Ancient intron insertion sites and palindromic genomic duplication evolutionally shapes an elementally functioning membrane protein family

Background  

In spite of the recent accumulation of genomic data, the evolutionary pathway in the individual genes of present-day living taxa is still elusive for most genes. Among ion channels, inward K⁺ rectifier (IRK) channels are the fundamental and well-defined protein group. We analyzed the genomic structures of this group and compared them among a phylogenetically wide range with our sequenced Halocynthia roretzi, a tunicate, IRK genomic genes.     

Group II intron retroelements: function and diversity

Group II introns are a class of retroelements capable of carrying out both self-splicing and retromobility reactions. In recent years, the number of known group II introns has increased dramatically, particularly in bacteria, and the new information is altering our understanding of these intriguing elements. Here we review the basic properties of group II introns, and summarize the differences between the organellar and bacterial introns with regard to structures, insertion patterns and inferred behaviors. We also discuss the evolution of group II introns, as they are the putative ancestors of spliceosomal introns and possibly non-LTR retroelements, and may have played an important role in the development of eukaryote genomes.     

Metal ion binding sites in a group II intron core

Group II introns are catalytic RNA molecules that require divalent metal ions for folding, substrate binding, and chemical catalysis. Metal ion binding sites in the group II core have now been elucidated by monitoring the site-specific RNA hydrolysis patterns of bound ions such as Tb(3+) and Mg(2+). Major sites are localized near active site elements such as domain 5 and its surrounding tertiary interaction partners. Numerous sites are also observed at intron substructures that are involved in binding and potentially activating the splice sites. These results highlight the locations of specific metal ions that are likely to play a role in ribozyme catalysis.     

Group II intron splicing factors derived by diversification of an ancient RNA-binding domain

Group II introns are ribozymes whose catalytic mechanism closely resembles that of the spliceosome. Many group II introns have lost the ability to splice autonomously as the result of an evolutionary process in which the loss of self-splicing activity was compensated by the recruitment of host-encoded protein cofactors. Genetic screens previously identified CRS1 and CRS2 as host-encoded proteins required for the splicing of group II introns in maize chloroplasts. Here, we describe two additional host-encoded group II intron splicing factors, CRS2-associated factors 1 and 2 (CAF1 and CAF2). We show that CRS2 functions in the context of intron ribonucleoprotein particles that include either CAF1 or CAF2, and that CRS2-CAF1 and CRS2-CAF2 complexes have distinct intron specificities. CAF1, CAF2 and the previously described group II intron splicing factor CRS1 are characterized by similar repeated domains, which we name here the CRM (chloroplast RNA splicing and ribosome maturation) domains. We propose that the CRM domain is an ancient RNA-binding module that has diversified to mediate specific interactions with various highly structured RNAs.     

Cytochemical localization of photosystem II donor sites

Summary Tetramethylbenzidene, an artificial donor of electrons to the photosystem (PS) II reaction center, is oxidized to an osmiophilic polymer that allows for the localization of the donor site of PS II. Mesophyll chloroplasts of Sorghum bicolor (L.) Moench. contain 0.1–0.2 m deposits only in the grana stacks and on the lumen side of the thylakoid. Agranal mature bundle sheath plastids (known to be devoid of PS II activity) show little deposition and the products appear randomly distributed along the lamellae. The cytochemical localization of PS II donor sites on the lumen side is consistent with flash photolysis and diaminobenzenesulfonic acid inactivation studies as well as the chemiosmotic theory for the generation of a proton gradient.     

Group II intron RNA-catalyzed recombination of RNA in vitro.

We report the first evidence for a novel reaction mediated by the self-splicing yeast mitochondrial group II intron bl1; the site-specific recombination of RNA molecules in vitro. Upon incubation of the intron lariat with two different RNAs, each harbouring a short sequence complementary to exon binding site 1 (EBS1) of the intron, novel recombined RNAs are formed. As a result of this intron-mediated shuffling of gene segments, the 5' part of RNA1 is ligated to the 3' part of RNA2 and, reciprocally, the 5' part of RNA2 to the 3' part of RNA1. Sequence analysis of the recombinant junction shows that the site of recombination is precisely located 3' to intron binding site 1 (IBS1). The hypothesized mechanism of recombination involves exchange of RNA 5' parts after the first step of a reverse splicing reaction. The possible role of this mechanism in vivo and during prebiotic evolution is discussed.     

Group II intron domain 5 facilitates a trans-splicing reaction.

A self-splicing group II intron of yeast mitochondrial DNA (aI5g) was divided within intron domain 4 to yield two RNAs that trans-spliced in vitro with associated trans-branching of excised intron fragments. Reformation of the domain 4 secondary structure was not necessary for the trans reaction, since domain 4 sequences were shown to be dispensable. Instead, the trans reaction depended on a previously unpredicted interaction between intron domain 5, the most highly conserved region of group II introns, and another region of the RNA. Domain 5 was shown to be essential for cleavage at the 5' splice site. It stimulated that cleavage when supplied as a trans-acting RNA containing only 42 nucleotides of intron sequence. The relevance of our findings to in vivo trans-splicing mechanisms is discussed.     

Group II intron splicing in chloroplasts: identificationof mutations determining intron stability and fate of exon RNA.

In order to investigate in vivo splicing of group II introns in chloroplasts, we previously have integrated the mitochondrial intron rI1 from the green alga Scenedesmus obliquus into the Chlamydomonas chloroplast tscA gene. This construct allows a functional analysis of conserved intron sequences in vivo, since intron rI1 is correctly spliced in chloroplasts. Using site-directed mutagenesis, deletions of the conserved intron domains V and VI were performed. In another set of experiments, each possible substitution of the strictly conserved first intron nucleotide G1 was generated, as well as each possible single and double mutation of the tertiary base pairing gamma-gamma ' involved in the formation of the intron's tertiary RNA structure. In most cases, the intron mutations showed the same effect on in vivo intron splicing efficiency as they did on the in vitro self-splicing reaction, since catalytic activity is provided by the intron RNA itself. In vivo, all mutations have additional effects on the chimeric tscA -rI1 RNA, most probably due to the role played by trans -acting factors in intron processing. Substitutions of the gamma-gamma ' base pair lead to an accumulation of excised intron RNA, since intron stability is increased. In sharp contrast to autocatalytic splicing, all point mutations result in a complete loss of exon RNA, although the spliced intron accumulates to high levels. Intron degradation and exon ligation only occur in double mutants with restored base pairing between the gamma and gamma' sites. Therefore, we conclude that intron degradation, as well as the ligation of exon-exon molecules, depends on the tertiary intron structure. Furthermore, our data suggest that intron excision proceeds in vivo independent of ligation of exon-exon molecules.     

Group II intron self-splicing. Alternative reaction conditions yield novel products

Reaction parameters were modified to enhance the in vitro reaction rate and to reveal partial and novel reactions of the group II intron 5g of the mitochondrial gene from Saccharomyces cerevisiae encoding cytochrome c oxidase subunit I. One alteration yields separate 5'- and 3'-exons plus linear excised intron as the main products. A linear reaction intermediate, containing intron and 3'-exon, and products resulting from cleavages at two unexpected sites were identified. Spliced exon "reopening," a novel reaction between excised intron and spliced exons, appears responsible for separate 5'- and 3'-exon products.     

Group II twintron: an intron within an intron in a chloroplast cytochrome b-559 gene.

The psbF gene of chloroplast DNAs encodes the beta-subunit of cytochrome b-559 of the photosystem II reaction center. The psbF locus of Euglena gracilis chloroplast DNA has an unusual 1042 nt group II intron that appears to be formed from the insertion of one group II intron into structural domain V of a second group II intron. Using both direct primer extension cDNA sequencing and cDNA cloning and sequencing, we have determined that a 618 nt internal intron is first excised from the 1042 nt intron of psbF pre-mRNA, resulting in a partially spliced pre-mRNA containing a 424 nt group II intron with a spliced domain V. The 424 nt intron is then removed to yield the mature psbF mRNA. Therefore, the 1042 nt intron of psbF is a group II intron within another group II intron. We use the term 'twintron' to define this new type of genetic element. Intermediates in the splicing pathway were detected by northern hybridization. Splicing of both the internal and external introns occurs via lariat intermediates. Twintron splicing was found to proceed by a sequential pathway, the internal intron being removed prior to the excision of the external intron. A possible mechanism for twintron formation by intron transposition is discussed.     

Restriction for gene insertion within the Lactococcus lactis Ll.LtrB group II intron

The Ll.LtrB intron, from the low G+C gram-positive bacterium Lactococcus lactis, was the first bacterial group II intron shown to splice and mobilize in vivo. The detailed retrohoming and retrotransposition pathways of Ll.LtrB were studied in both L. lactis and Escherichia coli. This bacterial retroelement has many features that would make it a good gene delivery vector. Here we report that the mobility efficiency of Ll.LtrB expressing LtrA in trans is only slightly affected by the insertion of fragments <100 nucleotides within the loop region of domain IV. In contrast, Ll.LtrB mobility efficiency is drastically decreased by the insertion of foreign sequences >1 kb. We demonstrate that the inhibitory effect caused by the addition of expression cassettes on Ll.LtrB mobility efficiency is not sequence specific, and not due to the expression, or the toxicity, of the cargo genes. Using genetic screens, we demonstrate that in order to maintain intron mobility, the loop region of domain IV, more specifically domain IVb, is by far the best region to insert foreign sequences within Ll.LtrB. Poisoned primer extension and Northern blot analyses reveal that Ll.LtrB constructs harboring cargo sequences splice less efficiently, and show a significant reduction in lariat accumulation in L. lactis. This suggests that cargo-containing Ll.LtrB variants are less stable. These results reveal the potential, yet limitations, of the Ll.LtrB group II intron to be used as a gene delivery vector, and validate the random insertion approach described in this study to create cargo-containing Ll.LtrB variants that are mobile.     

Evolution of the EF-hand calcium-binding protein family: Evidence for exon shuffling and intron insertion

Summary The evolutionary history of the intracellular calcium-binding protein superfamily is well documented. The members of this gene family are all believed to be derived from a common ancestor, which, itself, was the product of two successive gene duplications. In this study, we have compared and analyzed the structures of the recently described genes coding for these proteins. We propose a series of evolutionary events, which include exon shuffling and intron insertion, that could account for the evolutionary origin of all the members of this super-family. According to this hypothesis, the ancestral gene, a product of two successive duplications, consisted of at least four exons. Each exon coding for a peptide (a calcium-binding domain) was separated by an intron that had mediated the duplication. Each distinct lineage evolved from this ancestor by genomic rearrangement, with insertion of introns being a prominent feature.     

Group I intron located in PR protein homologue gene in Youngia japonica

A Youngia japonica strain had a group I intron that was suggested to have been transferred from Protomyces inouyei, a pathogenic fungus of Y. japonica. It was located in the miraculin homologue coding gene by reverse complementation. The deduced amino acid sequence of this miraculin homologue of Y. japonica was similar to the amino acid sequences of tobacco and tomato pathogenesis-related proteins.     

Cytochrome oxidase subunit II gene of rice has an insertion sequence within the intron.

We have isolated and sequenced the cytochrome oxidase subunit II gene from rice (Oryza sativa L. var Labelle). The overall structural organization of this gene is very similar to that of the maize gene. This gene contains an intron in a position identical to the intron in the maize gene. However, the intron in the rice gene is longer than that of the maize gene largely due to a 461 bp insertion sequence, which has inverted repeats at its termini and is flanked by direct repeats, characteristic of transposable elements. Apart from this insertion sequence, the remainder of the intron sequence is strikingly homologous to that of maize (98.6% homology), suggesting a possible functional or structural role. The coding regions of the two genes exhibit 99.5% nucleotide sequence homology and their deduced amino acid sequences are identical. Similarly, the 3'-noncoding regions, except for several small insertions and deletions, show complete sequence homology. On the contrary, no sequence homology is detected in the 5'-noncoding regions.     

The two steps of group II intron self-splicing are mechanistically distinguishable.

The two transesterification reactions catalyzed by self-splicing group II introns take place in either two active sites or two conformations of a single active site involving rearrangements of the positions of the reacting groups. We have investigated the effects on the rates of the chemical steps of the two reactions due to sulfur substitution of nonbridging oxygens at both the 5' and 3' splice sites as well as the deoxyribose substitution of the ribose 2' hydroxyl group at the 5' splice site. The data suggest that the two active sites differ in their interactions with several of these groups. Specifically, sulfur substitution of the pro-Sp nonbridging oxygen at the 5' splice site reduces the chemical rate of the step one branching reaction by at least 250-fold, whereas substitution of the pro-Sp oxygen at the 3' splice site has only a 4.5-fold effect on the chemical rate of step two. Previous work demonstrated that the Rp phosphorothioate substitutions at both the 5' and 3' splice sites reduced the rate of both steps of splicing to an undetectable level. These results suggest that either two distinct active sites catalyze the two steps or that more significant alterations must be made in a single bifunctional active site to accommodate the two different reactions.