Group IIB Intron Research Articles

A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in Chlamydomonas reinhardtii

In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont.

The Receptor for Branch-Site Docking within a Group II Intron Active Site

The distinguishing feature of group II introns, and the property that links them with spliceosomal catalysis, is their ability to undergo splicing through branching. In this reaction, the 2'-hydroxyl group of a specific adenosine within intron domain 6 serves as the nucleophile for attack on the 5' splice site. We know less about branching than any other feature of group II intron catalysis, largely because the receptor structure for activating the branch site is unknown. Here, we identify the intronic region that binds the branch site of a group IIB intron. Located in domain 1, close to receptors for intron domain 5 and both splice sites, we demonstrate that the branch-site receptor is a functional element required for transesterification. Furthermore, we show that crosslinked branch sites can carry out both steps of splicing, suggesting that the conformational state of the intron core is set early and that it persists throughout the entire splicing process.

Coevolution of group II intron RNA structures with their intron-encoded reverse transcriptases.

Catalytic RNAs are often regarded as molecular fossils from the RNA World, yet it is usually difficult to get more specific information about their evolution. Here we have investigated the coevolution of group II intron RNA structures with their intron-encoded reverse transcriptases (RTs). Unlike group I introns, there has been no obvious reshuffling between intron RNA structures and ORFs. Of the six classes of intron structures that encode ORFs, three are conventional forms of group II A1, B1, and B2 secondary structures, whereas the remaining classes are bacterial, are possibly associated with the most primitive ORFs, and have unusual features and hybrid features of group IIA and group IIB intron structures. Based on these data, we propose a new model for the evolution of group II introns, designated the retroelement ancestor hypothesis, which predicts that the major RNA structural forms of group II introns developed through coevolution with the intron-encoded protein rather than as independent catalytic RNAs, and that most ORF-less introns are derivatives of ORF-containing introns. The model is supported by the distribution of ORF-containing and ORF-less introns, and by numerous examples of ORF-less introns that contain ORF remnants.

Group II intron splicing in Escherichia coli: phenotypes of cis-acting mutations resemble splicing defects observed in organelle RNA processing.

The mitochondrial group IIB intron rI1, from the green algae Scenedesmus obliquus ' LSUrRNA gene, has been introduced into the lacZ gene encoding beta-galacto-sidase. After DNA-mediated transformation of the recombinant lacZ gene into Escherichia coli, we observed correct splicing of the chimeric precursor RNA in vivo. In contrast to autocatalytic in vitro self-splicing, intron processing in vivo is independent of the growth temperature, suggesting that in E.coli, trans -acting factors are involved in group II intron splicing. Such a system would seem suitable as a model for analyzing intron processing in a prokaryotic host. In order to study further the effect of cis -mutations on intron splicing, different rI1 mutants were analyzed (with respect to their splicing activity) in E.coli. Although the phenotypes of these E. coli intron splicing mutants were identical to those which can be observed during organellar splicing of rI1, they are different to those observed in in vitro self-splicing experiments. Therefore, in both organelles and prokaryotes, it is likely that either similar splicing factors or trans -acting factors exhibiting similar functions are involved in splicing. We speculate that ubiquitous trans -acting factors, via recent horizontal transfer, have contributed to the spread of group II introns.

The reverse-transcriptase-like proteins encoded by group II introns in the mitochondrial genome of the brown alga Pylaiella littoralis belong to two different lineages which apparently coevolved with the group II ribosyme lineages.

The mitochondrial genome of the brown alga Pylaiella littoralis contains two different types of group II introns. They each encode complete complex proteins, i.e., with a reverse transcriptase domain, a maturase or X domain, and an endonuclease or H-N-H/zinc finger domain. To our knowledge, this is the first example of the presence in the same genome of introns belonging to subgroups IIA and IIB which both contain multidomained RT-like proteins. We describe the group IIA introns that interrupt the cox1 gene. The RT-like proteins contained in these introns were compared to those of the LSU rDNA group IIB introns. The phylogenetic relationships of these intron ORFs were investigated and the possible evolution of group II introns is discussed.

The Mitochondrial LSU rDNA of the Brown Alga Pylaiella littoralisReveals α-Proteobacterial Features and is Split by Four Group IIB Introns with an Atypical Phylogeny

The mitochondrial DNA region coding for the larger ribosomal RNA subunit from the brown alga Pylaiella littoralis(L.) Kjellm was sequenced. The LSU rRNA was folded into a secondary structure and aligned with homologous, mitochondrial and eubacterial sequences. Taking into account the primary and secondary structure levels, the mitochondrial LSU rRNA of P. littoralisshares more structural features with α-proteobacterial genes than do those of the green alga Prototheca wickerhamiiand land plants. In phylogenetic trees, branches leading to brown algae, red algae, the protozan Acanthamoeba castellaniiand land plants, respectively, emerge approximately at the same time, as they do in nuclear-gene based phylogenies. This suggests that there is only one origin for the mitochondrial rRNA genes found in these lineages. The LSU rDNA is split by four group IIB introns. The first two introns each contain one open reading frame which encodes a reverse transcriptase-like protein. Comparison of their amino acid sequences with those of other reverse transcriptase-like genes contained in group II introns shows that these genes are more closely related to plastid and cyanobacterial homologous genes than to any known mitochondrial intronic reverse transcriptase.

Complete Sequence of the Mitochondrial DNA of the Rhodophyte Chondrus crispus(Gigartinales). Gene Content and Genome Organization

The complete nucleotide sequence of the circular mitochondrial (mt) DNA from the red alga Chondrus crispuswas determined (25, 836 nucleotides, A+T content 72.1%). Fifty one genes were identified. They include genes encoding three subunits of the cytochrome oxidase ( cox1 to 3), apocytochrome b ( cob), seven subunits of the NADH dehydrogenase complex ( nad1to 6, nad4L), two ATPase subunits ( atp6and atp9), three ribosomal RNAs ( rrn5, srnand Irn), 23 tRNAs and four ribosomal proteins ( rps3, rps11, rps12and rpl16). Two subunits of the succinate dehydrogenase complex ( sdhBand sdhC), usually found on nuclear genomes, are also located on the mtDNA of C. crispus.One group IIb intron is inserted in the tRNA IIegene. Six potentially functional open reading frames were identified, four of them having counterparts among green plant mtDNAs. The use of a modified genetic code and the absence of RNA editing, previously reported for the cox3gene, appears as a general characteristic of this molecule. Mitochondrial genes are encoded on both DNA strands, in two opposite major transcriptional directions, suggesting the existence of two main transcriptional units. Two long and stable stem-loops were identified in intergenic regions, which are believed to be involved with transcription and replication. The main structural features of this genome are compared with the overall organization of mtDNAs and are discussed in view of the evolution of mitochondria.

Self-splicing of a Podospora anserina Group IIA Intron in vitro: Effects of 3′-Terminal Intron Alterations on Cleavage at the 5′ and 3′ Splice Site

A shortened derivative of the group IIA intron from the mitochondrial cytochrome-c-oxidase subunit I gene (COI II) of the ascomycete Podospora anserina can undergo self-splicing in vitro. When compared to self-splicing group IIB introns from yeast mitochondria (aI5c, bII) the autocatalytic reaction shows a lower efficiency and 5′ cleavage takes place predominantly by hydrolysis. In order to test the influence on reaction efficiency and mode of 5′ cleavage of the long peripheral structure of domain VI (dVI) we generated mutant Podospora introns that have different structural forms of shortened dVI. Our results show that: (1) in general the size and structure of dVI distal from the branch site is essential for 5′ transesterification and influences the efficiency of the second splicing step; (2) 5′ transesterification as well as the complete self-splicing reaction is more efficient when the structure of dVI is adapted to that of yeast group IIB introns. Moreover, our data indicate that the postulated γ-γ′ tertiary interaction is also functional for group IIA introns. A weakening or disruption of this interaction in the Podospora intron leads to a greatly reduced cleavage at the 3′ splice site and to a selection of cryptic sites downstream in the 3′ exon that almost exclusively restore the strong wild-type γ-γ′ pairing. The so-called "guide" interaction seems to support the selection of 3′ cleavage sites but is of secondary importance in relation to the γ-γ′ interaction.

A maturase-encoding group IIA intron of yeast mitochondria self-splices in vitro.

Intron 1 of the coxI gene of yeast mitochondrial DNA (aI1) is a group IIA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, aI1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4Cl or KCl or 2M (NH4)2SO4) for ready detection of splicing activity. In KCl-stimulated reactions, splicing occurs with little normal branch formation; a post-splicing reaction of linear excised intron RNA that forms shorter lariat RNAs with branches at cryptic sites was evident in those samples. At low levels of added NH4Cl or KCl, the precursor RNA carries out the first reaction step but appears blocked in the splicing step. AI1 RNA is most reactive at 37-42 degrees C, as compared with 45 degrees C for the group IIB introns; and it lacks the KCl- or NH4Cl-dependent spliced-exon reopening reaction that is evident for the self-splicing group IIB introns of yeast mitochondria. Like the group IIB intron aI5 gamma, the domain 4 of aI1 can be largely deleted in cis, without blocking splicing; also, trans-splicing of half molecules interrupted in domain 4 occurs. This is the first report of a maturase-encoding intron of either group I or group II that self-splices in vitro.

The group IIB intron from the green alga Scenedesmus obliquus mitochondrion: molecular characterization of the in vitro splicing products

In the presence of high molar salt concentrations, the mitochondrial group IIB intron (rI1) from the green alga Scenedesmus obliquus is capable of splicing in vitro. After establishing the optimal conditions for RNA processing the in vitro splicing products were unequivocally identified in self-splicing experiments by Northern hybridization analysis employing 3'end-labelled RNAs or exon- and/or intron-specific probes. Finally, two trans-esterification products were identified by sequencing of the spliced RNA. From our data we conclude that the processing of group II introns from both algal and yeast mitochondria is preceded by identical consecutive trans-esterification steps. The predicted secondary and tertiary structure of intron rI1 of S. obliquus contains all the motifs necessary for optimal self-splicing and which are characteristic of other group IIB introns from different species.