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Intron-lariat spliceosomes convert lariats to true circles: implications for intron transposition.

ABSTRACT: Rare, full length circular intron RNAs distinct from lariats have been reported in several species, but their biogenesis is not understood. We envision and test a hypothesis for their formation using Saccharomyces cerevisiae, documenting full length and novel processed circular RNAs from multiple introns. Evidence implicates a previously undescribed catalytic activity of the intron-lariat spliceosome (ILS) in which the 3'-OH of the lariat tail (with optional trimming and adenylation by the nuclear 3' processing machinery) attacks the branch, joining the intron 3' end to the 5' splice site in a 3'-5' linked circle. Human U2 and U12 spliceosomes produce analogous full length and processed circles. Post-splicing catalytic activity of the spliceosome may promote intron transposition during eukaryotic genome evolution.

SUBMITTER: Ares M

PROVIDER: S-EPMC10996645 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Intron-lariat spliceosomes convert lariats to true circles: implications for intron transposition.

Ares Manuel M Igel Haller H Katzman Sol S Donohue John P JP

bioRxiv : the preprint server for biology 20240327

Rare, full length circular intron RNAs distinct from lariats have been reported in several species, but their biogenesis is not understood. We envision and test a hypothesis for their formation using <i>Saccharomyces cerevisiae</i>, documenting full length and novel processed circular RNAs from multiple introns. Evidence implicates a previously undescribed catalytic activity of the intron-lariat spliceosome (ILS) in which the 3'-OH of the lariat tail (with optional trimming and adenylation by th ...[more]

PMID: 38585890

Similar Datasets

Intron lariat spliceosomes convert lariats to true circles: implications for intron transposition.

Project description:Rare, full-length circular intron RNAs distinct from lariats have been reported in several species, but their biogenesis is not understood. We envisioned and tested a hypothesis for their formation using Saccharomyces cerevisiae, documenting full-length and novel processed circular RNAs from multiple introns. Evidence implicates a previously undescribed catalytic activity of the intron lariat spliceosome (ILS) in which the 3'-OH of the lariat tail (with optional trimming and adenylation by the nuclear 3' processing machinery) attacks the branch, joining the intron 3' end to the 5' splice site in a 3'-5' linked circle. Human U2 and U12 spliceosomes produce analogous full-length and processed circles. Postsplicing catalytic activity of the spliceosome may promote intron transposition during eukaryotic genome evolution.

| S-EPMC11146597 | biostudies-literature

Endogenous U2·U5·U6 snRNA complexes in S. pombe are intron lariat spliceosomes.

Project description:Excision of introns from pre-mRNAs is mediated by the spliceosome, a multi-megadalton complex consisting of U1, U2, U4/U6, and U5 snRNPs plus scores of associated proteins. Spliceosome assembly and disassembly are highly dynamic processes involving multiple stable intermediates. In this study, we utilized a split TAP-tag approach for large-scale purification of an abundant endogenous U2·U5·U6 complex from Schizosaccharomyces pombe. RNAseq revealed this complex to largely contain excised introns, indicating that it is primarily ILS (intron lariat spliceosome) complexes. These endogenous ILS complexes are remarkably resistant to both high-salt and nuclease digestion. Mass spectrometry analysis identified 68, 45, and 43 proteins in low-salt-, high-salt-, and micrococcal nuclease-treated preps, respectively. The protein content of a S. pombe ILS complex strongly resembles that previously reported for human spliced product (P) and Saccharomyces cerevisiae ILS complexes assembled on single pre-mRNAs in vitro. However, the ATP-dependent RNA helicase Brr2 was either substoichiometric in low-salt preps or completely absent from high-salt and MNase preps. Because Brr2 facilitates spliceosome disassembly, its relative absence may explain why the ILS complex accumulates logarithmically growing cultures and the inability of S. pombe extracts to support in vitro splicing.

| S-EPMC3923126 | biostudies-literature

Crystal structure of a eukaryotic group II intron lariat.

Project description:The formation of branched lariat RNA is an evolutionarily conserved feature of splicing reactions for both group II and spliceosomal introns. The lariat is important for the fidelity of 5' splice-site selection and consists of a 2'-5' phosphodiester bond between a bulged adenosine and the 5' end of the intron. To gain insight into this ubiquitous intramolecular linkage, we determined the crystal structure of a eukaryotic group IIB intron in the lariat form at 3.7 Å. This revealed that two tandem tetraloop-receptor interactions, η-η' and π-π', place domain VI in the core to position the lariat bond in the post-catalytic state. On the basis of structural and biochemical data, we propose that π-π' is a dynamic interaction that mediates the transition between the two steps of splicing, with η-η' serving an ancillary role. The structure also reveals a four-magnesium-ion cluster involved in both catalysis and positioning of the 5' end. Given the evolutionary relationship between group II and nuclear introns, it is likely that this active site configuration exists in the spliceosome as well.

| S-EPMC4197185 | biostudies-literature

Isolation and characterization of post-splicing lariat-intron complexes.

Project description:Pre-mRNA splicing occurs in a large complex spliceosome. The steps of both spliceosome assembly and splicing reaction have been extensively analyzed, and many of the factors involved have been identified. However, the post-splicing intron turnover process, especially in vertebrates, remains to be examined. In this paper, we developed a two-tag affinity purification method for purifying lariat intron RNA-protein complexes obtained from an in vitro splicing reaction. Glycerol gradient sedimentation analyses revealed that there are at least two forms of post-splicing intron complexes, which we named the 'Intron Large (IL)' and the 'Intron Small (IS)' complexes. The IL complex contains U2, U5 and U6 snRNAs and other protein splicing factors, whereas the IS complex contains no such U snRNAs or proteins. We also showed that TFIP11, a human homolog of yeast Ntr1, is present in the IL complex and the TFIP11 mutant protein, which lacks the interaction domain with hPrp43 protein, caused accumulation of the IL complex and reduction of IS complex formation in vitro. Taken together, our results strongly suggest that TFIP11 in cooperation with hPrp43 mediates the transition from the IL complex to the IS complex, leading to efficient debranching and turnover of excised introns.

| S-EPMC2647322 | biostudies-literature

The debranching enzyme Dbr1 regulates lariat turnover and intron splicing.

Project description:The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of the first viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5' splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectroscopy. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping.

| S-EPMC10312976 | biostudies-literature

Speciation of a group I intron into a lariat capping ribozyme.

Project description:The lariat-capping (LC) ribozyme is a natural ribozyme isolated from eukaryotic microorganisms. Despite apparent structural similarity to group I introns, the LC ribozyme catalyzes cleavage by a 2',5' branching reaction, leaving the 3' product with a 3-nt lariat cap that functionally substitutes for a conventional mRNA cap in the downstream pre-mRNA encoding a homing endonuclease. We describe the crystal structures of the precleavage and postcleavage LC ribozymes, which suggest that structural features inherited from group I ribozymes have undergone speciation due to profound changes in molecular selection pressure, ultimately giving rise to an original branching ribozyme family. The structures elucidate the role of key elements that regulate the activity of the LC ribozyme by conformational switching and suggest a mechanism by which the signal for branching is transmitted to the catalytic core. The structures also show how conserved interactions twist residues, forming the lariat to join chemical groups involved in branching.

| S-EPMC4040574 | biostudies-literature

Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1.

Project description:The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2',5'-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2',5'-phosphodiester recognition and explain why the enzyme lacks activity toward 3',5'-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents to treat retroviral infections and neurodegenerative disease.

| S-EPMC4176325 | biostudies-literature

Intron Lariat RNA Inhibits MicroRNA Biogenesis by Sequestering the Dicing Complex in Arabidopsis.

Project description:Lariat RNAs formed as by-products of splicing are quickly degraded by the RNA debranching enzyme 1 (DBR1), leading to their turnover. Null dbr1 mutants in both animals and plants are embryo lethal, but the mechanism underlying the lethality remains unclear. Here we characterized a weak mutant allele of DBR1 in Arabidopsis, dbr1-2, and showed that a global increase in lariat RNAs was unexpectedly accompanied by a genome-wide reduction in miRNA accumulation. The dbr1-2 mutation had no effects on expression of miRNA biogenesis genes or primary miRNAs (pri-miRNAs), but the association of pri-miRNAs with the DCL1/HYL1 dicing complex was impaired. Lariat RNAs were associated with the DCL1/HYL1 dicing complex in vivo and competitively inhibited the binding of HYL1 with pri-miRNA. Consistent with the impacts of lariat RNAs on miRNA biogenesis, over-expression of lariat RNAs reduced miRNA accumulation. Lariat RNAs localized in nuclear bodies, and partially co-localize with HYL1, and both DCL1 and HYL1 were mis-localized in dbr1-2. Together with our findings that nearly four hundred lariat RNAs exist in wild type plants and that these lariat RNAs also associate with the DCL1/HYL1 dicing complex in vivo, we thus propose that lariat RNAs, as decoys, inhibit miRNA processing, suggesting a hitherto unknown layer of regulation in miRNA biogenesis.

| S-EPMC5147768 | biostudies-literature

Lariat formation and a hydrolytic pathway in plant chloroplast group II intron splicing.

Project description:Lariat formation has been studied intensively only with a few self-splicing group II introns, and little is known about how the numerous diverse introns in plant organelles are excised. Several of these introns have branch-points that are not a single bulge but are adjoined by A:A, A:C, A:G and G:G pairs. Using a highly sensitive in vivo approach, we demonstrate that all but one of the barley chloroplast introns splice via the common pathway that produces a branched product. RNA editing does not improve domain 5 and 6 structures of these introns. The conserved branch-point in tobacco rpl16 is chosen even if an adjacent unpaired adenosine is available, suggesting that spatial arrangements in domain 6 determine correct branch-point selection. Lariats were not detected for the chloroplast trnV intron, which lacks an unpaired adenosine in domain 6. Instead, this intron is released as linear molecules that undergo further polyadenylation. trnV, which is conserved throughout plant evolution, constitutes the first example of naturally occurring hydrolytic group II intron splicing in vivo.

| S-EPMC126105 | biostudies-literature

The Ll.LtrB intron from Lactococcus lactis excises as circles in vivo: insights into the group II intron circularization pathway.

Project description:Group II introns are large ribozymes that require the assistance of intron-encoded or free-standing maturases to splice from their pre-mRNAs in vivo. They mainly splice through the classical branching pathway, being released as RNA lariats. However, group II introns can also splice through secondary pathways like hydrolysis and circularization leading to the release of linear and circular introns, respectively. Here, we assessed in vivo splicing of various constructs of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis. The study of excised intron junctions revealed, in addition to branched intron lariats, the presence of perfect end-to-end intron circles and alternatively circularized introns. Removal of the branch point A residue prevented Ll.LtrB excision through the branching pathway but did not hinder intron circle formation. Complete intron RNA circles were found associated with the intron-encoded protein LtrA forming nevertheless inactive RNPs. Traces of double-stranded head-to-tail intron DNA junctions were also detected in L. lactis RNA and nucleic acid extracts. Some intron circles and alternatively circularized introns harbored variable number of non-encoded nucleotides at their splice junction. The presence of mRNA fragments at the splice junction of some intron RNA circles provides insights into the group II intron circularization pathway in bacteria.

| S-EPMC4478347 | biostudies-literature

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