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Fusion genes and transcripts can be biological markers as well as the reasons for tumor progression and development. Modern algorithms and high-throughput sequencing are the complementary clues to the question of the tumor origin and cancer detection as well as to the fundamental question of fusion genes origin and their influence on molecular processes of the cell. A wide range of algorithms for fusion genes detection was developed, with various differences in computing speed, sensitivity, specificity, and focus on the experimental design. Depending on the read length (50–300 bp—short reads, 5000–100000 bp—long reads), there are three main types of bioinformatic algorithms: those that focus on short-read sequencing or long-read sequencing exclusively and algorithms that combine the results of both short- and long-read sequencing. These algorithms are further subdivided into: 1) alignment-first approaches (STAR-Fusion, Arriba) that map reads to the genome or transcriptome directly and search the reads supporting the fusion gene or transcript; 2) assembly-first approaches (Fusion-Bloom) that assemble the genome or transcriptome from the overlapping reads de novo and then compare the results to the reference transcriptome or genome to find transcripts or genes not present in the reference and therefore raising questions; 3) pseudoalignment approaches that do not make local alignment, but just search for the closest transcript subsequence to the reads seed, following the precomputed index for all reference transcripts, and provide the results. This article describes the main classes of available software tools for fusion gene detection, provides the characteristics of these programs, their advantages and disadvantages. To date, assembly-first algorithms remain the most resource intensive and slowest. Mapping-first approaches are quite fast and rather accurate at fusion genes detection, yet the fastest and resource-saving are the pseudoalignment algorithms, however the speed of the search is increased at the expense of the search quality. [ABSTRACT FROM AUTHOR]

Adeno‐associated virus (AAV)‐based therapies are recognized as one of the most potent next‐generation treatments for inherited and genetic diseases. However, several biological and technological aspects of AAV vectors remain a critical issue for their widespread clinical application. Among them, the limited capacity of the AAV genome significantly hinders the development of AAV‐based gene therapy. In this context, genetically modified transgenes compatible with AAV are opening up new opportunities for unlimited gene therapies for many genetic disorders. Recent advances in de novo protein design and remodelling are paving the way for new, more efficient and targeted gene therapeutics. Using computational and genetic tools, AAV expression cassette and transgenic DNA can be split, miniaturized, shuffled or created from scratch to mediate efficient gene transfer into targeted cells. In this review, we highlight recent advances in AAV‐based gene therapy with a focus on its use in translational research. We summarize recent research and development in gene therapy, with an emphasis on large transgenes (>4.8 kb) and optimizing strategies applied by biomedical companies in the research pipeline. We critically discuss the prospects for AAV‐based treatment and some emerging challenges. We anticipate that the continued development of novel computational tools will lead to rapid advances in basic gene therapy research and translational studies. [ABSTRACT FROM AUTHOR]

Diverse splicing types in nuclear and chloroplast genes of protist Euglena gracilis have been recognized for decades. However, the splicing machinery responsible for processing nuclear precursor messenger RNA introns, including trans-splicing of the 5′ terminal outron and spliced leader (SL) RNA, remains elusive. Here, we identify 166 spliceosomal protein genes and two snRNA genes from E. gracilis by performing bioinformatics analysis from a combination of next-generation and full-length transcriptomic RNA sequencing (RNAseq) data as well as draft genomic data. With the spliceosomal proteins we identified in hand, the insensitivity of E. gracilis to some splicing modulators is revealed at the sequence level. The prevalence of SL RNA-mediated trans-splicing is estimated to be more than 70% from our full-length RNAseq data. Finally, the splicing proteomes between E. gracilis and its three evolutionary cousins within the same Excavata group are compared. In conclusion, our study characterizes the spliceosomal components in E. gracilis and provides the molecular basis for further exploration of underlying splicing mechanisms.

Shwachman–Diamond syndrome (SDS) represents one of the most common inherited bone marrow failure syndromes and is mainly caused by SBDS gene mutations. Only supportive treatments are available, with hematopoietic cell transplantation required when marrow failure occurs. Among all causative mutations, the SBDS c.258+2T>C variant at the 5′ splice site (ss) of exon 2 is one of the most frequent. Here, we investigated the molecular mechanisms underlying aberrant SBDS splicing and showed that SBDS exon 2 is dense in splicing regulatory elements and cryptic splice sites, complicating proper 5′ss selection. Studies ex vivo and in vitro demonstrated that the mutation alters splicing, but it is also compatible with tiny amounts of correct transcripts, which would explain the survival of SDS patients. Moreover, for the first time for SDS, we explored a panel of correction approaches at the RNA and DNA levels and provided experimental evidence that the mutation effect can be partially counteracted by engineered U1snRNA, trans-splicing, and base/prime editors, ultimately leading to correctly spliced transcripts (from barely detectable to 2.5–5.5%). Among them, we propose DNA editors that, by stably reverting the mutation and potentially conferring positive selection to bone-marrow cells, could lead to the development of an innovative SDS therapy. [ABSTRACT FROM AUTHOR]

Trans -splicing of a spliced leader (SL) to the 5′ ends of mRNAs is used to produce mature mRNAs in several phyla of great importance to human health and the marine ecosystem. One of the consequences of the addition of SL sequences is the change or disruption of the open reading frames (ORFs) in the recipient transcripts. Given that most SL sequences have one or more of the trinucleotide NUG, including AUG in flatworms, trans -splicing of SL sequences can potentially supply a start codon to create new ORFs, which we refer to as slORFs, in the recipient mRNAs. Due to the lack of a tool to precisely detect them, slORFs were usually neglected in previous studies. In this work, we present the tool slORFfinder, which automatically links the SL sequences to the recipient mRNAs at the trans -splicing sites identified from SL-containing reads of RNA-Seq and predicts slORFs according to the distribution of ribosome-protected footprints (RPFs) on the trans -spliced transcripts. By applying this tool to the analyses of nematodes, ascidians and euglena, whose RPFs are publicly available, we find wide existence of slORFs in these taxa. Furthermore, we find that slORFs are generally translated at higher levels than the annotated ORFs in the genomes, suggesting they might have important functions. Overall, this study provides a tool, slORFfinder (https://github.com/songbo446/slORFfinder), to identify slORFs, which can enhance our understanding of ORFs in taxa with SL machinery. [ABSTRACT FROM AUTHOR]

Extensive investigation of gene fusions in cancer has led to the discovery of novel biomarkers and therapeutic targets. To date, most studies have neglected chromosomal rearrangement-independent fusion transcripts and complex fusion structures such as double or triple-hop fusions, and fusion-circRNAs. In this review, we untangle fusion-related terminology and propose a classification system involving both gene and transcript fusions. We highlight the importance of RNA-level fusions and how long-read sequencing approaches can improve detection and characterization. Moreover, we discuss novel bioinformatic tools to identify fusions in long-read sequencing data and strategies to experimentally validate and functionally characterize fusion transcripts. [ABSTRACT FROM AUTHOR]

Cysticerci or metacestodes of Taenia solium cause cysticercosis, being the neurocysticercosis (NCC) the main pathology. The characterization of genes is essential for the knowledge of parasite biology, the understanding of the parasite-host relationship, and the identification of possible targets for diagnosis, treatment, and protection. The objective of this work was the molecular and bioinformatic characterization of three news cDNAs (TsTF10, TsAAP8, and TsrGAP8), obtained by spliced leader-PCR screening of a Taenia solium cDNA library. The sequences of the three cDNA were compared with the sequences in the nucleic acid and protein databases (GenBank, EMBL) and analyzed by bioinformatic programs (CDD-Search of the National Center for Biotechnology Information, Interpro of the European Bioinformatics Institute, Motif scan and Expert Protein Analysis System of the Proteomics Server from Swiss Institute of Bioinformatics). Considering the high identities with similar molecules of related helminths (Taenia asiatica, Echinococcus granulosus, Echinococcus multilocularis, and Hymenolepis diminuta) and the functional domains found, the TsTF10, TsAAP8, and TsrGAP8 genes of Taenia could act as a nuclear transcription factor gamma, a putative vacuolar ATPase membrane sector associated protein and a Rho GTPase activating protein, respectively. Although few B epitopes could be predicted in the sequences, it could be relevant to evaluate them as possible candidates for diagnosis and protection in cysticercosis. The characterization and analysis of these sequences and the prediction of their possible usefulness as antigens, vaccines, or therapeutic targets contribute to the designing and planning of future studies.

Modern society faces many biomedical challenges that require urgent solutions. Two of the most important include the elucidation of mechanisms of socially significant diseases and the development of prospective drug treatments for these diseases. Experimental cell models are a convenient tool for addressing many of these problems. The power of cell models is further enhanced when combined with gene technologies, which allows the examination of even more subtle changes within the structure of the genome and permits testing of proteins in a native environment. The list and possibilities of these recently emerging technologies are truly colossal, which requires a rethink of a number of approaches for obtaining experimental cell models. In this review, we analyze the possibilities and limitations of promising gene technologies for obtaining cell models, and also give recommendations on the development and creation of relevant models. In our opinion, this review will be useful for novice cell biologists, as it provides some reference points in the rapidly growing universe of gene and cell technologies. [ABSTRACT FROM AUTHOR]

Export of RNA from the nucleus is essential for all eukaryotic cells and has emerged as a major step in the control of gene expression. mRNA molecules are required to complete a complex series of processing events and pass a quality control system to protect the cytoplasm from the translation of aberrant proteins. Many of these events are highly conserved across eukaryotes, reflecting their ancient origin, but significant deviation from a canonical pathway as described from animals and fungi has emerged in the trypanosomatids. With significant implications for the mechanisms that control gene expression and hence differentiation, responses to altered environments and fitness as a parasite, these deviations may also reveal additional, previously unsuspected, mRNA export pathways. mRNA export is a highly complex process at the core of gene expression controlling mRNA abundance. Steps include maturation, quality control ensuring that mRNAs are correctly coding, and the export process itself. Trypanosomes have distinct mechanisms for producing mRNA which sets them apart from animals and fungi. These include trans -splicing, absence of introns within open reading frames and polycistronic transcription. These require distinct processes to produce mature mRNAs, which are near identical in structure to higher eukaryotes. We describe how distinct mechanisms for mRNA processing have resulted in changes to mRNA export mechanisms, encompassing nuclear pore complex composition and organization, mRNA maturation, and quality control mechanisms. [ABSTRACT FROM AUTHOR]

Inteins are unique single-turnover enzymes that can excise themselves from the precursor protein without the aid of any external cofactors or energy. In most cases, inteins are covalently linked with the extein sequences and protein splicing happens spontaneously. In this study, a novel protein ligation system was developed based on two atypical split inteins without cross reaction, in which the large segments of one S1 and one S11 split intein fusion protein acted as a protein ligase, the small segments (only several amino acids long) was fused to the N-extein and C-extein, respectively. The splicing activity was demonstrated in E. coli and in vitro with different extein sequences, which showed ∼15% splicing efficiency in vitro. The protein trans -splicing in vitro was further optimized, and possible reaction explanations were explored. As a proof of concept, we expect this approach to expand the scope of trans -splicing-based protein engineering and provide new clues for intein based protein ligase. • Two atypical split inteins were fused together to act as a protein ligase. • A novel protein ligation system mediated by dual split intein was established. • This protein ligation system worked with different exteins in E. coli and in vitro. [ABSTRACT FROM AUTHOR]

Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Genome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genome and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar transcripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3'-end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C. reinhardtii identified 2658 variants in the organellar genomes, which is 1/10th as much genetic diversity as is found in the nucleus.

Tau is a microtubule-associated protein predominantly expressed in neurons, which participates in microtubule polymerization and axonal transport. Abnormal tau metabolism leads to neurodegenerative diseases named tauopathies, such as Alzheimer’s disease and frontotemporal dementia. The alternative splicing of exon 10 (E10) in the primary transcript produces tau protein isoforms with three (3R) or four (4R) microtubule binding repeats, which are found in equal amounts in the normal adult human brain. Several tauopathies are associated with abnormal E10 alternative splicing, leading to an imbalance between 3R and 4R isoforms, which underlies disease. Correction of such imbalance represents a potential disease-modifying therapy for those tauopathies. We have previously optimized a trans-splicing RNA reprogramming strategy to modulate the 3R:4R tau content in a mouse model of tauopathy related to tau mis-splicing (htau mice), and showed that local modulation of E10 inclusion in the prefrontal cortex prevents cognitive decline, neuronal firing impairments and hyperphosphorylated tau accumulation. Furthermore, local shifting of 3R–4R tau into the striatum of htau mice prevented motor coordination deficits. However, a major bottleneck of our previous work is that local splicing regulation was performed in young mice, before the onset of pathological phenotypes. Here we tested whether regulation of tau E10 splicing could rescue tau pathology phenotypes in htau mice, after the onset of cognitive and motor impairments, comparable to early stages of human tauopathies. To determine phenotypic time course and affected brain nuclei, we assessed htau mice using behavioural tests and microPET FDG imaging over time, similarly to diagnosis methods used in patients. Based on these analyses, we performed local delivery of pre-trans splicing molecules to regulate E10 inclusion either into the medial prefrontal cortex (mPFC) or the striatum at 6-month-old once behavioral phenotypes and metabolic changes were detected. Tau isoforms modulation into the mPFC restored cognitive performance in mice that previously showed mild to severe memory impairment while motor coordination deficit was rescued after striatal injection of trans-splicing molecules. Our data suggest that tau regulation could recover pathological phenotypes early after phenotypic onset, raising promising perspectives for the use of RNA based therapies in tauopathies related to MAPT abnormal splicing.

The haptophyte Isochrysís galbana is considered as a promising source for food supplements due to its rich fucoxanthin and polyunsaturated fatty acids content. Here, the I. galbana mitochondrial genome (mitogenome) was sequenced using a combination of Illumina and PacBio sequencing platforms. This 39,258bp circular mitogenome has a total of 46 genes, including 20 protein-coding genes, 24 tRNA genes and two rRNA genes. A large block of repeats (~12.7kb) was segregated in one region of the mitogenome, accounting for almost one third of the total size. A trans-spliced gene coxl was first identified in I. galbana mitogenome and was verified by RNA-seq and DNA-seq data. The massive expansion of tandem repeat size and cis- to trans-splicing shift could be explained by the high mitogenome rearrangement rates in haptophytes. Strict SNP calling based on deep transcriptome sequencing data suggested the lack of RNA editing in both organelles in this species, consistent with previous studies in other algal lineages. To gain insight into haptophyte mitogenome evolution, a comparative analysis of mitogenomes within haptophytes and among eight main algal lineages was performed. A core gene set of 15 energy and metabolism genes is present in haptophyte mitogenomes, consisting of 1 cob, 3 cox, 7 nad, 2 atp and 2 ribosomal genes. Gene content and order was poorly conserved in this lineage. Haptophyte mitogenomes have lost many functional genes found in many other eukaryotes including rps/rpl, sdh, tat, secY genes, which make it contain the smallest gene set among all algal taxa. All these implied the rapid-evolving and more recently evolved mitogenomes of haptophytes compared to other algal lineages. The phylogenetic tree constructed by cox1 genes of 204 algal mitogenomes yielded well-resolved internal relationships, providing new evidence for red-lineages that contained plastids of red algal secondary endosymbiotic origin. This newly assembled mitogenome will add to our knowledge of general trends in algal mitogenome evolution within haptophytes and among different algal taxa. [ABSTRACT FROM AUTHOR]

Simple Summary: Expression of circular RNAs is known to be deregulated in cancer. Here the most comprehensive set of differentially expressed RNA circles in medulloblastoma compared to cerebellum is provided. Additionally, fusion RNAs are also identified in both cancerous and normal cerebellar tissue. Some of the fusions detected in medulloblastoma are generated by genomic rearrangements that link different genes. However, fusion RNAs are also detected in normal cerebellum. In fact, there are cases where the same fusion RNA is also found in medulloblastoma. This observation underscores that the formation of fusion transcripts may not be limited to chromosomal events but could also result from mechanisms that act at the RNA level. These include read-through transcription of neighboring genes and intermolecular splicing of pre-mRNAs from different genes Importantly, these RNA "recombination" events occur not only in normal but also in cancerous tissue. Background. The cerebellar cancer medulloblastoma is the most common childhood cancer in the brain. Methods. RNA sequencing of 81 human biospecimens of medulloblastoma using pipelines to detect circular and fusion RNAs. Validation via PCR and Sanger sequencing. Results. 27, 56, 28 and 11 RNA circles were found to be uniquely up-regulated, while 149, 7, 20 and 15 uniquely down-regulated in the SHH, WNT, Group 3, and Group 4 medulloblastoma subtypes, respectively. Moreover, linear and circular fusion RNAs containing exons from distinct genes joined at canonical splice sites were also identified. These were generally expressed less than the circular RNAs, however the expression of both the linear and the circular fusions was comparable. Importantly, the expression of the fusions in medulloblastoma was also comparable to that of cerebellum. Conclusions. A significant number of fusions in tumor may be generated by mechanisms similar to the ones generating fusions in normal tissue. Some fusions could be rationalized by read-through transcription of two neighboring genes. However, for other fusions, e.g., a linear fusion with an exon from a downstream gene joined 5′ to 3′ with an exon from an upstream gene, more complicated splicing mechanisms, e.g., trans-splicing, have to be postulated. [ABSTRACT FROM AUTHOR]

The nematode Caenorhabditis briggsae is routinely used in comparative and evolutionary studies involving its well-known cousin Caenorhabditis elegans. The C. briggsae genome sequence has accelerated research by facilitating the generation of new resources, tools, and functional studies of genes. While substantial progress has been made in predicting genes and start sites, experimental evidence is still lacking in many cases. Here, we report an improved annotation of the C. briggsae genome using the trans-spliced exon coupled RNA end determination technique. In addition to identifying the 50 ends of expressed genes, we have discovered operons and paralogs. In summary, our analysis yielded 10,243 unique 50 end sequence tags with matches in the C. briggsae genome. Of these, 6,395 were found to represent 4,252 unique genes along with 362 paralogs and 52 previously unknown exons. These genes included 14 that are exclusively trans-spliced in C. briggsae when compared with C. elegans orthologs. A major contribution of this study is the identification of 492 high confidence operons, of which two-thirds are fully supported by tags. In addition, 2 SL1-type operons were discovered. Interestingly, comparisons with C. elegans showed that only 40% of operons are conserved. Of the remaining operons, 73 are novel, including 12 that entirely lack orthologs in C. elegans. Further analysis revealed that 4 of the 12 novel operons are conserved in Caenorhabditis nigoni. Altogether, the work described here has significantly advanced our understanding of the C. briggsae system and serves as a rich resource to aid biological studies involving this species. [ABSTRACT FROM AUTHOR]

RNA exon editing is a therapeutic strategy for correcting disease-causing mutations by inducing trans -splicing between a synthetic RNA molecule and an endogenous pre-mRNA target, resulting in functionally restored mRNA and protein. This approach enables the replacement of exons at the kilobase scale, addresses multiple mutations with a single therapy, and maintains native gene expression without changes to DNA. For genes larger than 5 kb, RNA exon editors can be delivered in a single vector despite AAV capacity limitations because only mutated exons need to be replaced. While correcting mutations by trans -splicing has been previously demonstrated, prior attempts were hampered by low efficiency or lack of translation in preclinical models. Advances in synthetic biology, next-generation sequencing, and bioinformatics, with a deeper understanding of mechanisms controlling RNA splicing, have triggered a re-emergence of trans -splicing and the development of new RNA exon editing molecules for treating human disease, including the first application in a clinical trial (this study was registered at ClinicalTrials.gov [NCT06467344]). Here, we provide an overview of RNA splicing, the history of trans -splicing, previously reported therapeutic applications, and how modern advances are enabling the discovery of RNA exon editing molecules for genetic targets unable to be addressed by conventional gene therapy and gene editing approaches., Competing Interests: All authors are shareholders of Ascidian Therapeutics., (© 2024 The Authors.)

Spliced leader trans-splicing of pre-mRNAs is a critical step in the gene expression of many eukaryotes. How the spliced leader RNA and its target transcripts are brought together to form the trans-spliceosome remains an important unanswered question. Using immunoprecipitation followed by protein analysis via mass spectrometry and RIP-Seq, we show that the nematode-specific proteins, SNA-3 and SUT-1, form a complex with a set of enigmatic non-coding RNAs, the SmY RNAs. Our work redefines the SmY snRNP and shows for the first time that it is essential for nematode viability and is involved in spliced leader trans-splicing. SNA-3 and SUT-1 are associated with the 5' ends of most, if not all, nascent capped RNA polymerase II transcripts, and they also interact with components of the major nematode spliced leader (SL1) snRNP. We show that depletion of SNA-3 impairs the co-immunoprecipitation between one of the SL1 snRNP components, SNA-2, and several core spliceosomal proteins. We thus propose that the SmY snRNP recruits the SL1 snRNP to the 5' ends of nascent pre-mRNAs, an instrumental step in the assembly of the trans-spliceosome., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Protein complexes are involved in many vital biological processes. Therefore, researchers need these protein complexes for biochemical and biophysical studies. Several methods exist for expressing multi-subunit proteins in eukaryotic cells, such as 2A sequences, IRES, or intein. Nevertheless, each of these elements has several disadvantages that limit their usage. In this article, we suggest a new system for expressing multi-subunit proteins, which have several advantages over existing methods meanwhile it, lacks most of their disadvantages. Leishmania is a unicellular eukaryote and member of the Trypanosomatidae family. In the expression system of Leishmania, pre-long RNAs that contain several protein sequences transcribe. Then these long RNAs separate into mature mRNAs in the process named trans splicing. For producing multi-subunit protein, Leishmania transformed with a vector containing the sequences of all subunits. Therefore, those subunits translate and form the complex under eukaryotic cell conditions. The sequence of each protein must separate by the spatial sequence needed for trans splicing. Based on a Leishmania expression pattern, not only is it possible to produce the complexes with the correct structures and post-translational modifications, but also it is possible to overcome previous method problems.

Spliced-leader trans-splicing (SLTS) has been described in distantly related eukaryotes and acts to mark mRNAs with a short 5′ exon, giving different mRNAs identical 5′ sequence-signatures. The function of these systems is obscure. Perkinsozoa encompasses a diversity of parasitic protists that infect bivalves, toxic-tide dinoflagellates, fish and frog tadpoles. Here, we report considerable sequence variation in the SLTS-system across the Perkinsozoa and find that multiple variant SLTS-systems are encoded in parallel in the ecologically important Perkinsozoa parasite Parvilucifera sinerae. These results demonstrate that the transcriptome of P. sinerae is segregated based on the addition of different spliced-leader (SL) exons. This segregation marks different gene categories, suggesting that SL-segregation relates to functional differentiation of the transcriptome. By contrast, both sets of gene categories are present in the single SL-transcript type sampled from Maranthos, implying that the SL-segregation of the Parvilucifera transcriptome is a recent evolutionary innovation. Furthermore, we show that the SLTS-system marks a subsection of the transcriptome with increased mRNA abundance and includes genes that encode the spliceosome system necessary for SLTS-function. Collectively, these data provide a picture of how the SLTS-systems can vary within a major evolutionary group and identify how additional transcriptional-complexity can be achieved through SL-segregation.

Plastid primary endosymbiosis has occurred twice, once in the Archaeplastida ancestor and once in the Paulinella (Rhizaria) lineage. Both events precipitated massive evolutionary changes, including the recruitment and activation of genes that are horizontally acquired (HGT) and the redeployment of existing genes and pathways in novel contexts. Here we address the latter aspect in Paulinella micropora KR01 (hereafter, KR01) that has independently evolved spliced leader (SL) trans‐splicing (SLTS) of nuclear–derived transcripts. We investigated the role of this process in gene regulation, novel gene origination, and endosymbiont integration. Our analysis shows that 20% of KR01 genes give rise to transcripts with at least one (but in some cases, multiple) sites of SL addition. This process, which often occurs at canonical cis‐splicing acceptor sites (internal introns), results in shorter transcripts that may produce 5′‐truncated proteins with novel functions. SL–truncated transcripts fall into four categories that may show: (i) altered protein localization, (ii) altered protein function, structure, or regulation, (iii) loss of valid alternative start codons, preventing translation, or (iv) multiple SL addition sites at the 5′‐terminus. The SL RNA genes required for SLTS are putatively absent in the heterotrophic sister lineage of photosynthetic Paulinella species. Moreover, a high proportion of transcripts derived from genes of endosymbiotic gene transfer (EGT) and HGT origin contain SL sequences. We hypothesize that truncation of transcripts by SL addition may facilitate the generation and expression of novel gene variants and that SLTS may have enhanced the activation and fixation of foreign genes in the host genome of the photosynthetic lineages, playing a key role in primary endosymbiont integration. [ABSTRACT FROM AUTHOR]

Chimeric RNAs are transcripts that are generated by gene fusion and intergenic splicing events, thus comprising nucleotide sequences from different parental genes. In the past, Northern blot analysis and RT-PCR were used to detect chimeric RNAs. However, they are low-throughput and can be time-consuming, labor-intensive, and cost-prohibitive. With the development of RNA-seq and transcriptome analyses over the past decade, the number of chimeric RNAs in cancer as well as in rare inherited diseases has dramatically increased. Chimeric RNAs may be potential diagnostic biomarkers when they are specifically expressed in cancerous cells and/or tissues. Some chimeric RNAs can also play a role in cell proliferation and cancer development, acting as tools for cancer prognosis, and revealing new insights into the cell origin of tumors. Due to their abilities to characterize a whole transcriptome with a high sequencing depth and intergenically identify spliced chimeric RNAs produced with the absence of chromosomal rearrangement, RNA sequencing has not only enhanced our ability to diagnose genetic diseases, but also provided us with a deeper understanding of these diseases. Here, we reviewed the mechanisms of chimeric RNA formation and the utility of RNA sequencing for discovering chimeric RNAs in several types of cancer and rare inherited diseases. We also discussed the diagnostic, prognostic, and therapeutic values of chimeric RNAs. [ABSTRACT FROM AUTHOR]

[Display omitted] • The complete mitochondrial genome of Panax quinquefolius has been successfully assembled. • Compared to the previous version, our new version has improved the quality of tRNA gene annotation. • Recombination via short repetitive sequences causes cleavage of the mitochondrial intron cox2i373 in Panax genus. • Horizontal gene transfer from chloroplast to mitochondrial is frequent in Panax species. Panax quinquefolius is a perennial plant with medicinal values. In this study, we assembled the complete mitochondrial genome (mitogenome) of P. quinquefoliu s using PMAT assembler. The total length of P. quinquefoliu s mitogenome is 573,154 bp. We annotated a total of 34 protein-coding genes (PCGs), 35 tRNA genes, and 6 rRNA genes in this mitogenome. The analysis of repetitive elements shows that there are 153 SSRs, 24 tandem repeats and 242 pairs of dispersed repeats this mitogenome. Also, we found 24 homologous sequences with a total length of 64,070 bp among its mitogenome and plastome, accounting for 41.05 % of the plastome, and 11.18 % of the mitogenome, showing a remarkable frequent sequence dialogue between plastome and mitogenomes. Besides, a total of 583 C to U RNA editing sites on 34 PCGs of high confidence were predicted by using Deepred-mt. We also inferred the phylogenetic relationships of P. quinquefolius and other angiosperms based on mitochondrial PCGs. Finally, we observed a shift from cis - to trans -splicing in P. quinquefolius for two mitochondrial introns, namely cox2i373 and nad1i728 , and a pair of 48 bp short repetitive sequences may be associated with the breaking and rearrangement of the cox2i373 intron. The fragmentation of the cox2i373 intron was further confirmed by our PCR amplification experiments. In summary, our report on the P. quinquefolius mitogenome provides a new perspective on the intron evolution of the mitogenome. [ABSTRACT FROM AUTHOR]

The African trypanosome Trypanosoma brucei is a parasite of the mammalian bloodstream and tissues, where an antigenically variable Variant Surface Glycoprotein (VSG) coat protects it from immune attack. This dense layer comprised of ∼107 VSG proteins, makes VSG by far the most abundant mRNA (7–10% total) and protein (∼10% total) in the bloodstream form trypanosome. How can such prodigious amounts of VSG be produced from a single VSG gene? Extremely high levels of RNA polymerase I (Pol I) transcription of the active VSG provide part of the explanation. However, recent discoveries highlight the role of pre-mRNA processing, both in maintaining high levels of VSG transcription, as well as its monoallelic expression. Trypanosome mRNAs are matured through trans-splicing a spliced leader (SL) RNA to the 5’ end of precursor transcripts, meaning abundant SL RNA is required throughout the nucleus. However, requirement for SL RNA in the vicinity of the active VSG gene is so intense, that the cell reconfigures its chromatin architecture to facilitate interaction between the SL RNA genes and the active VSG. This presumably ensures that sufficient localised SL RNA is available, and not limiting for VSG mRNA expression. Recently, novel nuclear splicing bodies which appear to provide essential trans-splicing components, have been identified associating with the active VSG. These observations highlight the underappreciated role of pre-mRNA processing in modulating gene expression in trypanosomes. Dissecting the function of these nuclear RNA processing bodies should help us elucidate the mechanisms of both VSG expression and monoallelic exclusion in T. brucei.

In eukaryotes, trans-splicing is a process of nuclear pre-mRNA maturation where two different RNA molecules are joined together by the spliceosomal machinery utilizing mechanisms similar to cis-splicing. In diverse taxa of lower eukaryotes, spliced leader (SL) trans-splicing is the most frequent type of trans-splicing, when the same sequence derived from short small nuclear RNA molecules, called SL RNAs, is attached to the 5’ ends of different non-processed pre-mRNAs. One of the functions of SL trans-splicing is processing polycistronic pre-mRNA molecules transcribed from operons, when several genes are transcribed as one pre-mRNA molecule. However, only a fraction of trans-spliced genes reside in operons, suggesting that SL trans-splicing must also have some other, less understood functions. Regenerative flatworms are informative model organisms which hold the keys to understand the mechanism of stem cell regulation and specialization during regeneration and homeostasis. Their ability to regenerate is fueled by the division and differentiation of the adult somatic stem cell population called neoblasts. Macrostomum lignano is a flatworm model organism where substantial technological advances have been achieved in recent years, including the development of transgenesis. Although a large fraction of genes in M. lignano were estimated to be SL trans-spliced, SL trans-splicing was not studied in detail in M. lignano before. Here, we performed the first comprehensive study of SL trans-splicing in M. lignano. By reanalyzing the existing genome and transcriptome data of M. lignano, we estimate that 30 % of its genes are SL trans-spliced, 15 % are organized in operons, and almost 40 % are both SL trans-spliced and in operons. We annotated and characterized the sequence of SL RNA and characterized conserved cis- and SL transsplicing motifs. Finally, we found that a majority of SL trans-spliced genes are evolutionarily conserved and signif icantly over-represented in neoblast-specific genes. Our findings suggest an important role of SL trans-splicing in the regulation and maintenance of neoblasts in M. lignano.

Shifts from cis-to trans-splicing of mitochondrial introns tend to correlate with relative genome rearrangement rates during vascular plant evolution, as is particularly apparent in some lineages of gymnosperms. However, although many angiosperms have also relatively high mitogenomic rearrangement rates, very few cisto trans-splicing shifts except for five trans-spliced introns shared in seed plants have been reported. In this study, we sequenced and characterized the mitogenome of Tolypanthus maclurei, a hemiparasitic plant from the family Loranthaceae (Santalales). The mitogenome was assembled into a circular chromosome of 256,961 bp long, relatively small compared with its relatives from Santalales. It possessed a gene content of typical angiosperm mitogenomes, including 33 protein-coding genes, three rRNA genes and ten tRNA genes. Plastid-derived DNA fragments took up 9.1% of the mitogenome. The mitogenome contained one group I intron (cox1i729) and 23 group II introns. We found shifts from cis-to trans-splicing of five additional introns in its mitogenome, of which two are specific in T. maclurei. Moreover, atp1 is a chimeric gene and phylogenetic analysis indicated that a 356 bp region near the 3' end of atp1 of T. maclurei was acquired from Lamiales via horizontal gene transfer. Our results suggest that shifts to trans-splicing of mitochondrial introns may not be uncommon among angiosperms. [ABSTRACT FROM AUTHOR]

The causative agent of opisthorchiasis, the liver fluke Opisthorchis felineus (Rivolta, 1884) is one of the helminths of humans and animals in Russia. Together with closely related species of trematodes O. viverrini (Poirier, 1886) and Clonorchis sinensis (Loos, 1907), O. felineus is a part of a triad of epidemiologically important trematodes in the family Opisthorchiidae. Adult O. felineus worms infest the hepatobiliary system of warm-blooded animals and might provoke the development of severe pathologies, including malignancy of bile duct epithelium. The high medical importance of O. felineus attracts the attention of researchers. This review briefly summarizes the data about O. felineus genomics and proteomics. The review provides a comparative analysis of the number of genes and sizes of nuclear genomes of a number of flatworms, the distribution of intron lengths, as well as results of synteny between the O. felineus, O. viverrini and C. sinensis genomes. Special attention is paid to a particular form of RNA processing known as trans-splicing, widely presented in the opisthorchiid genomes. We also provide the results of a comparative analysis of the xenobiotic metabolizing system between parasitic and free-living flatworms. Moreover, data on parasitic granulins, which are potential promoters of cholangiocyte neoplasia, are also presented. Data on the O. felineus genomics and proteomics provide first insights into the structural and functional organization of the genome of this parasitic flatworm with a complex life cycle as well as provide a significant contribution to our understanding of “host-parasite” interaction and evolution of this group of parasitic flatworms.

Modern society faces many biomedical challenges that require urgent solutions. Two of the most important include the elucidation of mechanisms of socially significant diseases and the development of prospective drug treatments for these diseases. Experimental cell models are a convenient tool for addressing many of these problems. The power of cell models is further enhanced when combined with gene technologies, which allows the examination of even more subtle changes within the structure of the genome and permits testing of proteins in a native environment. The list and possibilities of these recently emerging technologies are truly colossal, which requires a rethink of a number of approaches for obtaining experimental cell models. In this review, we analyze the possibilities and limitations of promising gene technologies for obtaining cell models, and also give recommendations on the development and creation of relevant models. In our opinion, this review will be useful for novice cell biologists, as it provides some reference points in the rapidly growing universe of gene and cell technologies.

New insights into molecular genetics and pathomechanisms in epidermolysis bullosa (EB), methodological and technological advances in molecular biology as well as designated funding initiatives and facilitated approval procedures for orphan drugs have boosted translational research perspectives for this devastating disease. This is echoed by the increasing number of clinical trials assessing innovative molecular therapies in the field of EB. Despite remarkable progress, gene-corrective modalities, aimed at sustained or permanent restoration of functional protein expression, still await broad clinical availability. This also reflects the methodological and technological shortcomings of current strategies, including the translatability of certain methodologies beyond preclinical models as well as the safe, specific, efficient, feasible, sustained and cost-effective delivery of therapeutic/corrective information to target cells. This review gives an updated overview on status, prospects, challenges and limitations of current gene-targeted therapies. [ABSTRACT FROM AUTHOR]

The group I intron ribozyme from Tetrahymena was recently reengineered into a trans-splicing variant that is able to remove 100-nt introns from pre-mRNA, analogous to the spliceosome. These spliceozymes were improved in this study by 10 rounds of evolution in Escherichia coli cells. One clone with increased activity in E. coli cells was analyzed in detail. Three of its 10 necessary mutations extended the substrate binding duplexes, which led to increased product formation and reduced cleavage at the 5'-splice site. One mutation in the conserved core of the spliceozyme led to a further reduction of cleavage at the 5'-splice site but an increase in cleavage side products at the 3'-splice site. The latter was partially reduced by six additional mutations. Together, the mutations increased product formation while reducing activity at the 5'-splice site and increasing activity at the 3'-splice site. These results show the adaptation of a ribozyme that evolved in nature for cis-splicing to trans-splicing, and they highlight the interdependent function of nucleotides within group I intron ribozymes. Implications for the possible use of spliceozymes as tools in research and therapy, and as a model for the evolution of the spliceosome, are discussed.

Group I introns are ribozymes (catalytic RNAs) that excise themselves from RNA primary transcripts by catalyzing two successive transesterification reactions. These cis-splicing ribozymes can be converted into trans-splicing ribozymes, which can modify the sequence of a separate substrate RNA, both in vitro and in vivo. Previous work on trans-splicing ribozymes has mostly focused on the 16S rRNA group I intron ribozyme from Tetrahymena thermophila. Here, we test the trans-splicing potential of the tRNA(Ile) group I intron ribozyme from the bacterium Azoarcus. This ribozyme is only half the size of the Tetrahymena ribozyme and folds faster into its active conformation in vitro. Our results showed that in vitro, the Azoarcus and Tetrahymena ribozymes favored the same set of splice sites on a substrate RNA. Both ribozymes showed the same trans-splicing efficiency when containing their individually optimized 5' terminus. In contrast to the previously optimized 5'-terminal design of the Tetrahymena ribozyme, the Azoarcus ribozyme was most efficient with a trans-splicing design that resembled the secondary structure context of the natural cis-splicing Azoarcus ribozyme, which includes base-pairing between the substrate 5' portion and the ribozyme 3' exon. These results suggested preferred trans-splicing interactions for the Azoarcus ribozyme under near-physiological in vitro conditions. Despite the high activity in vitro, however, the splicing efficiency of the Azoarcus ribozyme in Escherichia coli cells was significantly below that of the Tetrahymena ribozyme.

How does a non-coding RNA evolve in cells? To address this question experimentally we evolved a trans-splicing variant of the group I intron ribozyme from Tetrahymena over 21 cycles of evolution in E.coli cells. Sequence variation was introduced during the evolution by mutagenic and recombinative PCR, and increasingly active ribozymes were selected by their repair of an mRNA mediating antibiotic resistance. The most efficient ribozyme contained four clustered mutations that were necessary and sufficient for maximum activity in cells. Surprisingly, these mutations did not increase the trans-splicing activity of the ribozyme. Instead, they appear to have recruited a cellular protein, the transcription termination factor Rho, and facilitated more efficient translation of the ribozyme's trans-splicing product. In addition, these mutations affected the expression of several other, unrelated genes. These results suggest that during RNA evolution in cells, four mutations can be sufficient to evolve new protein interactions, and four mutations in an RNA molecule can generate a large effect on gene regulation in the cell.

Abstract Background The three epidemiologically important Opisthorchiidae liver flukes Opisthorchis felineus, O. viverrini, and Clonorchis sinensis, are believed to harbour similar potencies to provoke hepatobiliary diseases in their definitive hosts, although their populations have substantially different ecogeographical aspects including habitat, preferred hosts, population structure. Lack of O. felineus genomic data is an obstacle to the development of comparative molecular biological approaches necessary to obtain new knowledge about the biology of Opisthorchiidae trematodes, to identify essential pathways linked to parasite-host interaction, to predict genes that contribute to liver fluke pathogenesis and for the effective prevention and control of the disease. Results Here we present the first draft genome assembly of O. felineus and its gene repertoire accompanied by a comparative analysis with that of O. viverrini and Clonorchis sinensis. We observed both noticeably high heterozygosity of the sequenced individual and substantial genetic diversity in a pooled sample. This indicates that potency of O. felineus population for rapid adaptive response to control and preventive measures of opisthorchiasis is higher than in O. viverrini and C. sinensis. We also have found that all three species are characterized by more intensive involvement of trans-splicing in RNA processing compared to other trematodes. Conclusion All revealed peculiarities of structural organization of genomes are of extreme importance for a proper description of genes and their products in these parasitic species. This should be taken into account both in academic and applied research of epidemiologically important liver flukes. Further comparative genomics studies of liver flukes and non-carcinogenic flatworms allow for generation of well-grounded hypotheses on the mechanisms underlying development of cholangiocarcinoma associated with opisthorchiasis and clonorchiasis as well as species-specific mechanisms of these diseases.

Background. The cerebellar cancer medulloblastoma is the most common childhood cancer in the brain. Methods. RNA sequencing of 81 human biospecimens of medulloblastoma using pipelines to detect circular and fusion RNAs. Validation via PCR and Sanger sequencing. Results. 27, 56, 28 and 11 RNA circles were found to be uniquely up-regulated, while 149, 7, 20 and 15 uniquely down-regulated in the SHH, WNT, Group 3, and Group 4 medulloblastoma subtypes, respectively. Moreover, linear and circular fusion RNAs containing exons from distinct genes joined at canonical splice sites were also identified. These were generally expressed less than the circular RNAs, however the expression of both the linear and the circular fusions was comparable. Importantly, the expression of the fusions in medulloblastoma was also comparable to that of cerebellum. Conclusions. A significant number of fusions in tumor may be generated by mechanisms similar to the ones generating fusions in normal tissue. Some fusions could be rationalized by read-through transcription of two neighboring genes. However, for other fusions, e.g., a linear fusion with an exon from a downstream gene joined 5′ to 3′ with an exon from an upstream gene, more complicated splicing mechanisms, e.g., trans-splicing, have to be postulated.

Chimeric RNAs are transcripts that are generated by gene fusion and intergenic splicing events, thus comprising nucleotide sequences from different parental genes. In the past, Northern blot analysis and RT-PCR were used to detect chimeric RNAs. However, they are low-throughput and can be time-consuming, labor-intensive, and cost-prohibitive. With the development of RNA-seq and transcriptome analyses over the past decade, the number of chimeric RNAs in cancer as well as in rare inherited diseases has dramatically increased. Chimeric RNAs may be potential diagnostic biomarkers when they are specifically expressed in cancerous cells and/or tissues. Some chimeric RNAs can also play a role in cell proliferation and cancer development, acting as tools for cancer prognosis, and revealing new insights into the cell origin of tumors. Due to their abilities to characterize a whole transcriptome with a high sequencing depth and intergenically identify spliced chimeric RNAs produced with the absence of chromosomal rearrangement, RNA sequencing has not only enhanced our ability to diagnose genetic diseases, but also provided us with a deeper understanding of these diseases. Here, we reviewed the mechanisms of chimeric RNA formation and the utility of RNA sequencing for discovering chimeric RNAs in several types of cancer and rare inherited diseases. We also discussed the diagnostic, prognostic, and therapeutic values of chimeric RNAs.

Fusion RNAs are a hallmark of some cancers. They result either from chromosomal rearrangements or from splicing mechanisms that are non-chromosomal rearrangements. Chromosomal rearrangements that result in gene fusions are particularly prevalent in sarcomas and hematopoietic malignancies; they are also common in solid tumors. The splicing process can also give rise to more complex RNA patterns in cells. Gene fusions frequently affect tyrosine kinases, chromatin regulators, or transcription factors, and can cause constitutive activation, enhancement of downstream signaling, and tumor development, as major drivers of oncogenesis. In addition, some fusion RNAs have been shown to function as noncoding RNAs and to affect cancer progression. Fusion genes and RNAs will therefore become increasingly important as diagnostic and therapeutic targets for cancer development. Here, we discuss the function, biogenesis, detection, clinical relevance, and therapeutic implications of oncogenic fusion genes and RNAs in cancer development. Further understanding the molecular mechanisms that regulate how fusion RNAs form in cancers is critical to the development of therapeutic strategies against tumorigenesis. [ABSTRACT FROM AUTHOR]

Group II introns are large self-splicing RNA enzymes with a broad but somewhat irregular phylogenetic distribution. These ancient retromobile elements are the proposed ancestors of approximately half the human genome, including the abundant spliceosomal introns and non-long terminal repeat retrotransposons. In contrast to their eukaryotic derivatives, bacterial group II introns have largely been considered as harmful selfish mobile retroelements that parasitize the genome of their host. As a challenge to this view, we recently uncovered a new intergenic trans -splicing pathway that generates an assortment of mRNA chimeras. The ability of group II introns to combine disparate mRNA fragments was proposed to increase the genetic diversity of the bacterial host by shuffling coding sequences. Here, we show that the Ll.LtrB and Ef.PcfG group II introns from Lactococcus lactis and Enterococcus faecalis respectively can both use the intergenic trans -splicing pathway to catalyze the formation of chimeric relaxase mRNAs and functional proteins. We demonstrated that some of these compound relaxase enzymes yield gain-of-function phenotypes, being significantly more efficient than their precursor wild-type enzymes at supporting bacterial conjugation. We also found that relaxase enzymes with shuffled functional domains are produced in biologically relevant settings under natural expression levels. Finally, we uncovered examples of lactococcal chimeric relaxase genes with junctions exactly at the intron insertion site. Overall, our work demonstrates that the genetic diversity generated by group II introns, at the RNA level by intergenic trans -splicing and at the DNA level by recombination, can yield new functional enzymes with shuffled exons, which can lead to gain-of-function phenotypes. [ABSTRACT FROM AUTHOR]

Type VI CRISPR enzymes have been developed as programmable RNA-guided Cas proteins for eukaryotic RNA editing. Notably, Cas13 has been utilized for site-targeted single base edits, demethylation, RNA cleavage or knockdown and alternative splicing. However, the ability to edit large stretches of mRNA transcripts remains a significant challenge. Here, we demonstrate that CRISPR-Cas13 systems can be repurposed to assist trans-splicing of exogenous RNA fragments into an endogenous pre-mRNA transcript, a method termed CRISPR Assisted mRNA Fragment Trans-splicing (CRAFT). Using split reporter-based assays, we evaluate orthogonal Cas13 systems, optimize guide RNA length and screen for optimal trans-splicing site(s) across a range of intronic targets. We achieve markedly improved editing of large 5' and 3' segments in different endogenous mRNAs across various mammalian cell types compared to other spliceosome-mediated trans-splicing methods. CRAFT can serve as a versatile platform for attachment of protein tags, studying the impact of multiple mutations/single nucleotide polymorphisms, modification of untranslated regions (UTRs) or replacing large segments of mRNA transcripts., (© 2024. The Author(s).)

A purification system was constructed with the N-segment of the Npu DnaE split intein as an affinity ligand immobilized onto an epoxy-activated medium and the C-segment used as the cleavable tag fusing target protein. The affinity properties of C-tagged proteins adsorbed on IN affinity chromatography medium were studied with GFP as a model target protein. The saturated adsorption capacity and dynamic adsorption capacity reached 51.9–21.0 mg mL−1, respectively. With this system, two model proteins, GFP and alcohol dehydrogenase (ADH), has been successfully taglessly purified with regulation of Zn2+ and DTT. The yield, purification factor and purity of purified tagless GFP reached 39, 11.7 and 97%, respectively; while these values for purified tagless ADH were 38.2, 6.8 and 91%, respectively. These results showed that the system for Npu DnaE split intein-mediated affinity adsorption and in situ cleavage is a potential platform for recombinant protein production. [ABSTRACT FROM AUTHOR]

We assessed the potential of Lmna-mRNA repair by spliceosome-mediated RNA trans-splicing as a therapeutic approach for LMNA-related congenital muscular dystrophy. This gene therapy strategy leads to reduction of mutated transcript expression for the benefit of corresponding wild-type (WT) transcripts. We developed 5′-RNA pre-trans-splicing molecules containing the first five exons of Lmna and targeting intron 5 of Lmna pre-mRNA. Among nine pre-trans-splicing molecules, differing in the targeted sequence in intron 5 and tested in C2C12 myoblasts, three induced trans-splicing events on endogenous Lmna mRNA and confirmed at protein level. Further analyses performed in primary myotubes derived from an LMNA-related congenital muscular dystrophy (L-CMD) mouse model led to a partial rescue of the mutant phenotype. Finally, we tested this approach in vivo using adeno-associated virus (AAV) delivery in newborn mice and showed that trans-splicing events occurred in WT mice 50 days after AAV delivery, although at a low rate. Altogether, while these results provide the first evidence for reprogramming LMNA mRNA in vitro, strategies to improve the rate of trans-splicing events still need to be developed for efficient application of this therapeutic approach in vivo.

Chimeric RNAs are often associated with chromosomal rearrangements in cancer. In addition, they are also widely detected in normal tissues, contributing to transcriptomic complexity. Despite their prevalence, little is known about the characteristics and functions of chimeric RNAs. Here, we examine the genetic structure and biological roles of CLEC12A-MIR223HG, a novel chimeric transcript produced by the fusion of the cell surface receptor CLEC12A and the miRNA-223 host gene (MIR223HG), first identified in chronic myeloid leukemia (CML) patients. Surprisingly, we observed that CLEC12A-MIR223HG is not just expressed in CML, but also in a variety of normal tissues and cell lines. CLEC12A-MIR223HG expression is elevated in pro-monocytic cells resistant to chemotherapy and during monocyte-to-macrophage differentiation. We observed that CLEC12A-MIR223HG is a product of trans-splicing rather than a chromosomal rearrangement and that transcriptional activation of CLEC12A with the CRISPR/Cas9 Synergistic Activation Mediator (SAM) system increases CLEC12A-MIR223HG expression. CLEC12A-MIR223HG translates into a chimeric protein, which largely resembles CLEC12A but harbours an altered C-type lectin domain altering key disulphide bonds. These alterations result in differences in post-translational modifications, cellular localization, and protein–protein interactions. Taken together, our observations support a possible involvement of CLEC12A-MIR223HG in the regulation of CLEC12A function. Our workflow also serves as a template to study other uncharacterized chimeric RNAs.

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. [ABSTRACT FROM AUTHOR]

Hundreds of plant mitogenomes have been sequenced from angiosperms, but relatively few mitogenomes are available from its sister lineage, gymnosperms. To examine mitogenomic diversity among extant gymnosperms, we generated draft mitogenomes from 11 diverse species and compared them with four previously published mitogenomes. Examined mitogenomes from Pinaceae and cycads retained all 41 protein genes and 26 introns present in the common ancestor of seed plants, whereas gnetophyte and cupressophyte mitogenomes experienced extensive gene and intron loss. In Pinaceae and cupressophyte mitogenomes, an unprecedented number of exons are distantly dispersed, requiring trans -splicing of 50–70% of mitochondrial introns to generate mature transcripts. RNAseq data confirm trans- splicing of these dispersed exons in Pinus. The prevalence of trans -splicing in vascular plant lineages with recombinogenic mitogenomes suggests that genomic rearrangement is the primary cause of shifts from cis- to trans- splicing in plant mitochondria. [ABSTRACT FROM AUTHOR]

Phytoene synthase (PSY) has been considered as an important regulatory enzyme in carotenoids biosynthesis pathway. Previous study finds that the yellow fruit in Solanum lycopersicum var. cerasiforme accession PI 114490 is caused by loss-of-function of SlPSY1 due to trans-splicing between SlPsy1 and an unknown gene transcribed from neighbour opposite strand DNA of SlPsy1. The genomic DNA sequences of SlPsy1 between red and yellow-fruited tomato lines have one single-nucleotide polymorphism (SNP) in the fourth intron and one SSR in the intergenic region. In the current study, the cause of trans-splicing event was further investigated. The data showed that the previously defined unknown gene was a putative long non-coding RNA ACoS-AS1 with three variants in many yellow-fruited tomato lines. The intronic SNP and intergenic SSR were tightly associated with trans-splicing event SlPsy1-ACoS-AS1. However, transgenic tomato lines carrying the genomic DNA of SlPsy1 from PI 114490 did not generate transcripts of ACoS-AS1and SlPsy1-ACoS-AS1 suggesting that only the intronic SNP could not cause the trans-splicing event. Over-expression of SlPsy1-ACoS-AS1 in red-fruited tomato line M82 did not have any phenotype change while over-expression of wild type SlPsy1 resulted in altered leaf colour. Sub-cellular localization analysis showed that SlPSY1-ACoS-AS1 could not enter plastids where SlPSY1 has its enzyme activity. Mutation of ACoS-AS1 in PI 114490 generated by CRISPR/Cas9 techniques resulted in red fruits implying that ACoS-AS1 was essential to trans-splicing event SlPsy1-ACoS-AS1. The results obtained here will extend knowledge to understand the mechanism of trans-splicing event SlPsy1-ACoS-AS1 and provide additional information for the regulation of carotenoids biosynthesis. [ABSTRACT FROM AUTHOR]

A long-standing mystery of genomic/transcriptomic structure involves spliced leader trans-splicing (SLTS), in which short RNA "tags" transcribed from a distinct genomic locus is added near the 5′ end of RNA transcripts by the spliceosome. SLTS has been observed in diverse eukaryotes in a phylogenetic pattern implying recurrent independent evolution. This striking convergence suggests important functions for SLTS, however no general novel function is known. Recent findings of frequent alternative SLTS (ALT-TS) suggest that ALT-TS could impart widespread functionality. Here, we tested the hypothesis that ALT-TS diversifies proteomes by comparing splicing patterns in orthologous genes between two deeply diverged trypanosome parasites. We also tested proteome diversification functions of ALT-TS by utilizing ribosome profiling sequence data. Finally, we investigated ALT-TS as a mechanism to regulate the expression of unproductive transcripts. Although our results indicate the functional importance of some cases of trans-splicing, we find no evidence for the hypothesis that proteome diversification is a general function of trans-splicing. [ABSTRACT FROM AUTHOR]