiquitous eukaryotic feature. Introns are rare and spliceosomal machinery is extremely reduced in C. merolae, in contrast to G. sulphuraria. Previous studies also revealed divergent spliceosomes in the mesophilic red alga Porphyridium purpureum and the red algal derived plastid of Guillardia theta (Cryptophyta), along with unusually high levels of unspliced transcripts. To further examine the evolution of splicing in red algae, we compared C. merolae and G. sulphuraria, investigating splicing levels, intron position, intron sequence features, and the composition of the spliceosome. In addition to identifying 11 additional introns in C. merolae, our transcriptomic analysis also revealed typical eukaryotic splicing in G. sulphuraria, whereas most transcripts in C. merolae remain unspliced. The distribution of intron positions within their host genes was examined to provide insight into patterns of intron loss in red algae. We observed increasing variability of 5' splice sites and branch donor regions with increasing intron richness. We also found these relationships to be connected to reductions in and losses of corresponding parts of the spliceosome. Our findings highlight patterns of intron and spliceosome evolution in related red algae under the pressures of genome reduction.

Characterization of Pre-mRNA Splicing and Spliceosomal Machinery in Porphyridium purpureum and Evolutionary Implications for Red Algae.

Pre-mRNA splicing is a highly conserved eukaryotic process, but our understanding of it is limited by a historical focus on well-studied organisms such as humans and yeast. There is considerable diversity in mechanisms and components of pre-mRNA splicing, especially in lineages that have evolved under the pressures of genome reduction. The ancestor of red algae is thought to have undergone genome reduction prior to the lineage's radiation, resulting in overall gene and intron loss in extant groups. Previous studies on the extremophilic red alga Cyanidioschyzon merolae revealed an intron-sparse genome with a highly reduced spliceosome. To determine whether these features applied to other red algae, we investigated multiple aspects of pre-mRNA splicing in the mesophilic red alga Porphyridium purpureum. Through strand-specific RNA-Seq, we observed high levels of intron retention across a large number of its introns, and nearly half of the transcripts for these genes are not spliced at all. We also discovered a relationship between variability of 5' splice site sequences and levels of splicing. To further investigate the connections between intron retention and splicing machinery, we bioinformatically assembled the P. purpureum spliceosome, and biochemically verified the presence of snRNAs. While most other core spliceosomal components are present, our results suggest highly divergent or missing U1 snRNP proteins, despite the presence of an uncharacteristically long U1 snRNA. These unusual aspects highlight the diverse nature of pre-mRNA splicing that can be seen in lesser-studied eukaryotes, raising the importance of investigating fundamental eukaryotic processes outside of model organisms.

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.

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.

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.

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.

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.

Preparation of yeast whole cell splicing extract.

Pre-mRNA splicing, the removal of introns from pre-messenger RNA, is an essential step in eukaryotic gene expression. In humans, it has been estimated that 60 % of noninfectious diseases are caused by errors in splicing, making the study of pre-mRNA splicing a high priority from a health perspective. Pre-mRNA splicing is also complicated: the molecular machine that catalyzes the reaction, the spliceosome, is composed of five small nuclear RNAs, and over 100 proteins, making splicing one of the most complex processes in the cell.An important tool for studying pre-mRNA splicing is the in vitro splicing assay. With an in vitro assay, it is possible to test the function of each splicing component by removing the endogenous version and replacing it (or reconstituting it) with a modified one. This assay relies on the ability to produce an extract-either whole cell or nuclear-that contains all of the activities required to convert pre-mRNA to mRNA. To date, splicing extracts have only been produced from human and S. cerevisiae (yeast) cells. We describe a method to produce whole cell extracts from yeast that support splicing with efficiencies up to 90 %. These extracts have been used to reconstitute snRNAs, screen small molecule libraries for splicing inhibitors, and purify a variety of splicing complexes.

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.

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.

Secondary structure of U6 small nuclear RNA: implications for spliceosome assembly.

U6 snRNA (small nuclear RNA), one of five RNA molecules that are required for the essential process of pre-mRNA splicing, is notable for its high level of sequence conservation and the important role it is thought to play in the splicing reaction. Nevertheless, the secondary structure of U6 in the free snRNP (small nuclear ribonucleoprotein) form has remained elusive, with predictions changing substantially over the years. In the present review we discuss the evidence for existing models and critically evaluate a fundamental assumption of these models, namely whether the important 3' ISL (3' internal stem-loop) is present in the free U6 particle, as well as in the active splicing complex. We compare existing models of free U6 with a newly proposed model lacking the 3' ISL and evaluate the implications of the new model for the structure and function of U6's base-pairing partner U4 snRNA. Intriguingly, the new model predicts a role for U4 that was unanticipated previously, namely as an activator of U6 for assembly into the splicing machinery.

Endoribonuclease activity of human apurinic/apyrimidinic endonuclease 1 revealed by a real-time fluorometric assay.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme with a well-established abasic DNA cleaving activity in the base excision DNA repair pathway and in providing redox activity to several well-known transcription factors. APE1 has recently been shown to cleave at the UA, CA, and UG sites of c-myc RNA in vitro and regulates c-myc messenger RNA (mRNA) in cells. To further understand this new endoribonuclease activity of APE1, we have developed an accurate, sensitive, and rapid real-time endonuclease assay based on a fluorogenic oligodeoxynucleotide substrate with a single ribonucleotide. Using this substrate, we carried out the first kinetic analysis of APE1 endoribonuclease activity. We found that the purified native APE1 cleaves the fluorogenic substrate efficiently, as revealed by a k(cat)/K(m) of 2.62x10(6)M(-1)s(-1), a value that is only 71-fold lower than that obtained with the potent bovine pancreatic RNase A. Ion concentrations ranging from 0.2 to 2mM Mg2+ promoted catalysis, whereas 10 to 20mM Mg2+ was inhibitory to the RNA-cleaving activity of APE1. The monovalent cation K+ was inhibitory except at 20mM, where it significantly stimulated recombinant APE1 activity. These results demonstrate rapid and specific endoribonucleolytic cleavage by APE1 and support the notion that this activity is a previously undefined function of APE1.

Small molecule inhibitors of yeast pre-mRNA splicing.

The spliceosome catalyzes pre-messenger RNA (pre-mRNA) splicing, an essential process in eukaryotic gene expression in which non-protein-coding sequences are removed from pre-mRNA. The spliceosome is a large, molecular complex composed of five small nuclear RNAs (snRNAs) and over 100 proteins. Large-scale rearrangements of the snRNAs and their associated proteins, including changes in base-pairing partners, are required to properly identify the intron-containing pre-mRNA, position it within the spliceosome, and complete the cleavage and ligation reactions of splicing. Despite detailed knowledge of the composition of the spliceosome at various stages of assembly, the critical signals and conformational changes that drive the dynamic rearrangements required for pre-mRNA splicing remain largely unknown. Just as ribosome-binding antibiotics facilitated mechanistic studies of the ribosome, study of the catalytic mechanisms of the spliceosome could be enhanced by the availability of small molecule inhibitors that block spliceosome assembly and splicing at defined stages. We sought to identify inhibitors of Saccharomyces cerevisiae splicing by screening for small molecules that block yeast splicing in vitro. We identified 10 small molecule inhibitors of yeast splicing, including four antibiotics, one kinase inhibitor, and five oxaspiro compounds. We also report that a subset of the oxaspiro derivatives permitted assembly of spliceosomal complexes onto pre-mRNA but blocked splicing prior to the first cleavage reaction.

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.

Targeting of U4/U6 small nuclear RNP assembly factor SART3/p110 to Cajal bodies.

The spliceosomal small nuclear RNAs (snRNAs) are distributed throughout the nucleoplasm and concentrated in nuclear inclusions termed Cajal bodies (CBs). A role for CBs in the metabolism of snRNPs has been proposed but is not well understood. The SART3/p110 protein interacts transiently with the U6 and U4/U6 snRNPs and promotes the reassembly of U4/U6 snRNPs after splicing in vitro. Here we report that SART3/p110 is enriched in CBs but not in gems or residual CBs lacking coilin. The U6 snRNP Sm-like (LSm) proteins, also involved in U4/U6 snRNP assembly, were localized to CBs as well. The levels of SART3/p110 and LSm proteins in CBs were reduced upon treatment with the transcription inhibitor alpha-amanitin, suggesting that CB localization reflects active processes dependent on transcription/splicing. The NH2-terminal HAT domain of SART3/p110 was necessary and sufficient for specific protein targeting to CBs. Overexpression of truncation mutants containing the HAT domain had dominant negative effects on U6 snRNP localization to CBs, indicating that endogenous SART3/p110 plays a role in targeting the U6 snRNP to CBs. We propose that U4 and U6 snRNPs accumulate in CBs for the purpose of assembly into U4/U6 snRNPs by SART3/p110.

A conserved Lsm-interaction motif in Prp24 required for efficient U4/U6 di-snRNP formation.

The assembly of the U4 and U6 snRNPs into the U4/U6 di-snRNP is necessary for pre-mRNA splicing, and in Saccharomyces cerevisiae requires the splicing factor Prp24. We have identified a family of Prp24 homologs that includes the human protein SART3/p110nrb, which had been identified previously as a surface antigen in several cancers. Sequence conservation among the Prp24 homologs reveals the existence of a fourth previously unidentified RNA recognition motif (RRM) in Prp24, which we demonstrate is necessary for growth of budding yeast at 37 degrees C. The family is also characterized by a highly conserved 12-amino-acid motif at the extreme C terminus. Deletion of this motif in Prp24 causes a cold-sensitive growth phenotype and a decrease in base-paired U4/U6 levels in vivo. The mutant protein also has a reduced association with U6 snRNA in extract, and is unable to interact with the U6 Lsm proteins by two-hybrid assay. In vitro annealing assays demonstrate that deletion of the motif causes a defect in U4/U6 formation by reducing binding of Prp24 to its substrate. We conclude that the conserved C-terminal motif of Prp24 interacts with the Lsm proteins to promote U4/U6 formation.