In vivo Validation of Hsp90 Trans-splicing in Giardia lamblia: Highlighting the Role of Cis-elements.

Giardia lambliacauses giardiasis, one of the most common human infectious diseases globally. Previous studies from our lab have shown that hsp90 gene ofGiardia is split into two halves, namely hspN and hspC. The independent pre-mRNAs of these split genes join by trans-splicing, producing a full-length Hsp90 (FlHsp90) mRNA. Genetic manipulation of the participating genes is necessary to understand the mechanism and significance of such trans-splicing based expression of Hsp90. In this study, we have performed transfection based exogenous expression of hspN and/or hspC in G. lamblia. We electroporated a plasmid containing the Avi-tagged hspN component of Hsp90 and examined its fate in G. lamblia. We show that the exogenously expressed hspN RNA gets trans-spliced to endogenously expressed hspC RNA, giving rise to a hybrid-FlHsp90. We highlight the importance of cis-elements in this trans-splicing reaction through mutational analysis. The episomal plasmid carrying deletions in the intronic region of hspN, showed inhibition of the trans-splicing reaction.Additionally, exogenous hspC RNA also followed the same fate as of exogenous hspN, while upon co-transfection with episomal hspN, they underwent trans-splicing with each other. Using eGFP as a test protein, we have shown that intronic sequences of hsp90 gene can guide trans-splicing mediated repair of any associated exonic sequences. Our study provides in vivo validation of Hsp90 trans-splicing, showing crucial role of cis-elements and importantly highlights the potential of hsp90 intronic sequences to function as a minimal splicing tool.

mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy.

Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.

The SNAPc complex mediates starvation-induced trans-splicing in Caenorhabditis elegans.

Dietary restriction usually suppresses biosynthesis but activates catabolic pathways in animals. However, the short-term starvation enhances biosynthetic activities and promotes ribosomal biogenesis in adult Caenorhabditis elegans. The mechanism underlying the processes remains largely unknown. Here, we find that the short-term starvation enhances the SL1 trans-splicing of translation-related genes in adult C. elegans by transcriptome analysis. The small nuclear RNA-activating protein complex (SNAPc) promotes SL RNA production and mediates starvation-induced trans-splicing. TOFU-5, a core factor in the upstream sequence transcription complex (USTC) essential for piRNA production, is also involved in the starvation-induced trans-splicing processes. Knocking down components of the SNAPc complex and tofu-5 extends worm survival under starvation conditions. Taken together, our study highlights the importance of SL trans-splicing in the nutrition response and reveals a mechanism of the survival regulation by food deprivation via SNAPc and TOFU-5.

SLIDR and SLOPPR: flexible identification of spliced leader trans-splicing and prediction of eukaryotic operons from RNA-Seq data.

BACKGROUND: Spliced leader (SL) trans-splicing replaces the 5' end of pre-mRNAs with the spliced leader, an exon derived from a specialised non-coding RNA originating from elsewhere in the genome. This process is essential for resolving polycistronic pre-mRNAs produced by eukaryotic operons into monocistronic transcripts. SL trans-splicing and operons may have independently evolved multiple times throughout Eukarya, yet our understanding of these phenomena is limited to only a few well-characterised organisms, most notably C. elegans and trypanosomes. The primary barrier to systematic discovery and characterisation of SL trans-splicing and operons is the lack of computational tools for exploiting the surge of transcriptomic and genomic resources for a wide range of eukaryotes. RESULTS: Here we present two novel pipelines that automate the discovery of SLs and the prediction of operons in eukaryotic genomes from RNA-Seq data. SLIDR assembles putative SLs from 5' read tails present after read alignment to a reference genome or transcriptome, which are then verified by interrogating corresponding SL RNA genes for sequence motifs expected in bona fide SL RNA molecules. SLOPPR identifies RNA-Seq reads that contain a given 5' SL sequence, quantifies genome-wide SL trans-splicing events and predicts operons via distinct patterns of SL trans-splicing events across adjacent genes. We tested both pipelines with organisms known to carry out SL trans-splicing and organise their genes into operons, and demonstrate that (1) SLIDR correctly detects expected SLs and often discovers novel SL variants; (2) SLOPPR correctly identifies functionally specialised SLs, correctly predicts known operons and detects plausible novel operons. CONCLUSIONS: SLIDR and SLOPPR are flexible tools that will accelerate research into the evolutionary dynamics of SL trans-splicing and operons throughout Eukarya and improve gene discovery and annotation for a wide range of eukaryotic genomes. Both pipelines are implemented in Bash and R and are built upon readily available software commonly installed on most bioinformatics servers. Biological insight can be gleaned even from sparse, low-coverage datasets, implying that an untapped wealth of information can be retrieved from existing RNA-Seq datasets as well as from novel full-isoform sequencing protocols as they become more widely available.

Resolution of polycistronic RNA by SL2 trans-splicing is a widely conserved nematode trait.

Spliced leader trans-splicing is essential for the processing and translation of polycistronic RNAs generated by eukaryotic operons. In C. elegans, a specialized spliced leader, SL2, provides the 5' end for uncapped pre-mRNAs derived from polycistronic RNAs. Studies of other nematodes suggested that SL2-type trans-splicing is a relatively recent innovation, confined to Rhabditina, the clade containing C. elegans and its close relatives. Here we conduct a survey of transcriptome-wide spliced leader trans-splicing in Trichinella spiralis, a distant relative of C. elegans with a particularly diverse repertoire of 15 spliced leaders. By systematically comparing the genomic context of trans-splicing events for each spliced leader, we identified a subset of T. spiralis spliced leaders that are specifically used to process polycistronic RNAs-the first examples of SL2-type spliced leaders outside of Rhabditina. These T. spiralis spliced leader RNAs possess a perfectly conserved stem-loop motif previously shown to be essential for SL2-type trans-splicing in C. elegans We show that genes trans-spliced to these SL2-type spliced leaders are organized in operonic fashion, with short intercistronic distances. A subset of T. spiralis operons show conservation of synteny with C. elegans operons. Our work substantially revises our understanding of nematode spliced leader trans-splicing, showing that SL2 trans-splicing is a major mechanism for nematode polycistronic RNA processing, which may have evolved prior to the radiation of the Nematoda. This work has important implications for the improvement of genome annotation pipelines in nematodes and other eukaryotes with operonic gene organization.

Evidence of constraint in the 3D genome for trans-splicing in human cells.

Fusion transcripts are commonly found in eukaryotes, and many aberrant fusions are associated with severe diseases, including cancer. One class of fusion transcripts is generated by joining separate transcripts through trans-splicing. However, the mechanism of trans-splicing in mammals remains largely elusive. Here we showed evidence to support an intuitive hypothesis that attributes trans-sphcing to the spatial proximity between premature transcripts. A novel trans-splicing detection tool (TSD) was developed to reliably identify intra-chromosomal trans-splicing events (iTSEs) from RNA-seq data. TSD can maintain a remarkable balance between sensitivity and accuracy, thus distinguishing it from most state-of-the-art tools. The accuracy of TSD was experimentally demonstrated by excluding potential false discovery from mosaic genome or template switching during PCR. We showed that iTSEs identified by TSD were frequently found between genomic regulatory elements, which are known to be more prone to interact with each other. Moreover, iTSE sites may be more physically adjacent to each other than random control in the tested human lymphoblastoid cell line according to Hi-C data. Our results suggest that trans-splicing and 3D genome architecture may be coupled in mammals and that our pipeline, TSD, may facilitate investigations of trans-splicing on a systematic and accurate level previously thought impossible.

Small Animal PET Imaging of hTERT RNA-Targeted HSV1-tk Gene Expression with Trans-Splicing Ribozyme.

Background: Trans-splicing ribozymes (TSR) are useful anticancer agents targeting cancer-specific transcripts and replacing the RNA to induce anticancer gene expression specifically and selectively in cancer cells. Similar to other gene therapy methods, it is also important to evaluate the transgene expression for target specificity and ribozyme activity. Materials and Methods: In this study, the authors performed in vivo small animal positron emission tomography (PET) imaging and biodistribution assay to evaluate human telomerase reverse transcriptase (hTERT) RNA-targeting-specific TSR, which directs the expression of herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene selectively in hTERT-positive tumors through targeted RNA replacement of the hTERT transcript. Results: The hTERT RNA-targeted HSV1-tk expression with TSR was monitored by PET imaging with 124I labeled 2'-fluoro-2'-deoxy-1-ß-D-arabinofuranosyl-5-iodouracil, which is one of the thymidine derivatives acting as substrates for HSV1-tk, in hTERT-positive tumor-bearing mice. Conclusions: Imaging of hTERT RNA-targeted HSV1-tk expression by TSR could be used in the development of advanced gene therapy using tumor-specific TSR.

ChiTaRS 5.0: the comprehensive database of chimeric transcripts matched with druggable fusions and 3D chromatin maps.

Chimeric RNA transcripts are formed when exons from two genes fuse together, often due to chromosomal translocations, transcriptional errors or trans-splicing effect. While these chimeric RNAs produce functional proteins only in certain cases, they play a significant role in disease phenotyping and progression. ChiTaRS 5.0 (http://chitars.md.biu.ac.il/) is the latest and most comprehensive chimeric transcript repository, with 111 582 annotated entries from eight species, including 23 167 known human cancer breakpoints. The database includes unique information correlating chimeric breakpoints with 3D chromatin contact maps, generated from public datasets of chromosome conformation capture techniques (Hi-C). In this update, we have added curated information on druggable fusion targets matched with chimeric breakpoints, which are applicable to precision medicine in cancers. The introduction of a new section that lists chimeric RNAs in various cell-lines is another salient feature. Finally, using text-mining techniques, novel chimeras in Alzheimer's disease, schizophrenia, dyslexia and other diseases were collected in ChiTaRS. Thus, this improved version is an extensive catalogue of chimeras from multiple species. It extends our understanding of the evolution of chimeric transcripts in eukaryotes and contributes to the analysis of 3D genome conformational changes and the functional role of chimeras in the etiopathogenesis of cancers and other complex diseases.

Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing.

Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system with potential for regulation of site-specific recombination events at the protein level in vivo. The ϕC31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in Escherichia coli generated a fully functional ϕC31 integrase. We showed that protein splicing is necessary for recombination activity; deletion of intein domains or mutation of key intein residues inactivated recombination. We used an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system in building reversible genetic switches. We used the same split inteins to control the reconstitution of a split Integrase-Recombination Directionality Factor fusion (Integrase-RDF) that efficiently catalysed the reverse attR x attL recombination. This demonstrates the potential for split-intein regulation of the forward and reverse reactions using the integrase and the integrase-RDF fusion, respectively. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.

The vault RNA of Trypanosoma brucei plays a role in the production of trans-spliced mRNA.

The vault ribonucleoprotein (RNP), comprising vault RNA (vtRNA) and telomerase-associated protein 1 (TEP1), is found in many eukaryotes. However, previous studies of vtRNAs, for example in mammalian cells, have failed to reach a definitive conclusion about their function. vtRNAs are related to Y RNAs, which are complexed with Ro protein and influence Ro's function in noncoding RNA (ncRNA) quality control and processing. In Trypanosoma brucei, the small noncoding TBsRNA-10 was first described in a survey of the ncRNA repertoire in this organism. Here, we report that TBsRNA-10 in T. brucei is a vtRNA, based on its association with TEP1 and sequence similarity to those of other known and predicted vtRNAs. We observed that like vtRNAs in other species, TBsRNA-10 is transcribed by RNA polymerase III, which in trypanosomes also generates the spliceosomal U-rich small nuclear RNAs. In T. brucei, spliced leader (SL)-mediated trans-splicing of pre-mRNAs is an obligatory step in gene expression, and we found here that T. brucei's vtRNA is highly enriched in a non-nucleolar locus in the cell nucleus implicated in SL RNP biogenesis. Using a newly developed permeabilized cell system for the bloodstream form of T. brucei, we show that down-regulated vtRNA levels impair trans-spliced mRNA production, consistent with a role of vtRNA in trypanosome mRNA metabolism. Our results suggest a common theme for the functions of vtRNAs and Y RNAs. We conclude that by complexing with their protein-binding partners TEP1 and Ro, respectively, these two RNA species modulate the metabolism of various RNA classes.

The coordinated action of PPR4 and EMB2654 on each intron half mediates trans-splicing of rps12 transcripts in plant chloroplasts.

The pentatricopeptide repeat proteins PPR4 and EMB2654 have been shown to be required for the trans-splicing of plastid rps12 transcripts in Zea mays (maize) and Arabidopsis, respectively, but their roles in this process are not well understood. We investigated the functions of the Arabidopsis and Oryza sativa (rice) orthologs of PPR4, designated AtPPR4 (At5g04810) and OsPPR4 (Os4g58780). Arabidopsis atppr4 and rice osppr4 mutants are embryo-lethal and seedling-lethal 3 weeks after germination, respectively, showing that PPR4 is essential in the development of both dicot and monocot plants. Artificial microRNA-mediated mutants of AtPPR4 displayed a specific defect in rps12 trans-splicing, with pale-green, yellowish or albino phenotypes, according to the degree of knock-down of AtPPR4 expression. Comparison of RNA footprints in atppr4 and emb2654 mutants showed a similar concordant loss of extensive footprints at the 3' end of intron 1a and at the 5' end of intron 1b in both cases. EMB2654 is known to bind within the footprint region in intron 1a and we show that AtPPR4 binds to the footprint region in intron 1b, via its PPR motifs. Binding of both PPR4 and EMB2654 is essential to juxtapose the two intron halves and to maintain the RNAs in a splicing-competent structure for the efficient trans-splicing of rps12 intron 1, which is crucial for chloroplast biogenesis and plant development. The similarity of EMB2654 and PPR4 orthologs and their respective binding sites across land plant phylogeny indicates that their coordinate function in rps12 trans-splicing has probably been conserved for 500 million years.

Evolution of intron splicing towards optimized gene expression is based on various Cis- and Trans-molecular mechanisms.

Splicing expands, reshapes, and regulates the transcriptome of eukaryotic organisms. Despite its importance, key questions remain unanswered, including the following: Can splicing evolve when organisms adapt to new challenges? How does evolution optimize inefficiency of introns' splicing and of the splicing machinery? To explore these questions, we evolved yeast cells that were engineered to contain an inefficiently spliced intron inside a gene whose protein product was under selection for an increased expression level. We identified a combination of mutations in Cis (within the gene of interest) and in Trans (in mRNA-maturation machinery). Surprisingly, the mutations in Cis resided outside of known intronic functional sites and improved the intron's splicing efficiency potentially by easing tight mRNA structures. One of these mutations hampered a protein's domain that was not under selection, demonstrating the evolutionary flexibility of multi-domain proteins as one domain functionality was improved at the expense of the other domain. The Trans adaptations resided in two proteins, Npl3 and Gbp2, that bind pre-mRNAs and are central to their maturation. Interestingly, these mutations either increased or decreased the affinity of these proteins to mRNA, presumably allowing faster spliceosome recruitment or increased time before degradation of the pre-mRNAs, respectively. Altogether, our work reveals various mechanistic pathways toward optimizations of intron splicing to ultimately adapt gene expression patterns to novel demands.

Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina.

Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.

Rice FLOURY ENDOSPERM10 encodes a pentatricopeptide repeat protein that is essential for the trans-splicing of mitochondrial nad1 intron 1 and endosperm development.

Endosperm, the major storage organ in cereal grains, determines grain yield and quality. Despite the fact that a role for P-type pentatricopeptide repeat (PPR) proteins in the regulation of endosperm development has emerged, molecular functions of many P-type PPR proteins remain obscure. Here, we report a rice endosperm defective mutant, floury endosperm10 (flo10), which developed smaller starch grains in starchy endosperm and abnormal cells in the aleurone layer. Map-based cloning and rescued experiments showed that FLO10 encodes a P-type PPR protein with 26 PPR motifs, which is localized to mitochondria. Loss of function of FLO10 affected the trans-splicing of the mitochondrial nad1 intron 1, which was accompanied by the increased accumulation of the nad1 exon 1 and exons 2-5 precursors. The failed formation of mature nad1 led to a dramatically decreased assembly and activity of complex I, reduced ATP production, and changed mitochondrial morphology. In addition, loss of function of FLO10 significantly induced an alternative respiratory pathway involving alternative oxidase. These results reveal that FLO10 plays an important role in the maintenance of mitochondrial function and endosperm development through its effect on the trans-splicing of the mitochondrial nad1 intron 1 in rice.

Heterodera glycines utilizes promiscuous spliced leaders and demonstrates a unique preference for a species-specific spliced leader over C. elegans SL1.

Spliced leader trans-splicing (SLTS) plays a part in the maturation of pre-mRNAs in select species across multiple phyla but is particularly prevalent in Nematoda. The role of spliced leaders (SL) within the cell is unclear and an accurate assessment of SL occurrence within an organism is possible only after extensive sequencing data are available, which is not currently the case for many nematode species. SL discovery is further complicated by an absence of SL sequences from high-throughput sequencing results due to incomplete sequencing of the 5'-ends of transcripts during RNA-seq library preparation, known as 5'-bias. Existing datasets and novel methodology were used to identify both conserved SLs and unique hypervariable SLs within Heterodera glycines, the soybean cyst nematode. In H. glycines, twenty-one distinct SL sequences were found on 2,532 unique H. glycines transcripts. The SL sequences identified on the H. glycines transcripts demonstrated a high level of promiscuity, meaning that some transcripts produced as many as nine different individual SL-transcript combinations. Most uniquely, transcriptome analysis revealed that H. glycines is the first nematode to demonstrate a higher SL trans-splicing rate using a species-specific SL over well-conserved Caenorhabditis elegans SL-like sequences.

Light-control of the ultra-fast Gp41-1 split intein with preserved stability of a genetically encoded photo-caged amino acid in bacterial cells.

Inteins change the structure and function of their host protein in a unique way and the Gp41-1 split intein is the fastest protein trans-splicing intein known to date. To design a photo-activatable variant, we have incorporated ortho-nitrobenzyl-tyrosine (ONBY) at the position of a structurally conserved phenylalanine in the Gp41-1-N fragment. Using irradiation at 365 nm, the splicing reaction was triggered with virtually unchanged rates. The partial cellular reduction of the nitro group in ONBY, previously observed during bacterial protein expression for several photo-caged amino acids, was overcome by periplasmatic expression and by using an E. coli K12(DE3) strain instead of BL21(DE3). Together, our findings provide new tools for the artificial photo-control of proteins.

Trypanosomes can initiate nuclear export co-transcriptionally.

The nuclear envelope serves as important messenger RNA (mRNA) surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualized nuclear export in trypanosomes by intra- and intermolecular multi-colour single molecule FISH. We found that, in striking contrast to other eukaryotes, the initiation of nuclear export requires neither the completion of transcription nor splicing. Nevertheless, we show that unspliced mRNAs are mostly prevented from reaching the nucleus-distant cytoplasm and instead accumulate at the nuclear periphery in cytoplasmic nuclear periphery granules (NPGs). Further characterization of NPGs by electron microscopy and proteomics revealed that the granules are located at the cytoplasmic site of the nuclear pores and contain most cytoplasmic RNA-binding proteins but none of the major translation initiation factors, consistent with a function in preventing faulty mRNAs from reaching translation. Our data indicate that trypanosomes regulate the completion of nuclear export, rather than the initiation. Nuclear export control remains poorly understood, in any organism, and the described way of control may not be restricted to trypanosomes.

The Leishmania PABP1-eIF4E4 interface: a novel 5'-3' interaction architecture for trans-spliced mRNAs.

Trans-splicing of trypanosomatid polycistronic transcripts produces polyadenylated monocistronic mRNAs modified to form the 5' cap4 structure (m7Gpppm36,6,2'Apm2'Apm2'Cpm23,2'U). NMR and X-ray crystallography reveal that Leishmania has a unique type of N-terminally-extended cap-binding protein (eIF4E4) that binds via a PAM2 motif to PABP1. This relies on the interactions of a combination of polar and charged amino acid side-chains together with multiple hydrophobic interactions, and underpins a novel architecture in the Leishmania cap4-binding translation factor complex. Measurements using microscale thermophoresis, fluorescence anisotropy and surface plasmon resonance characterize the key interactions driving assembly of the Leishmania translation initiation complex. We demonstrate that this complex can accommodate Leishmania eIF4G3 which, unlike the standard eukaryotic initiation complex paradigm, binds tightly to eIF4E4, but not to PABP1. Thus, in Leishmania, the chain of interactions 5'cap4-eIF4E4-PABP1-poly(A) bridges the mRNA 5' and 3' ends. Exceptionally, therefore, by binding tightly to two protein ligands and to the mRNA 5' cap4 structure, the trypanosomatid N-terminally extended form of eIF4E acts as the core molecular scaffold for the mRNA-cap-binding complex. Finally, the eIF4E4 N-terminal extension is an intrinsically disordered region that transitions to a partly folded form upon binding to PABP1, whereby this interaction is not modulated by poly(A) binding to PABP1.

Massive mitochondrial DNA content in diplonemid and kinetoplastid protists.

The mitochondrial DNA of diplonemid and kinetoplastid protists is known for its suite of bizarre features, including the presence of concatenated circular molecules, extensive trans-splicing and various forms of RNA editing. Here we report on the existence of another remarkable characteristic: hyper-inflated DNA content. We estimated the total amount of mitochondrial DNA in four kinetoplastid species (Trypanosoma brucei, Trypanoplasma borreli, Cryptobia helicis, and Perkinsela sp.) and the diplonemid Diplonema papillatum. Staining with 4',6-diamidino-2-phenylindole and RedDot1 followed by color deconvolution and quantification revealed massive inflation in the total amount of DNA in their organelles. This was further confirmed by electron microscopy. The most extreme case is the ∼260 Mbp of DNA in the mitochondrion of Diplonema, which greatly exceeds that in its nucleus; this is, to our knowledge, the largest amount of DNA described in any organelle. Perkinsela sp. has a total mitochondrial DNA content ~6.6× greater than its nuclear genome. This mass of DNA occupies most of the volume of the Perkinsela cell, despite the fact that it contains only six protein-coding genes. Why so much DNA? We propose that these bloated mitochondrial DNAs accumulated by a ratchet-like process. Despite their excessive nature, the synthesis and maintenance of these mtDNAs must incur a relatively low cost, considering that diplonemids are one of the most ubiquitous and speciose protist groups in the ocean. © 2018 IUBMB Life, 70(12):1267-1274, 2018.

Group I Intron-Based Therapeutics Through Trans-Splicing Reaction.

In 1982, the Cech group discovered that an intron structure in an rRNA precursor of Tetrahymena thermophila is sufficient to complete splicing without assistance from proteins. This was the first moment that scientists recognized RNAs can have catalytic activities derived from their own unique three-dimensional structures and thus play more various roles in biological processes than thought before. Several additional catalytic RNAs, called ribozymes, were subsequently identified in nature followed by intense studies to reveal their mechanisms of action and to engineer them for use in fields such as molecular cell biology, therapeutics, imaging, etc. Naturally occurring RNA-targeting ribozymes can be broadly classified into two categories by their abilities: Self-cleavage and self-splicing. Since ribozymes use base-pairing to recognize cleavage sites, identification of the catalytic center of naturally occurring ribozymes enables to engineer from "self" to "trans" acting ones which has accelerated to design and use ribozyme as valuable tools in gene therapy fields. Especially, group I intron-based trans-splicing ribozyme has unique property to use as a gene therapeutic agent. It can destroy and simultaneously repair (and/or reprogram) target RNAs to yield the desired therapeutic RNAs, maintaining endogenous spatial and temporal gene regulation of target RNAs. There have been progressive improvements in trans-splicing ribozymes and successful applications of these elements in gene therapy and molecular imaging approaches for various pathogenic conditions. In this chapter, current status of trans-splicing ribozyme therapeutics, focusing on Tetrahymena group I intron-based ribozymes, and their future prospects will be discussed.