Circular Intron Research Articles

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

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.

Nuclear export and translation of circular repeat-containing intronic RNA in C9ORF72-ALS/FTD

C9ORF72 hexanucleotide GGGGCC repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-containing RNA mediates toxicity through nuclear granules and dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG translation. However, it remains unclear how the intron-localized repeats are exported and translated in the cytoplasm. We use single molecule imaging approach to examine the molecular identity and spatiotemporal dynamics of the repeat RNA. We demonstrate that the spliced intron with G-rich repeats is stabilized in a circular form due to defective lariat debranching. The spliced circular intron, instead of pre-mRNA, serves as the translation template. The NXF1-NXT1 pathway plays an important role in the nuclear export of the circular intron and modulates toxic DPR production. This study reveals an uncharacterized disease-causing RNA species mediated by repeat expansion and demonstrates the importance of RNA spatial localization to understand disease etiology.

Free circular introns with an unusual branchpoint in neuronal projections.

The polarized structure of axons and dendrites in neuronal cells depends in part on RNA localization. Previous studies have looked at which polyadenylated RNAs are enriched in neuronal projections or at synapses, but less is known about the distribution of non-adenylated RNAs. By physically dissecting projections from cell bodies of primary rat hippocampal neurons and sequencing total RNA, we found an unexpected set of free circular introns with a non-canonical branchpoint enriched in neuronal projections. These introns appear to be tailless lariats that escape debranching. They lack ribosome occupancy, sequence conservation, and known localization signals, and their function, if any, is not known. Nonetheless, their enrichment in projections has important implications for our understanding of the mechanisms by which RNAs reach distal compartments of asymmetric cells.

Accumulation of Stable Full-Length Circular Group I Intron RNAs during Heat-Shock.

Group I introns in nuclear ribosomal RNA of eukaryotic microorganisms are processed by splicing or circularization. The latter results in formation of full-length circular introns without ligation of the exons and has been proposed to be active in intron mobility. We applied qRT-PCR to estimate the copy number of circular intron RNA from the myxomycete Didymium iridis. In exponentially growing amoebae, the circular introns are nuclear and found in 70 copies per cell. During heat-shock, the circular form is up-regulated to more than 500 copies per cell. The intron harbours two ribozymes that have the potential to linearize the circle. To understand the structural features that maintain circle integrity, we performed chemical and enzymatic probing of the splicing ribozyme combined with molecular modeling to arrive at models of the inactive circular form and its active linear counterpart. We show that the two forms have the same overall structure but differ in key parts, including the catalytic core element P7 and the junctions at which reactions take place. These differences explain the relative stability of the circular species, demonstrate how it is prone to react with a target molecule for circle integration and thus supports the notion that the circular form is a biologically significant molecule possibly with a role in intron mobility.

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

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.

In vitro characterization of the splicing efficiency and fidelity of the RmInt1 group II intron as a means of controlling the dispersion of its host mobile element.

Group II introns are catalytic RNAs that are excised from their precursors in a protein-dependent manner in vivo. Certain group II introns can also react in a protein-independent manner under nonphysiological conditions in vitro. The efficiency and fidelity of the splicing reaction is crucial, to guarantee the correct formation and expression of the protein-coding mRNA. RmInt1 is an efficient mobile intron found within the ISRm2011-2 insertion sequence in the symbiotic bacterium Sinorhizobium meliloti. The RmInt1 intron self-splices in vitro, but this reaction generates side products due to a predicted cryptic IBS1* sequence within the 3′ exon. We engineered an RmInt1 intron lacking the cryptic IBS1* sequence, which improved the fidelity of the splicing reaction. However, atypical circular forms of similar electrophoretic mobility to the lariat intron were nevertheless observed. We analyzed a run of four cytidine residues at the 3′ splice site potentially responsible for a lack of fidelity at this site leading to the formation of circular intron forms. We showed that mutations of residues base-pairing in the tertiary EBS3–IBS3 interaction increased the efficiency and fidelity of the splicing reaction. Our results indicate that RmInt1 has developed strategies for decreasing its splicing efficiency and fidelity. RmInt1 makes use of unproductive splicing reactions to limit the transposition of the insertion sequence into which it inserts itself in its natural context, thereby preventing potentially harmful dispersion of ISRm2011-2 throughout the genome of its host.

Predicted group II intron lineages E and F comprise catalytically active ribozymes

Group II introns are self-splicing, retrotransposable ribozymes that contribute to gene expression and evolution in most organisms. The ongoing identification of new group II introns and recent bioinformatic analyses have suggested that there are novel lineages, which include the group IIE and IIF introns. Because the function and biochemical activity of group IIE and IIF introns have never been experimentally tested and because these introns appear to have features that distinguish them from other introns, we set out to determine if they were indeed self-splicing, catalytically active RNA molecules. To this end, we transcribed and studied a set of diverse group IIE and IIF introns, quantitatively characterizing their in vitro self-splicing reactivity, ionic requirements, and reaction products. In addition, we used mutational analysis to determine the relative role of the EBS-IBS 1 and 2 recognition elements during splicing by these introns. We show that group IIE and IIF introns are indeed distinct active intron families, with different reactivities and structures. We show that the group IIE introns self-splice exclusively through the hydrolytic pathway, while group IIF introns can also catalyze transesterifications. Intriguingly, we observe one group IIF intron that forms circular intron. Finally, despite an apparent EBS2-IBS2 duplex in the sequences of these introns, we find that this interaction plays no role during self-splicing in vitro. It is now clear that the group IIE and IIF introns are functional ribozymes, with distinctive properties that may be useful for biotechnological applications, and which may contribute to the biology of host organisms.

Multiple physical forms of excised group II intron RNAs in wheat mitochondria

Plant mitochondrial group II introns do not all possess hallmark ribozymic features such as the bulged adenosine involved in lariat formation. To gain insight into their splicing pathways, we have examined the physical form of excised introns in germinating wheat embryos. Using RT–PCR and cRT–PCR, we observed conventional lariats consistent with a two-step transesterification pathway for introns such as nad2 intron 4, but this was not the case for the cox2 intron or nad1 intron 2. For cox2, we detected full-length linear introns, which possess non-encoded 3′terminaladenosines, as well as heterogeneous circular introns, which lack 3′ nucleotide stretches. These observations are consistent with hydrolytic splicing followed by polyadenylation as well as an in vivo circularization pathway, respectively. The presence of both linear and circular species in vivo is supported by RNase H analysis. Furthermore, the nad1 intron 2, which lacks a bulged nucleotide at the branchpoint position, comprised a mixed population of precisely full-length molecules and circular ones which also include a short, discrete block of non-encoded nucleotides. The presence of these various linear and circular forms of excised intron molecules in plant mitochondria points to multiple novel group II splicing mechanisms in vivo.

The ability to form full-length intron RNA circles is a general property of nuclear group I introns.

In addition to splicing, group I intron RNA is capable of an alternative two-step processing pathway that results in the formation of full-length intron circular RNA. The circularization pathway is initiated by hydrolytic cleavage at the 3' splice site and followed by a transesterification reaction in which the intron terminal guanosine attacks the 5' splice site presented in a structure analogous to that of the first step of splicing. The products of the reactions are full-length circular intron and unligated exons. For this reason, the circularization reaction is to the benefit of the intron at the expense of the host. The circularization pathway has distinct structural requirements that differ from those of splicing and appears to be specifically suppressed in vivo. The ability to form full-length circles is found in all types of nuclear group I introns, including those from the Tetrahymena ribosomal DNA. The biological function of the full-length circles is not known, but the fact that the circles contain the entire genetic information of the intron suggests a role in intron mobility.

Excision of Group II Introns as Circles

Group II introns are usually removed from precursor RNAs as lariats comprised of a circular component and a short 3′ tail. We find that group II introns can also be excised as complete circles. Circle formation requires release of the 3′ exon of a splicing substrate, apparently by a trans splicing mechanism. After 3′ exon release, the terminal uridine of the intron attacks the 5′ splice site, releasing the 5′ exon and joining the first and last intron residues by a 2′–5′ phosphodiester bond. RNA isolated from yeast mitochondria also contains circles, indicating that at least one group II intron (aI2) forms circles in vivo. Furthermore, analysis of RNA and DNA from certain mutant yeast strains shows that circular DNA introns exist and are produced by reverse transcription of RNA, rather than by ectopic homing.

Effects of divalent cations on the splicing of phage T4 thymidylate synthase intron RNA

Effects of divalent cations such as Mg2+, Mn2+, Ca2+, and Zn2 on splicing activity of phage T4 thymidylate synthase intron RNA have been investigated. At the concentration of 0.5 mM Mn2+ in the absence of Mg2+, a very small amount of pre‐RNA was cleaved into ligation products (E1‐E2) but no circular or linear intron was produced. As the concentration of Mn2+ was increased from 1 to 5 mM. the pre‐RNA was completely hydrolyzed. In the presence of 5 mM Mg2+, both the linear intron and circular intron were produced but no E1‐E2 ligation product was produced. At both 3 and 5 mM Mn2+, the RNA was hydrolyzed completely as observed with no Mg2+ being present. In the case of Zn2+, even at 0.5 mM concentration, the pre‐RNA was completely hydrolyzed. This observation suggested that Zn2+ facilitates RNA hydrolysis more rapidly than Mn2+ does. At 5mM Ca2+, the RNA was not hydrolyzed and remained intact as a primary transcript.

Purification and characterization of two forms of I-DmoI, a thermophilic site-specific endonuclease encoded by an archaeal intron.

The archaeal intron in the 23 S rRNA gene of the hyperthermophile Desulfurococcus mobilis has previously been shown to encode a site-specific DNA endonuclease that contains the LAGLIDADG motif. The enzyme, I-DmoI, has been shown to be active in two forms when expressed in vitro, from RNAs representing either the linear (I-DmoIl) or circular (I-DmoIc) intron. In this study we have overexpressed I-DmoIl and I-DmoIc and purified the enzymes from Escherichia coli. The optimal conditions for the enzymatic activity in vitro were determined, and the enzyme was used to delimit the recognition boundary on its DNA substrate (14-20 nucleotides), an intronless 23 S rRNA gene. Despite belonging to the archaeal kingdom, and being the product of a hyperthermophile, I-DmoI shares many properties with LAGLIDADG intron and intein endonucleases in other kingdoms. These results support the view that these phylogenetically diverse enzymes, which function to mobilize the DNA sequences that encode them, share a common ancestry.

DNA Double-strand Break in vivo at the 3′ Extremity of Exons Located Upstream of Group II Introns: Senescence and Circular DNA Introns in Podospora Mitochondria

In the filamentous fungus Podospora anserina, the unavoidable phenomenon of senescence is associated with the amplification of the first intron of the mitochondrial cox1 that accumulates as circular DNA molecules consisting of tandem repeats. This group II intron (cox1-i1 or α) is able to transpose and contains an open reading frame with significant amino acid similarity with reverse transcriptases. The generation of these intronic circular DNA molecules, their amplification and their involvement in the senescence process are unresolved questions. We demonstrate here that: (1) another group II intron, the fourth intron of gene cox1, cox1-i4, is also able to give precise DNA end to end junctions; (2) this intronic sequence can be found amplified during senescence, although to a lesser extent that cox1-i1; (3) the amplification of the DNA multimeric cox1-i1 molecules likely does not proceed by autonomous replication; (4) the generation of the DNA intronic circles does not require efficient intron splicing; (5) a DNA double-strand break occurs in vivo at the 3′ extremity of the cox1 -e1 and cox1-e4 exons preceding the group II introns that form circular DNAs.On the whole, these results show that the ability to form DNA circular molecules is a property of some group II introns and they demonstrate the occurrence of a specific DNA cleavage at or near the integration site of these group II introns. The results strongly suggest that this cleavage is involved in the formation of the group II intronic DNA circles and could also be involved in the phenomenon of group II intron homing.

The NAM1/MTF2 nuclear gene product is selectively required for the stability and/or processing of mitochondrial transcripts of the atp6 and of the mosaic, cox1 and cytb genes in Saccharomyces cerevisiae.

The NAM1/MTF2 gene was firstly isolated as a multicopy suppressor of mitochondrial splicing deficiencies and independently as a gene of which a thermosensitive allele affects mitochondrial transcription in organello. To determine which step in mitochondrial RNA metabolism is controlled in vivo by the NAM1 gene, mitochondrial transcripts of seven transcription units from strains carrying an inactive nam1::URA3 gene disruption in various mitochondrial genetic backgrounds were analysed by Northern blot hybridisations. In a strain carrying an intron-containing mitochondrial genome, the inactivation of the NAM1 gene led to a strong decrease in (or total absence of) the mosaic cytb and cox1 mRNAs and in transcripts of the atp6-rf3/ens2 genes, which are co-transcribed with cox1. Neither the accumulation of unspliced cytb or cox1 pre-mRNAs, nor that of excised circular intron molecules of ai1 or ai2 were observed, but the abundance of the bi1 and ai7 lariats was comparable to that observed in the wild-type strain, thus demonstrating that transcription of the cytb and cox1 genes does occur. In strains carrying the intron-less mitochondrial genome with or without the rf3/ens2 sequence, wild-type amounts of cytb and cox1 mRNAs were detected while the amount of the atp6 mRNA was always strongly decreased. The abundance of transcripts from five other genes was either slightly (21S rRNA) or not at all (cox2, cox3, atp9 and 15S rRNA) affected by the nam1 inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Splicing of COB intron 5 requires pairing between the internal guide sequence and both flanking exons.

Group I introns are characterized by a set of conserved sequence elements and secondary structures. Evidence supporting the pairing of certain of these sequences has come from the comparison of intron sequences and from the analysis of mutations that disrupt splicing by interfering with pairing. One of the structures proposed for all group I introns is an internal guide sequence that base pairs with the upstream and the downstream exons, bringing them into alignment for ligation. We made specific mutations in the internal guide sequence and the flanking exons of the fifth intron in the yeast mitochondrial gene for apocytochrome b (COB). Mutations that disrupted the pairing between the internal guide sequence and the upstream exon (the P1 pairing) blocked addition of guanosine to the 5' end of the intron during autocatalytic reactions and prevented formation of the full-length circular intron. In contrast, transcripts containing mutations that disrupted the pairing between the guide sequence and the downstream exon (the P10 helix) initiated splicing but failed to ligate exons. Compensatory mutations that restored helices of normal stability mitigated the effects of the original mutations. These data provide direct evidence for the importance of the base pairing between the internal guide sequence and the downstream exon in the splicing of a wild-type group I intron.

Novel splicing mechanism for the ribosomal RNA intron in the archaebacterium desulfurococcus mobilis

The intron of the 23S rRNA gene of D. mobilis is excised from the pre-23S RNA at specific sites in vivo and subsequently ligated to form a stable circular RNA, with a normal 5'-3' phosphodiester bond, containing the entire intron sequence; 95% of this RNA codes for a protein of 194 amino acids that can be expressed in E. coli. Crude cell extracts from D. mobilis also induce a two-step slicing reaction in vitro, producing the same circular intron RNA but a low yield of ligated exons. Cleavage depends on the RNA structure adjacent to the cleavage site and yields a 3'-terminal phosphate. Splicing is enhanced by GTP, but does not require divalent metal ions. The cleavage and exon-splicing reactions resemble those found for tRNA introns in eukaryotes and a possible structural rationale for this similarity is considered together with its possible implications for the origin of eukaryotic rRNA and tRNA introns.

Interlocked RNA circle formation by a self-splicing yeast mitochondrial group I intron

RNA containing the al3 group I intron of the yeast mitochondrial gene encoding cytochrome oxidase subunit I shows self-splicing in vitro. The excised intron, comprising 1514 nucleotides, is partially split into an upstream portion, containing the intronic reading frame, and a downstream portion, containing the typical group I conserved sequence elements. Full-length intron RNA and intron parts occur in linear and circular form. In the transesterification reactions leading to circle formation, only the guanosine nucleotide added during splicing is removed. Reincubation of isolated, complete circular intron RNA under self-splicing conditions leads to formation of free subintronic RNA circles. Under similar conditions, purified linear intron RNA gives rise to a number of circular and linear products, one of which consists of interlocked subintronic RNA circles. These observations suggest that the intron RNA possesses a dynamic structure in which subtle alterations in folding result in the formation of RNA products with different topology.

Processing of phage T4 td-encoded RNA is analogous to the eukaryotic group I splicing pathway.

Several features of the split td gene of phage T4 suggest an RNA processing mechanism analogous to that of the self-splicing rRNA of Tetrahymena and other group I eukaryotic introns. Previous work has revealed conserved sequence elements and the ability of td-encoded RNA to self-splice in vitro. We show here that a noncoded guanosine residue is covalently joined to the 5' end of the intron during processing. Further, we demonstrate the existence of linear and circular intron forms in RNA extracted from T4-infected cells and from uninfected Escherichia coli expressing the cloned td gene. Sequence analysis of the intron cyclization junction indicates that the noncoded guanosine and one additional nucleotide are lost from the 5' end of the intron upon cyclization. This analysis places a uridine residue upstream of the cyclization site, in analogy to three other group I cyclization junctions. These striking similarities to the splicing intermediates of eukaryotic group I introns point not only to an analogous processing pathway and conserved features of cyclization site recognition but also to a common ancestry between this prokaryotic intervening sequence and the group I eukaryotic introns.

RNA splicing in Neurospora mitochondria : Defective splicing of mitochondrial mRNA precursors in the nuclear mutant cyt18-1

cyt18-1 (299-9) is a nuclear mutant of Neurospora crassa that has been shown to have a temperature-sensitive defect in splicing the mitochondrial large rRNA intron. In the present work, we investigate the effect of the cyt18-1 mutation on splicing of mitochondrial mRNA introns. Two genes were studied in detail; the cytochrome b ( cob) gene, which contains two introns, and a “long form” of the cytochrome oxidase subunit I ( coI) gene, which contains four introns. We found that splicing of both cob introns and splicing of at least two of the coI introns are strongly inhibited in the mutant, whereas splicing of col intron 1, which is excised as a 2.6 × 10 3 base circle, is relatively unaffected. The rRNA intron and both cob introns are group I introns, whereas the circular coI intron may belong to another structural class. Control experiments showed that the degree of inhibition of splicing is greater in the mutant than can be accounted for by severe inhibition of mitochondrial protein synthesis. Finally, experiments in which mutant cells were shifted from 25 °C to 37 °C showed that splicing of the large rRNA precursor and splicing of the coI mRNA precursor are inhibited with similar kinetics. Considered together, our results suggest that the cyt18-1 gene encodes a trans-acting component that is required for the splicing of group I mitochondrial DNA introns or some subclass thereof. Since Neurospora cob intron 1 has been shown to be self-splicing in vitro, defective splicing of this intron in cyt18-1 indicates that an essentially RNA-catalyzed splicing reaction must be facilitated by a trans-acting factor, presumably a protein, in vivo.