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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.

Summary Background Generalized severe epidermolysis bullosa simplex (EBS‐gen sev) is a genetic blistering skin disease in which autosomal dominant mutations in either the keratin KRT5 or KRT14 genes lead to impaired function of the intermediate filament cytoskeleton in the basal epidermis. Here we present an ex vivo RNA trans‐splicing‐based therapeutic approach to correct the phenotype. Objectives To correct a mutation within exon 1 of the KRT14 gene, using a 5′‐trans‐splicing approach, where any mutation within the first seven exons could be replaced by a single therapeutic molecule. Methods A therapeutic RNA trans‐splicing molecule containing wild‐type exons 1–7 was stably transduced into an EBS patient‐derived keratinocyte line. Trans‐splicing was confirmed via reverse‐transcriptase polymerase chain reaction, Western blotting and immunofluorescence microscopy. Skin equivalents generated from corrected keratinocytes were grafted onto nude mice and analysed about 8 weeks post‐transplantation for regular epidermal stratification, trans‐splicing‐induced green fluorescent protein expression and blistering. Results Transplanted skin equivalents generated from trans‐splicing‐corrected patient keratinocytes showed a stable and blister‐free epidermis. KRT14 correction disrupted EBS‐gen sev‐associated proinflammatory signalling, as shown at the mRNA and protein levels. Disruption of the pathogenic feedback loop in addition to overall downregulation of KRT14 expression highlighted the effect of KRT14 correction on the EBS pathomechanism. Conclusions Our data demonstrate that trans‐splicing‐mediated mRNA therapy is an effective method for the correction of dominantly inherited KRT14 mutations at the transcriptional level. This results in the rescue of the EBS‐gen sev phenotype and stabilization of the epidermis in a xenograft mouse model., What's already known about this topic? RTM163, described in this study, was previously used in a transient in vitro transfection system, where the ability to correct KRT14 at the mRNA level was demonstrated. What does this study add? In this study, we stably transduced RTM163 in a second patient‐derived keratinocyte line.Successful trans‐splicing was confirmed in this cell line.The expression of disease‐related marker genes, which are characteristically deregulated in epidermolysis bullosa simplex, were analysed.For the first time this study showed that RNA trans‐splicing molecule‐transduced patient keratinocytes can differentiate into a phenotypically normal and blister‐free epidermis in a xenograft mouse model. What is the translational message? This study shows the feasibility of using spliceosome‐mediated RNA trans‐splicing to generate a stable and blister‐free epidermis in vivo.Combined with pre‐existing ex vivo gene therapeutic methods, this might be a valid option for future treatments of dominantly inherited genodermatoses. Linked Comment: Bremer and van den Akker. Br J Dermatol 2019; 180:17–19.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) has been identified as a cancer-specific transcript in various solid cancers, including colorectal cancer. Given its excellent cancer-specific expression profile, we hypothesized that Ct-OATP1B3 could represent a promising target for cancer-specific expression of the suicide gene, herpes simplex virus 1 thymidine kinase (HSV-tk), via a spliceosome-mediated RNA trans-splicing (SMaRT) approach. SMaRT technology is used to recombine two RNA molecules to generate a chimeric transcript. In this study, we engineered an RNA trans-splicing molecule carrying a translation-defective HSV-tk sequence (RTM44), which was capable of inducing its own trans-splicing to the desired Ct-OATP1B3 pre-mRNA target. RTM44 expression in LS180 cells resulted in generation of Ct-OATP1B3/HSV-tk fusion mRNA. A functional translation start site contributed by the target pre-mRNA restored HSV-tk protein expression, rendering LS180 cells sensitive to ganciclovir treatment in vitro and in xenografted mice. The observed effects are ascribed to accurate and efficient trans-splicing, as they were absent in cells carrying a splicing-deficient mutant of RTM44. Collectively, our data highlights Ct-OATP1B3 as an ideal target for the HSV-tk SMaRT suicide system, which opens up new translational avenues for Ct-OATP1B3-targeted cancer therapy.

Summary 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 The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology., 70(12):1267–1274, 2018

Ribozymes are catalytic RNAs that are attractive platforms for the construction of nanoscale objects with biological functions. We designed a dimeric form of the Tetrahymena group I ribozyme as a unit structure in which two ribozymes were connected in a tail-to-tail manner with a linker element. We introduced a kink-turn motif as a bent linker element of the ribozyme dimer to design a closed trimer with a triangular shape. The oligomeric states of the resulting ribozyme dimers (kUrds) were analyzed biochemically and observed directly by atomic force microscopy (AFM). Formation of kUrd oligomers also triggered trans-splicing reactions, which could be monitored with a reporter system to yield a fluorescent RNA aptamer as the trans-splicing product.

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.Возбудитель описторхоза, печеночный сосальщик Opisthorchis felineus (Rivolta, 1884) – один из наиболее распространенных видов гельминтов человека и животных на территории России. Вместе с близкородственными видами печеночных трематод O. viverrini (Poirier, 1886) и Clonorchis sinensis (Loos, 1907), ареалы которых расположены в Юго-Восточной Азии и на Дальнем Востоке, О. felineus составляет триаду эпидемиологически значимых трематод семейства Opisthorchiidae. Половозрелые особи (мариты) O. felineus паразитируют в гепатобилиарной системе теплокровных и при длительной инвазии провоцируют развитие тяжелых осложнений, включая малигнизацию эпителия желчных протоков. Высокая медицинская значимость O. felineus привлекает внимание исследователей, работающих в различных областях биологии и медицины. Так, в последнее время активно проводятся исследования молекулярной биологии этого представителя паразитических плоских червей. В настоящем обзоре кратко суммированы результаты исследований геномики и протеомики O. felineus, являющиеся, на наш взгляд, существенным вкладом в решение вопросов структурно-функциональной организации геномов многоклеточных паразитов со сложным жизненным циклом и изучение молекулярных механизмов взаимодействия паразит–хозяин. Приведены сравнительные данные по количеству генов и размерам ядерных геномов ряда плоских червей, распределению длин интронов, а также анализу синтении геномов описторхид O. felineus, O. viverrini и C. sinensis. Отдельное внимание уделено обсуждению особой формы процессинга РНК, известной как транс-сплайсинг, широко представленной в геноме O. felineus. В статье приводятся анализ литературных данных по генам системы метаболизма ксенобиотиков, сравнению представленности генов этой систе- мы между различными видами паразитических и свободноживущих плоских червей и обзор литературы по наличию и уровню экспрессии генов гранулинов – потенциальных промоторов неоплазии холангиоцитов. Данные по геномике и протеомике O. felineus пополняют базу знаний, обеспечивающую исследо- вание проблем эволюции трематод, патогенеза печеночных гельминтозов и гельминт-ассоциированного канцерогенеза.

Atypical S1 and S11 split inteins have been used for N-terminal or C-terminal protein labeling. Here we reported a novel site-specific internal protein labeling method based on two atypical split inteins, Ter DnaE3 S11 and Rma DnaB S1. Protein-peptide trans-splicing activity was first demonstrated in vitro between a short peptide (Flag tag, FLAG) and two recombinant proteins (Maltose binding protein, MBP, and Thioredoxin, Trx) by trans-splicing between MBP-TE3S11N (MBP-N fragment of Ter DnaE3 S11), TE3S11C-FLAG-RBS1N (C fragment of Ter DnaE3 S11-FLAG-N fragment of Rma DnaB S1), and RBS1C-Trx (C fragment of Rma DnaB S1-Trx). To minimize the middle synthetic peptide (TE3S11C-linker-RBS1N), we reduced the number of native extein amino acids, which may play a role in protein trans-splicing. The results showed at least 3 (CKG) native extein amino acids were required for detectable trans-splicing activity. This method was further demonstrated to be effective in facilitating the incorporation of fluorescent probe (FITC) to the internal site of recombinant protein, generating the FITC-labeled protein. Besides the fluorescent group, these two split inteins can also be useful for adding any desirable chemical groups into a protein of interest, which may include biotin, modified and unnatural amino acids, or drug molecules.

Chimeric RNAs are hybrid transcripts containing exons from two separate genes. Chimeric RNAs are traditionally considered to be transcribed from fusion genes caused by chromosomal rearrangement. These canonical chimeric RNAs are well characterized to be expressed in a cancer-unique pattern and/or act as oncogene products. However, benefited by the development of advanced deep sequencing technologies, novel types of non-canonical chimeric RNAs have been discovered to be generated from intergenic splicing without genomic aberrations. They can be formed through trans-splicing or cis-splicing between adjacent genes (cis-SAGe) mechanisms. Non-canonical chimeric RNAs are widely detected in normal physiology, although several have been shown to have a cancer-specific expression pattern. Further studies have indicated that some of them play fundamental roles in controlling cell growth and motility, and may have functions independent of the parental genes. These discoveries are unveiling a new layer of the functional transcriptome and are also raising the possibility of utilizing non-canonical chimeric RNAs as cancer diagnostic markers and therapeutic targets. In this chapter, we will overview different categories of chimeric RNAs and their expression in various types of cancerous and normal samples. Acknowledging that chimeric RNAs are not unique to cancer, we will discuss both bioinformatic and biological methods to identify credible cancer-specific chimeric RNAs. Furthermore, we will describe downstream methods to explore their molecular processing mechanisms and potential functions. A better understanding of the biogenesis mechanisms and functional products of cancer-specific chimeric RNAs will pave ways for the development of novel cancer biomarkers and therapeutic targets.

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.

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.

A recent meta-analysis of alternative exon usage in C. elegans refined our comprehension of its transcriptome, especially regarding the splicing quantitative aspects of alternative splicing in messenger RNAs. However, Next-Generation Sequencing technologies like Illumina technology are proving to be limited to fully characterize one’s transcriptome. PCR-based sequencing methods are known to introduce amplification bias affecting the overall distribution of mRNAs detected in one experiment and short-reads are not suited to accurately predict the frequency of isoforms derived from multiple alternative splicing events.In this study, we exploited new possibilities offered by Oxford Nanopore Technology (ONT) to overcome those limitations. Nanopore-based sequencing allow us to directly sequence nucleic acids without any prior amplification step and generates long-reads covering up to the full-length of the molecule. Hence, permitting to further characterize C. elegans transcriptome by providing a more accurate measure of isoforms ratios, a better comprehension of exons associations during alternative splicing and by characterizing differentially trans-spliced mRNAs.We assessed the efficiency of different sequencing kits commercialized by ONT and our results indicates that direct-cDNA sequencing is better suited for performing transcriptome analysis in C. elegans, in regard to the quantity and quality of data generated. Following this analysis, several direct-cDNA sequencing experiments have been performed on different populations of mRNAs: libraries of poly(A) RNAs representing the whole-animal transcriptome and libraries of SL1-enriched mRNAs. Our findings indicates that trans-spliced RNAs have an atypical behaviour during ONT’s library preparation and trans-splicing of C. elegans mRNAs is more prevalent than previously reported. Finally, we also show that alternative promoters can lead to population of isoformes exhibiting different trans-splicing status., Une récente méta-analyse de l’utilisation des jonctions exon-exon chez C. elegans nous a permis d’améliorer notre compréhension de son transcriptome, en particulier pour la quantification des événements d’épissage alternatif au sein des ARNs messagers. Cependant, les technologies de séquençage NGS, comme l’Illumina, se montrent limitées pour l’étude des transcriptomes. Ces technologies, basées sur la PCR, entraînent l’apparition de biais d’amplifications qui affectent la distribution des ARNms détectés au sein d’une expérience, et la petite taille des fragments générés n’est pas adapté pour correctement déterminer la fréquence des isoformes issus des différents événements d’épissage alternatifs.Dans cette étude, nous avons utilisé les nouvelles possibilités offertes par les séquenceurs Oxford Nanopore Technology (ONT) pour nous affranchir de ces limitations. Le séquençage nanopore nous permet de séquencer directement des molécules d’acides nucléiques sans avoir recours à des étapes d’amplification, et permet de générer des fragments pouvant couvrir la taille totale de la molécule séquencée. Ceci nous permettant de mieux caractériser le transcriptome de C. elegans, en effectuant une mesure plus précise des fréquences d’isoformes, et en nous permettant une meilleure compréhension des événements d’épissage alternatif et de trans-épissage qui affectent les messagers.Nous avons évalué trois différents kits de séquençage commercialisés par ONT et nos résultats suggèrent que le kit de séquençage direct-cDNA est plus adapté aux études de transcriptomes, en termes de quantité et de qualité de données générées. À la suite de cette analyse, plusieurs séquençages direct-cDNA ont été réalisés sur différentes populations d’ARNs : des librairies d’ARNs poly(A) représentants le transcriptome entier de C. elegans, et des librairies enrichies en ARNs SL1. Nos résultats indiquent que les ARNs trans-épissés se comportent différemment lors de la préparation des librairies ONT et que les phénomènes de trans-épissage sont plus prédominants que précédemment rapporté. Enfin, nous montrons que les promoteurs alternatifs peuvent conduire à la génération de populations d’isoformes présentant différents statuts de trans-épissage.

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.

When expressing streptavidin recombinant polypeptide on magnetosomes (called bacterial magnetic nanoparticles, or BMPs), the presence of endogenous bacterial biotin might be detrimental. In the study, the streptavidin monomer fragment (S1-116) was fused with the intein N-terminal (termed precursor S1-116-IN), and S1-116-IN was expressed in E. coli (BL21). Meanwhile, the SA117-160 fragment was fused with the C-terminal intein, and then this chimeric polypeptide was expressed on magnetosomes by fusion with magnetosome membrance protein MamF. In the in vitro protein splicing system, the purified engineered magnetosomes (BMP-SA117-160-IC) and the S1-116-IN precursor were mixed. Intein-mediated trans-splicing reaction was induced to produce the functional magnetic beads BMP-SA. Our results indicate that intein-mediated protein trans-splicing may lead to efficient synthesis of the recombinant streptavidin on the magnetosomes, showing its promising potential to produce other functional magnetic nanoparticles.

Hepatitis B virus core protein (HBc) spontaneously assembles as Virus-like particles (VLPs) in Escherichia coli (E. coli) which is extensively used as a nanocarrier to boost antigen immunogenicity. Genetic fusion of cargo protein with HBc occasionally forms inclusion bodies instead of properly assembled VLPs. To this end, we devised HBc VLPs as a modular nanocarrier for antigen delivery by intein-mediated trans-splicing (TS). We introduced split inteinC (intC) to the C-terminus of split HBc N-core to employ intein-mediated TS technology to HBc VLPs. Split HBc with the insertion of intC at N-core C-terminus (designated as HBc N-intC-C) existed in inclusion bodies. Interestingly, introduction of a soluble tag, gb1, to intC C-terminus remarkably improved the solubility of recombinant protein (named HBc N-intC-gb1-C). Moreover, newly designed recombinant spontaneously assembled as VLPs and endowed efficiently coupling two different model antigens onto HBc N-intC-gb1-C VLPs. Furthermore, model antigens delivered by HBc VLPs induced a dramatically enhanced antigen-specific immune responses. Antigen proteins mainly elicited Th2 IgG responses while antigens delivered by HBc VLPs steered Th1/Th2 balanced IgG responses. Taken together, intein-mediated TS was amenable to decorate HBc VLPs with antigens and showed good potential for antigen delivery.

BACKGROUND: RNA trans-splicing joins exons from different pre-mRNA transcripts to generate a chimeric product. Trans-splicing can also occur at the protein level, with split inteins mediating the ligation of separate gene products to generate a mature protein. SOURCES OF DATA: Comprehensive literature search of published research papers and reviews using Pubmed. AREAS OF AGREEMENT: Trans-splicing techniques have been used to target a wide range of diseases in both in vitro and in vivo models, resulting in RNA, protein and functional correction. AREAS OF CONTROVERSY: Off-target effects can lead to therapeutically undesirable consequences. In vivo efficacy is typically low, and delivery issues remain a challenge. GROWING POINTS: Trans-splicing provides a promising avenue for developing novel therapeutic approaches. However, much more research needs to be done before developing towards preclinical studies. AREAS TIMELY FOR DEVELOPING RESEARCH: Increasing trans-splicing efficacy and specificity by rational design, screening and competitive inhibition of endogenous cis-splicing.

Group II introns are noncoding sequences that interrupt genes, and that must be removed or spliced-out at the RNA level during gene expression. Following the transcription of interrupted genes, group II introns self-splice while concurrently ligating their flanking exons to generate mature mRNAs ready for translation. Ll.LtrB, the model group II intron from the gram-positive bacterium Lactococcus lactis, interrupts the gene coding for a relaxase enzyme that initiates the transfer of mobile elements by conjugation. This functional link between group II intron splicing and conjugative transfer enabled us to engineer highly sensitive splicing assays using the native biological context of Ll.LtrB. The splicing efficiency/conjugation assay was developed to determine the splicing competence of various Ll.LtrB mutants, whereas the splicing selection/conjugation assay was established to isolate splicing-proficient variants from a randomly generated bank of mutated introns.

Monoclonal antibodies, engineered antibodies, and antibody fragments have become important biological therapeutic platforms. The IgG format with bivalent binding sites has a modular structure with different biological roles, i.e., effector and binding functions, in different domains. We demonstrated the reconstruction of an IgG-like domain structure in vitro by protein ligation using protein trans-splicing. We produced various binding domains to replace the binding domain of IgG from Escherichia coli and the Fc domain of human IgG from Brevibacillus choshinensis as split-intein fusions. We showed that in vitro protein ligation could produce various Fc-fusions at the N-terminus in vitro from the independently produced domains from different organisms. We thus propose an off-the-shelf approach for the combinatorial production of Fc fusions in vitro with several distinct binding domains, particularly from naturally occurring binding domains. Antiviral lectins from algae are known to inhibit virus entry of HIV and SARS coronavirus. We demonstrated that a lectin could be fused with the Fc-domain in vitro by protein ligation, producing an IgG-like molecule as a &ldquo, lectibody&rdquo, Such an Fc-fusion could be produced in vitro by this approach, which could be an attractive method for developing potential therapeutic agents against rapidly emerging infectious diseases like SARS coronavirus without any genetic fusion and expression optimization.

Manipulation of proteins by chemical modification is a powerful way to decipher their function. However, most ribosome-dependent and semi-synthetic methods have limitations in the number and type of modifications that can be introduced, especially in live cells. Here, we present an approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into proteins expressed in eukaryotic cells. We insert synthetic peptides into GFP, NaV1.5 and P2X2 receptors via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating protein function. We anticipate the approach will overcome some drawbacks of existing protein enigineering methods., Chemical modification of proteins can be used to decipher function or use that function for therapeutic purposes. Here, the authors insert synthetic peptides via tandem protein trans-splicing to add post-translational modifications or non-canonical amino acids.

Recombination is widespread among RNA viruses, but many molecular mechanisms of this phenomenon are still poorly understood. It was believed until recently that the only possible mechanism of RNA recombination is replicative template switching, with synthesis of a complementary strand starting on one viral RNA molecule and being completed on another. The newly synthesized RNA is a primary recombinant molecule in this case. Recent studies have revealed other mechanisms of replicative RNA recombination. In addition, recombination between the genomes of RNA viruses can be nonreplicative, resulting from a joining of preexisting parental molecules. Recombination is a potent tool providing for both the variation and conservation of the genome in RNA viruses. Replicative and nonreplicative mechanisms may contribute differently to each of these evolutionary processes. In the form of trans splicing, nonreplicative recombination of cell RNAs plays an important role in at least some organisms. It is conceivable that RNA recombination continues to contribute to the evolution of DNA genomes.

In recent years, split inteins have seen widespread use as molecular platforms for the design of a variety of peptide and protein chemistry technologies, most notably protein ligation. The development of these approaches is dependent on the identification and/or design of split inteins with robust activity, stability, and solubility. Here, we describe two approaches to characterize and compare the activities of newly identified or engineered split inteins. The first assay employs an E. coli-based selection system to rapidly screen the activities of many inteins and can be repurposed for directed evolution. The second assay utilizes reverse-phase high-performance liquid chromatography (RP-HPLC) to provide insights into individual chemical steps in the protein splicing reaction, information that can guide further engineering efforts. These techniques provide useful alternatives to common assays that utilize SDS-PAGE to analyze splicing reaction progress.

The functional content of the mitochondrial genome (mitogenome) is highly diverse across eukaryotes. Among land plants, our understanding of the variation in mitochondrial gene and intron content is improving from concerted efforts to densely sample mitogenomes from diverse land plants. Here I review the current state of knowledge regarding the diversity in content of protein genes and introns in the mitogenomes of all major land plant lineages. Mitochondrial protein gene content is largely conserved among mosses and liverworts, but it varies substantially among and within other land plant lineages due to convergent losses of genes encoding ribosomal proteins and, to a lesser extent, genes for proteins involved in cytochrome c maturation and oxidative phosphorylation. Mitochondrial intron content is fairly stable within each major land plant lineage, but highly variable among lineages, resulting from occasional gains and many convergent losses over time. Trans-splicing has evolved dozens of times in various vascular plant lineages, particularly those with relatively higher rates of mitogenomic rearrangement. Across eukaryotes, mitochondrial protein gene and intron content has been shaped massive convergent evolution.

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.

The abstract is available here: https://uscholar.univie.ac.at/o:1229895

Expressed protein ligation is a simple and powerful method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size. This methodology has been developed based on the knowledge obtained from protein splicing. Protein splicing is a multistep biochemical reaction that includes the concomitant cleavage and formation of peptide bonds carried out by self-processing domains named inteins. The natural substrates of protein splicing are essential proteins found in intein-containing organisms; inteins are also functional in nonnative frameworks and can be used to alter nearly any protein's primary amino acid sequence. Accordingly, different reactivity features of inteins have been largely exploited to manipulate proteins in countless methods encompassing fields from biochemical research to the development of biotechnological applications including the study of disease progression and validation of potential drug candidates. Here, we review almost three decades of research to uncover the chemical and biochemical enigmas of protein splicing and the development of inteins as potent protein engineering tools.

A recent meta-analysis of alternative exon usage in C. elegans refined our comprehension of its transcriptome, especially regarding the splicing quantitative aspects of alternative splicing in messenger RNAs. However, Next-Generation Sequencing technologies like Illumina technology are proving to be limited to fully characterize one’s transcriptome. PCR-based sequencing methods are known to introduce amplification bias affecting the overall distribution of mRNAs detected in one experiment and short-reads are not suited to accurately predict the frequency of isoforms derived from multiple alternative splicing events.In this study, we exploited new possibilities offered by Oxford Nanopore Technology (ONT) to overcome those limitations. Nanopore-based sequencing allow us to directly sequence nucleic acids without any prior amplification step and generates long-reads covering up to the full-length of the molecule. Hence, permitting to further characterize C. elegans transcriptome by providing a more accurate measure of isoforms ratios, a better comprehension of exons associations during alternative splicing and by characterizing differentially trans-spliced mRNAs.We assessed the efficiency of different sequencing kits commercialized by ONT and our results indicates that direct-cDNA sequencing is better suited for performing transcriptome analysis in C. elegans, in regard to the quantity and quality of data generated. Following this analysis, several direct-cDNA sequencing experiments have been performed on different populations of mRNAs: libraries of poly(A) RNAs representing the whole-animal transcriptome and libraries of SL1-enriched mRNAs. Our findings indicates that trans-spliced RNAs have an atypical behaviour during ONT’s library preparation and trans-splicing of C. elegans mRNAs is more prevalent than previously reported. Finally, we also show that alternative promoters can lead to population of isoformes exhibiting different trans-splicing status., Une récente méta-analyse de l’utilisation des jonctions exon-exon chez C. elegans nous a permis d’améliorer notre compréhension de son transcriptome, en particulier pour la quantification des événements d’épissage alternatif au sein des ARNs messagers. Cependant, les technologies de séquençage NGS, comme l’Illumina, se montrent limitées pour l’étude des transcriptomes. Ces technologies, basées sur la PCR, entraînent l’apparition de biais d’amplifications qui affectent la distribution des ARNms détectés au sein d’une expérience, et la petite taille des fragments générés n’est pas adapté pour correctement déterminer la fréquence des isoformes issus des différents événements d’épissage alternatifs.Dans cette étude, nous avons utilisé les nouvelles possibilités offertes par les séquenceurs Oxford Nanopore Technology (ONT) pour nous affranchir de ces limitations. Le séquençage nanopore nous permet de séquencer directement des molécules d’acides nucléiques sans avoir recours à des étapes d’amplification, et permet de générer des fragments pouvant couvrir la taille totale de la molécule séquencée. Ceci nous permettant de mieux caractériser le transcriptome de C. elegans, en effectuant une mesure plus précise des fréquences d’isoformes, et en nous permettant une meilleure compréhension des événements d’épissage alternatif et de trans-épissage qui affectent les messagers.Nous avons évalué trois différents kits de séquençage commercialisés par ONT et nos résultats suggèrent que le kit de séquençage direct-cDNA est plus adapté aux études de transcriptomes, en termes de quantité et de qualité de données générées. À la suite de cette analyse, plusieurs séquençages direct-cDNA ont été réalisés sur différentes populations d’ARNs : des librairies d’ARNs poly(A) représentants le transcriptome entier de C. elegans, et des librairies enrichies en ARNs SL1. Nos résultats indiquent que les ARNs trans-épissés se comportent différemment lors de la préparation des librairies ONT et que les phénomènes de trans-épissage sont plus prédominants que précédemment rapporté. Enfin, nous montrons que les promoteurs alternatifs peuvent conduire à la génération de populations d’isoformes présentant différents statuts de trans-épissage.

Background Spliced Leader trans-splicing is an important mechanism for the maturation of mRNAs in several lineages of eukaryotes, including several groups of parasites of great medical and economic importance. Nevertheless, its study across the tree of life is severely hindered by the problem of identifying the SL sequences that are being trans-spliced. Results In this paper we present SLFinder, a four-step pipeline meant to identify de novo candidate SL sequences making very few assumptions regarding the SL sequence properties. The pipeline takes transcriptomic de novo assemblies and a reference genome as input and allows the user intervention on several points to account for unexpected features of the dataset. The strategy and its implementation were tested on real RNAseq data from species with and without SL Trans-Splicing. Conclusions SLFinder is capable to identify SL candidates with good precision in a reasonable amount of time. It is especially suitable for species with unknown SL sequences, generating candidate sequences for further refining and experimental validation.

The development of expressed protein ligation (EPL) widened the scope of questions that could be addressed by mechanistic biochemistry. Protein trans-splicing (PTS) relies on the same basic chemical principles, but utilizes split inteins to tracelessly ligate distinct peptide or polypeptide fragments together with native peptide bonds. Here we present a method to adapt PTS methodologies for their use in live cells, in order to deliver synthetic or native histone modifications. As an example, we provide a protocol to incorporate a small molecule fluorophore into chromatinized histones. The protocol should be easily adaptable to incorporate other modifications to chromatin in vivo.