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The Tetrahymena group I intron was one of the two first catalytic RNAs (ribozymes) to be discovered. Group I introns are sequences that can exist between exons in pre-mRNAs, that are able to self-splice and remove themselves when forming mature mRNAs in the absence of the spliceosome. The Tetrahymena cis-splicing ribozyme was engineered to accept substrate RNAs in trans. The designed trans-splicing ribozyme was termed the “spliceozyme”. The spliceozyme could in principle be used in therapeutic applications. However, there are two hurdles to overcome; the delivery of the ribozyme into cells and its efficiency once delivered. The focus of this study is to improve the efficiency of the trans-splicing ribozyme in cells by evolving the ribozyme in E. coli. In a previous study, an evolution system in cells was developed for the spliceozyme which was used to generate a clone, W11, that increased product formation. However, this evolution used high ribozyme expression levels and focused on a single splice site. To improve the efficiency and sequence generality, the spliceozyme was evolved at low expression levels at two different splice sites with different flanking sequences. The results of the spliceozyme evolution in bacterial cells showed that specific mutations are able to improve spliceozyme efficiency in bacterial cells. This suggests that further analysis, and perhaps further evolution experiments, could generate spliceozymes with improved efficiency on a broad range of substrate sequences.

The Tetrahymena group I intron was one of the two first catalytic RNAs (ribozymes) to be discovered. Group I introns are sequences that can exist between exons in pre-mRNAs, that are able to self-splice and remove themselves when forming mature mRNAs in the absence of the spliceosome. The Tetrahymena cis-splicing ribozyme was engineered to accept substrate RNAs in trans. The designed trans-splicing ribozyme was termed the “spliceozyme”. The spliceozyme could in principle be used in therapeutic applications. However, there are two hurdles to overcome; the delivery of the ribozyme into cells and its efficiency once delivered. The focus of this study is to improve the efficiency of the trans-splicing ribozyme in cells by evolving the ribozyme in E. coli. In a previous study, an evolution system in cells was developed for the spliceozyme which was used to generate a clone, W11, that increased product formation. However, this evolution used high ribozyme expression levels and focused on a single splice site. To improve the efficiency and sequence generality, the spliceozyme was evolved at low expression levels at two different splice sites with different flanking sequences. The results of the spliceozyme evolution in bacterial cells showed that specific mutations are able to improve spliceozyme efficiency in bacterial cells. This suggests that further analysis, and perhaps further evolution experiments, could generate spliceozymes with improved efficiency on a broad range of substrate sequences.

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.

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.

Although mitochondria have evolved from a single endosymbiotic event, present day mitochondria of diverse eukaryotes display a great range of genome structures, content and features. Group I and group II introns are two features that are distributed broadly but patchily in mitochondrial genomes across branches of the tree of eukaryotes. While group I intron-mediated trans -splicing has been reported from some lineages distantly related to each other, findings of group II intron-mediated trans -splicing has been restricted to members of the Chloroplastida. In this study, we found the mitochondrial genome of the unicellular eukaryote Diphylleia rotans possesses currently the second largest gene repertoire. On the basis of a probable phylogenetic position of Diphylleia , which is located within Amorphea, current mosaic gene distribution in Amorphea must invoke parallel gene losses from mitochondrial genomes during evolution. Most notably, although the cytochrome c oxidase subunit ( cox ) 1 gene was split into four pieces which located at a distance to each other, we confirmed that a single mature mRNA that covered the entire coding region could be generated by group II intron-mediated trans -splicing. This is the first example of group II intron-mediated trans -splicing outside Chloroplastida. Similar trans- splicing mechanisms likely work for bipartitely split cox2 and nad3 genes to generate single mature mRNAs. We finally discuss origin and evolution of this type of trans -splicing in D. rotans as well as in eukaryotes.

Anopheles stephensi is a potent vector of malaria throughout the Indian subcontinent and Middle East. An. stephensi is emerging as a model for molecular and genetic studies of mosquito-parasite interactions. Here we conducted a series of genomic and transcriptomic studies to improve the understanding of the biology of Anopheles stephensi and mosquito in general. First we reported the genome sequence and annotation of the Indian strain of the type form of An. stephensi. The 221 Mb genome assembly was produced using a combination of 454, Illumina, and PacBio sequencing. This hybrid assembly method was significantly better than assemblies generated from a single data source. A total of 11,789 protein-encoding genes were annotated using a combination of homology and de novo prediction. Secondly, we demonstrated the presence of complete dosage compensation in An. stephensi by determining that autosomal and X-linked genes have very similar levels of expression in both males and females. The uniformity of average expression levels of autosomal and X-linked genes remained when An. stephensi gene expression was normalized by that of their Ae. aegypti orthologs, strengthening the conclusion of complete dosage compensation in Anopheles. Lastly, we investigated trans-splicing events in Anopheles stephensi. We identified six trans-splicing events and all the trans-splicing sites are conserved and present in Ae. aegypti. The proteins encoded by the trans-spliced mRNAs are also highly conserved and their orthologs are co-linearly transcribed in out-groups of family Culicidae. This finding indicates the need to preserve the intact mRNA and protein function of the broken-up genes by trans-splicing during evolution. In summary, we presented the first genome assembly of Anopheles stephensi and studied two interesting evolution events" dosage compensation and trans-splicing - via transcriptomic analysis.

Anopheles stephensi is a potent vector of malaria throughout the Indian subcontinent and Middle East. An. stephensi is emerging as a model for molecular and genetic studies of mosquito-parasite interactions. Here we conducted a series of genomic and transcriptomic studies to improve the understanding of the biology of Anopheles stephensi and mosquito in general. First we reported the genome sequence and annotation of the Indian strain of the type form of An. stephensi. The 221 Mb genome assembly was produced using a combination of 454, Illumina, and PacBio sequencing. This hybrid assembly method was significantly better than assemblies generated from a single data source. A total of 11,789 protein-encoding genes were annotated using a combination of homology and de novo prediction. Secondly, we demonstrated the presence of complete dosage compensation in An. stephensi by determining that autosomal and X-linked genes have very similar levels of expression in both males and females. The uniformity of average expression levels of autosomal and X-linked genes remained when An. stephensi gene expression was normalized by that of their Ae. aegypti orthologs, strengthening the conclusion of complete dosage compensation in Anopheles. Lastly, we investigated trans-splicing events in Anopheles stephensi. We identified six trans-splicing events and all the trans-splicing sites are conserved and present in Ae. aegypti. The proteins encoded by the trans-spliced mRNAs are also highly conserved and their orthologs are co-linearly transcribed in out-groups of family Culicidae. This finding indicates the need to preserve the intact mRNA and protein function of the broken-up genes by trans-splicing during evolution. In summary, we presented the first genome assembly of Anopheles stephensi and studied two interesting evolution events" dosage compensation and trans-splicing - via transcriptomic analysis.

Eukaryotic cells have several mRNA quality control checkpoints to avoid the production of aberrant proteins. Intron-containing mRNAs are actively degraded by the nuclear exosome, prevented from nuclear exit and, if these systems fail, degraded by the cytoplasmic NMD machinery. Trypanosomes have only two introns. However, they process mRNA5 from long polycistronic precursors by trans-splicing and polycistronic mRNA molecules frequently arise from any missed splice site. Here, we show that RNAi depletion of the trypanosome exosome, but not of the cytoplasmic 5'-3' exoribonuclease XRNA or the NMD helicase UPF1, causes accumulation of oligocistronic mRNA5. We have also revisited the localization of the trypanosome exosome by expressing eYFP-fusion proteins of the exosome subunits RRP44 and RRP6. Both proteins are significantly enriched in the nucleus. Together with published data, our data suggest a major nuclear function of the trypanosome exosome in rRNA, snoRNA and mRNA quality control. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.orgilicensesiby/4.0/).

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.

MicroRNAs (miRNAs) are small non-coding RNAs, ~22 nucleotides (nt) long, with major roles in gene regulation. MiRNAs bind imperfectly to complementary sequences in the 3’ untranslated region of target messenger RNAs (mRNAs) causing translational repression and destabilization. A single miRNA has the potential to regulate hundreds of different mRNA targets, highlighting the importance of miRNAs in almost all cellular pathways. Originally discovered as part of the Caenorhabditis elegans (C. elegans) developmental timing pathway, miRNAs were soon found in a multitude of other organisms, including humans. MiRBase, an online database for miRNAs, now lists >35,000 miRNAs, in >200 different species, including viruses, though many of their roles remain to be characterized. Because misregulation is often associated with disease, especially cancer, exploring miRNA biogenesis is critical for understanding the intricacies of disease development. Furthermore, the conserved temporal expression of various miRNAs highlights the importance of these regulators in pluripotency and development. Understanding how miRNAs are produced and regulated has been a major topic of study over the past 15 years. While the basic mechanisms of miRNA biogenesis and function have been uncovered, how these processes are regulated remains an outstanding problem.There are multiple instances of transcriptional and post-transcriptional regulation during miRNA biogenesis. In Chapter I, I introduce much of the latest understanding about the mechanisms of miRNA biogenesis and regulation. Details about the discovery of miRNAs, C. elegans as a model, as well as general information on biogenesis and targeting, can be found in Chapter II. I worked on several projects investigating post-transcriptional regulation of miRNA biogenesis in C. elegans. In Chapter III, I identify and characterize the primary lin-4 transcripts, and demonstrate how a conserved RNA binding protein, RBM-28, regulates mature lin-4 expr

MicroRNAs (miRNAs) are small non-coding RNAs, ~22 nucleotides (nt) long, with major roles in gene regulation. MiRNAs bind imperfectly to complementary sequences in the 3’ untranslated region of target messenger RNAs (mRNAs) causing translational repression and destabilization. A single miRNA has the potential to regulate hundreds of different mRNA targets, highlighting the importance of miRNAs in almost all cellular pathways. Originally discovered as part of the Caenorhabditis elegans (C. elegans) developmental timing pathway, miRNAs were soon found in a multitude of other organisms, including humans. MiRBase, an online database for miRNAs, now lists >35,000 miRNAs, in >200 different species, including viruses, though many of their roles remain to be characterized. Because misregulation is often associated with disease, especially cancer, exploring miRNA biogenesis is critical for understanding the intricacies of disease development. Furthermore, the conserved temporal expression of various miRNAs highlights the importance of these regulators in pluripotency and development. Understanding how miRNAs are produced and regulated has been a major topic of study over the past 15 years. While the basic mechanisms of miRNA biogenesis and function have been uncovered, how these processes are regulated remains an outstanding problem.There are multiple instances of transcriptional and post-transcriptional regulation during miRNA biogenesis. In Chapter I, I introduce much of the latest understanding about the mechanisms of miRNA biogenesis and regulation. Details about the discovery of miRNAs, C. elegans as a model, as well as general information on biogenesis and targeting, can be found in Chapter II. I worked on several projects investigating post-transcriptional regulation of miRNA biogenesis in C. elegans. In Chapter III, I identify and characterize the primary lin-4 transcripts, and demonstrate how a conserved RNA binding protein, RBM-28, regulates mature lin-4 expr

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.

Leishmania parasites cause leishmaniasis, a group of diseases that range in manifestations from skin lesions to fatal visceral disease. The parasite's life cycle is divided between its insect vector and its mammalian host, where it resides primarily inside of macrophages. Once intracellular, Leishmania parasites must avoid being killed by the innate and adaptive immune responses. We performed transcriptomic profiling using RNA-seq to simultaneously identify global changes in gene expression in Leishmania parasites across multiple lifecycle stages and in infected macrophages from both murine and human hosts. Using a novel approach based on a dual statistical test to identify genes that were differentially expressed relative to both uninfected macrophages and macrophages that had ingested inert particles, we were able to filter out genes that were differentially regulated as part of a general phagocytic response and thereby select genes specific to Leishmania infection. The most substantial and dynamic Leishmania-specific differential expression responses were observed during early infection, while changes observed later were common to phagocytosis more generally. An evaluation of RNA processing events within the parasite revealed precise UTR boundaries for a majority of genes and widespread alternative trans-splicing and polyadenylation. Collection of data from multiple biological replicates, the use of matched host control samples, careful statistical analysis of variation, and removal of batch effects enabled the detection of biological differences between samples and timepoints with high confidence and sensitivity. Pathway and gene ontology analyses provided insights into the higher level processes activated across parasite developmental stages and during intracellular infection to reveal signatures of Leishmania differentiation and infection.

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.

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.

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.

The Sm family of proteins is closely associated with RNA metabolismthroughout all life. These proteins form homomorphic and heteromorphic rings consisting of six or seven subunits with a characteristic central pore, the presence of which is critical for binding U-rich regions of single-stranded RNA. Eubacteria and Archaea typically carry one or two forms ofSm proteins and assemble one homomorphic ring per Sm protein. Eukaryotes typically carry 16 or more Sm proteins that assemble to form heteromorphic rings which lie at the center of a number of critical RNA-associated small nuclear ribonucleoproteins (snRNPs). High Sm protein diversity and heteromorphic Sm rings are features stretching back to the origin of eukaryotes; very deep phylogenetic divisions among existing Smproteins indicate simultaneous evolution across essentially all existing eukaryotic life. Two basic forms of heteromorphic Sm rings are found in eukaryotes. Fixed Sm rings are highly stable and static and are assembled around an RNA cofactor. Flexible Sm rings also stabilize and chaperone RNA but assemble in the absence of an RNA substrate and, more significantly, associate with and dissociate from RNA substrates more freely than fixed rings. This suggests that the conformation of flexible Smrings might be modified in some specific manner to facilitate association and dissociation with RNA. Diversification of eukaryotic Sm proteins may have been initiated by gene transfers and/or genome clashes that accompanied the origin of the eukaryotic cell itself, with further diversification driven by a greater need for steric specificity within increasingly complex snRNPs.

Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which can lead to the synthesis of two proteins. The occurrence of the three COT transcripts in rat has been found in all tissues examined and does not depend on sex, fat feeding, peroxisome proliferators or hyperinsulinaemia. Rat COT exon 2 contains a putative exonic splicing enhancer (ESE) sequence. Mutation of this ESE (GAAGAAG) to AAAAAAA decreased trans-splicing in vitro, from which it is deduced that this ESE sequence is partly responsible for the formation of the three transcripts. The protein encoded by cis-spliced mRNA of rat COT is inhibited by malonyl-CoA and etomoxir. cDNA species encoding full-length wild-type COT and one double mutant COT were expressed in Saccharomyces cerevisiae. The recombinant enzymes showed full activity towards both substrates, carnitine and decanoyl-CoA. The activity of the doubly mutated H131A/H340A enzyme was similar to that of the rat peroxisomal enzyme but was completely insensitive to malonyl-CoA and etomoxir. These results indicate that the histidine residues His-131 and His-340 are the sites responsible for the interaction of these two inhibitors, which inhibit COT by interacting with the same sites.

In man, activated N-, K- and H-ras oncogenes have been found in around 30% of the solid tumours tested. An exon known as IDX, which has been described previously and is located between exon 3 and exon 4A of the c-H-ras pre-mRNA, allows an alternative splicing process that results in the synthesis of the mRNA of a putative protein named p19. It has been suggested that this alternative pathway is less tumorigenic than that which results in the activation of p21. We have used the mammalian trans-splicing mechanism as a tool with which to modulate this particular pre-mRNA processing to produce mRNA similar to that of mature p19 RNA. The E4A exon of the activated H-ras gene was found to be a good target for external trans-splicing. We reprogrammed the rat carnitine octanoyltransferase exon 2 to specifically invade the terminal region of H-ras. Assays performed with this reprogrammed trans-exon showed that the trans-splicing product was obtained in competition with cis-splicing of the D intron of the H-ras gene, and was associated with concomitant down-modulation of D intron cis-splicing. We also found that the exon 4A of the human c-H-ras gene underwent successive trans-splicing rounds with an external exon.

Nowadays, a whole range of genetherapeutic methods is being used to restore a lost protein function due to mutation, a big number of preclinical and clinical studies of potential drugs that may allow to implement an etiotropic approach is being performed. One of the most prevalent and socially significant groups of genetic pathologies is musculardystrophy, including such diseases as Duchenne muscular dystrophy and dysfelinopathy. Despite a large number ofstudies in this field, there is no effective method of gene therapy for these diseases yet. This work is intended to review main genetherapeutic methods in myodystrophy treatment, especially pre- and posttranscriptional genetic (biosynthetic) information modification, and analyze most optimal of them.

© 2016, Springer Science+Business Media New York.Due to lack of effective therapies, muscular dystrophies became a focus for gene therapy. Multiple pre-clinical studies have shown successful restoration of dystrofin and dysferlin by RNA editing both in vivo and in vitro, but possibility of a clinical translation is still obscure. A number of new chemicals are being studied, and a search for new techniques is ongoing. This work is intended to give a brief overview of the current state of the RNA editing for treating muscular dystrophies.

© 2016, Springer Science+Business Media New York.Due to lack of effective therapies, muscular dystrophies became a focus for gene therapy. Multiple pre-clinical studies have shown successful restoration of dystrofin and dysferlin by RNA editing both in vivo and in vitro, but possibility of a clinical translation is still obscure. A number of new chemicals are being studied, and a search for new techniques is ongoing. This work is intended to give a brief overview of the current state of the RNA editing for treating muscular dystrophies.

Nowadays, a whole range of genetherapeutic methods is being used to restore a lost protein function due to mutation, a big number of preclinical and clinical studies of potential drugs that may allow to implement an etiotropic approach is being performed. One of the most prevalent and socially significant groups of genetic pathologies is musculardystrophy, including such diseases as Duchenne muscular dystrophy and dysfelinopathy. Despite a large number ofstudies in this field, there is no effective method of gene therapy for these diseases yet. This work is intended to review main genetherapeutic methods in myodystrophy treatment, especially pre- and posttranscriptional genetic (biosynthetic) information modification, and analyze most optimal of them.

Due to lack of effective therapies, muscular dystrophies became a focus for gene therapy. Multiple pre-clinical studies have shown successful restoration of dystrofin and dysferlin by RNA editing both in vivo and in vitro, but possibility of a clinical translation is still obscure. A number of new chemicals are being studied, and a search for new techniques is ongoing. This work is intended to give a brief overview of the current state of the RNA editing for treating muscular dystrophies.