Group II intron and repeat-rich red algal mitochondrial genomes demonstrate the dynamic recent history of autocatalytic RNAs

Background Group II introns are mobile genetic elements that can insert at specific target sequences, however, their origins are often challenging to reconstruct because of rapid sequence decay following invasion and spread into different sites. To advance understanding of group II intron spread, we studied the intron-rich mitochondrial genome (mitogenome) in the unicellular red alga, Porphyridium. Results Analysis of mitogenomes in three closely related species in this genus revealed they were 3–6-fold larger in size (56–132 kbp) than in other red algae, that have genomes of size 21–43 kbp. This discrepancy is explained by two factors, group II intron invasion and expansion of repeated sequences in large intergenic regions. Phylogenetic analysis demonstrates that many mitogenome group II intron families are specific to Porphyridium, whereas others are closely related to sequences in fungi and in the red alga-derived plastids of stramenopiles. Network analysis of intron-encoded proteins (IEPs) shows a clear link between plastid and mitochondrial IEPs in distantly related species, with both groups associated with prokaryotic sequences. Conclusion Our analysis of group II introns in Porphyridium mitogenomes demonstrates the dynamic nature of group II intron evolution, strongly supports the lateral movement of group II introns among diverse eukaryotes, and reveals their ability to proliferate, once integrated in mitochondrial DNA.

Distinct Expansion of Group II Introns Depends on the Type of Intron-encoded Protein and Genomic Signatures in Prokaryotes

Group II introns (G2Is) are self-splicing ribozymes that have retroelement characteristics in prokaryotes. Although G2Is are considered an important factor in the evolution of prokaryotes, comprehensive analyses of these introns among the tens of thousands of prokaryotic genomes currently available are still limited. Here, we developed a bioinformatic pipeline that systematically collects G2Is and applied it to prokaryotic genomes. We found that in bacteria, 25% (447 of 1,790) of the total representative species had an average of 5.3 G2Is, and in archaea, 9% (28 of 296) of the total representative species had an average of 3.0 G2Is. The greatest number of G2Is per species was 101 in Arthrospira platensis (phylum Cyanobacteriota). A comprehensive sequence analysis of the intron-encoded protein (IEP) in each G2I sequence was conducted and resulted in the addition of three new IEP classes (U1–U3) to the previous classification. This analysis suggested that about 30% of all IEPs are noncanonical IEPs. The number of G2Is per species was defined almost at the phylum level, and the type of IEP was associated as a factor in the G2I increase, i.e. there was an explosive increase in G2Is with bacterial C-type IEPs in the phylum Firmicutes and in G2Is with CL-type IEPs in the phylum Cyanobacteriota. We also systematically analyzed the relationship between genomic signatures and the mechanism of these increases in G2Is. This is the first study to systematically characterize G2Is in the prokaryotic phylogenies.

Organellar Introns in Fungi, Algae, and Plants

Introns are ubiquitous in eukaryotic genomes and have long been considered as ‘junk RNA’ but the huge energy expenditure in their transcription, removal, and degradation indicate that they may have functional significance and can offer evolutionary advantages. In fungi, plants and algae introns make a significant contribution to the size of the organellar genomes. Organellar introns are classified as catalytic self-splicing introns that can be categorized as either Group I or Group II introns. There are some biases, with Group I introns being more frequently encountered in fungal mitochondrial genomes, whereas among plants Group II introns dominate within the mitochondrial and chloroplast genomes. Organellar introns can encode a variety of proteins, such as maturases, homing endonucleases, reverse transcriptases, and, in some cases, ribosomal proteins, along with other novel open reading frames. Although organellar introns are viewed to be ribozymes, they do interact with various intron- or nuclear genome-encoded protein factors that assist in the intron RNA to fold into competent splicing structures, or facilitate the turn-over of intron RNAs to prevent reverse splicing. Organellar introns are also known to be involved in non-canonical splicing, such as backsplicing and trans-splicing which can result in novel splicing products or, in some instances, compensate for the fragmentation of genes by recombination events. In organellar genomes, Group I and II introns may exist in nested intronic arrangements, such as introns within introns, referred to as twintrons, where splicing of the external intron may be dependent on splicing of the internal intron. These nested or complex introns, with two or three-component intron modules, are being explored as platforms for alternative splicing and their possible function as molecular switches for modulating gene expression which could be potentially applied towards heterologous gene expression. This review explores recent findings on organellar Group I and II introns, focusing on splicing and mobility mechanisms aided by associated intron/nuclear encoded proteins and their potential roles in organellar gene expression and cross talk between nuclear and organellar genomes. Potential application for these types of elements in biotechnology are also discussed.

The association of group IIB intron with integrons in hypersaline environments

Background Group II introns are mobile genetic elements used as efficient gene targeting tools. They function as both ribozymes and retroelements. Group IIC introns are the only class reported so far to be associated with integrons. In order to identify group II introns linked with integrons and CALINS (cluster of attC sites lacking a neighboring integron integrase) within halophiles, we mined for integrons in 28 assembled metagenomes from hypersaline environments and publically available 104 halophilic genomes using Integron Finder followed by blast search for group II intron reverse transcriptases (RT)s. Results We report the presence of different group II introns associated with integrons and integron-related sequences denoted by UHB.F1, UHB.I2, H.ha.F1 and H.ha.F2. The first two were identified within putative integrons in the metagenome of Tanatar-5 hypersaline soda lake, belonging to IIC and IIB intron classes, respectively at which the first was a truncated intron. Other truncated introns H.ha.F1 and H.ha.F2 were also detected in a CALIN within the extreme halophile Halorhodospira halochloris, both belonging to group IIB introns. The intron-encoded proteins (IEP) s identified within group IIB introns belonged to different classes: CL1 class in UHB.I2 and bacterial class E in H.ha.Fa1 and H.ha.F2. A newly identified insertion sequence (ISHahl1) of IS200/605 superfamily was also identified adjacent to H. halochloris CALIN. Finally, an abundance of toxin-antitoxin (TA) systems was observed within the identified integrons. Conclusion So far, this is the first investigation of group II introns within integrons in halophilic genomes and metagenomes from hypersaline environments. We report the presence of group IIB introns associated with integrons or CALINs. This study provides the basis for understanding the role of group IIB introns in the evolution of halophiles and their potential biotechnological role.

The Chloroplast Trans-Splicing RNA–Protein Supercomplex from the Green Alga Chlamydomonas reinhardtii

In eukaryotes, RNA trans-splicing is a significant RNA modification process for the end-to-end ligation of exons from separately transcribed primary transcripts to generate mature mRNA. So far, three different categories of RNA trans-splicing have been found in organisms within a diverse range. Here, we review trans-splicing of discontinuous group II introns, which occurs in chloroplasts and mitochondria of lower eukaryotes and plants. We discuss the origin of intronic sequences and the evolutionary relationship between chloroplast ribonucleoprotein complexes and the nuclear spliceosome. Finally, we focus on the ribonucleoprotein supercomplex involved in trans-splicing of chloroplast group II introns from the green alga Chlamydomonas reinhardtii. This complex has been well characterized genetically and biochemically, resulting in a detailed picture of the chloroplast ribonucleoprotein supercomplex. This information contributes substantially to our understanding of the function of RNA-processing machineries and might provide a blueprint for other splicing complexes involved in trans- as well as cis-splicing of organellar intron RNAs.

Identification and Classification of Reverse Transcriptases in Bacterial Genomes and Metagenomes

ABSTRACTReverse Transcriptases (RTs) are found in different systems including group II introns, Diversity Generating Retroelements (DGRs), retrons, CRISPR-Cas systems, and Abortive Infection (Abi) systems in prokaryotes. Different classes of RTs can play different roles, such as template switching and mobility in group II introns, spacer acquisition in CRISPR-Cas systems, mutagenic retrohoming in DGRs, programmed cell suicide in Abi systems, and recently discovered phage defense in retrons. While some classes of RTs have been studied extensively, others remain to be characterized. There is a lack of computational tools for identifying and characterizing various classes of RTs. In this study, we built a tool (called myRT) for identification and classification of prokaryotic RTs. In addition, our tool provides information about the genomic neighborhood of each RT, providing potential functional clues. We applied our tool to predict and classify RTs in all complete and draft bacterial genomes, and created a collection that can be used for exploration of putative RTs. Application of myRT to gut metagenomes showed that gut metagenomes encode proportionally more RTs related to DGRs, outnumbering retron-related RTs, as compared to the collection of reference genomes. MyRT is both available as a standalone software and also through a website.