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At least 19 recordsLinked to original sources
PubMed1992

Trans-splicing in protozoa and helminths.

Trans-splicing is defined as the process whereby exons derived from two separately transcribed RNAs are joined together. In one type of trans-splicing, nuclear pre-mRNAs acquire their 5' terminal exon (the spliced leader) from a small spliced leader RNA (SL RNA) via an RNA processing reaction that is directly analagous to the removal of intervening sequences (cis-splicing). Such leader-addition by trans-splicing has been extensively studied in trypanosomatid protozoans and in nematodes. This review summarizes recent advances in research on trans-splicing in these two systems. Progress in elucidating functionally significant sequence elements within SL RNAs and progress in understanding the mechanism and biological role of trans-splicing is discussed.

Animals↗
PubMed1992

Conservation of gene organization and trans-splicing in the glyceraldehyde-3-phosphate dehydrogenase-encoding genes of Caenorhabditis briggsae.

The genes encoding body-wall-specific glyceraldehyde-3-phosphate dehydrogenase from Caenorhabditis briggsae were sequenced and compared to the homologous genes from Caenorhabditis elegans. The direct tandem organization of these genes, gpd-2 and gpd-3, and the size and location of the two introns in each gene are the same in C. elegans and C. briggsae. Primer-extension studies demonstrated that the two genes in C. briggsae are trans-splice differentially with the same splice leader (SL) RNAs as are observed in C. elegans. The gdp-2 gene is trans-spliced with SL1 while gdp-3 is trans-spliced with SL2. Significant sequence conservation was observed within the promoter regions of each species and may indicate those regions responsible for body-wall-muscle-specific gene expression and/or differential trans-splicing. Comparisons of the sequences suggest that the tandem repeat of the genes has been subjected to concerted evolution and that C. briggsae and C. elegans diverged much earlier than would be anticipated based on morphological similarities alone. Finally, an open reading frame found several hundred nucleotides upstream from gpd-2, in both species, appears to be homologous to the ATP synthase subunit, ATPase inhibitor protein, from bovine mitochondria.

Amino Acid Sequence↗
PubMed1992

Trans-splicing of an early embryo mRNA in Litomosoides carinii, coding for the major microfilarial sheath protein gp22.

Both genomic and cDNA clones have been isolated encoding the major sheath glycoprotein, gp22, of Litomosoides carinii microfilariae. The mature gp22 mRNA is shown to result from both trans-splicing of a 22-nucleotide 5'-leader sequence to an acceptor site at position 313 of the pre-mRNA, immediately upstream from the start codon, and from cis-splicing of a 117-nt intron located within the coding sequence. Cis-splicing precedes the trans-splicing reaction. The gp22 reading frame of 148 codons has the inferred structure of a prepro-protein and includes a leader peptide and a pro-segment ahead of the known N terminus of the mature, extracellular protein of 105 amino acids. The N-terminal part of that protein contains five repeats of an elastin-related pentapeptide sequence, which, together with a proline-threonine segment between two Cys clusters in the center and at its C terminus, may cause an elongated conformation with an apparent molecular size of 22 kDa in contrast to the calculated M(r) of 11,200.

Amino Acid Sequence↗
PubMed2026

Suppression of HIV-1 replication in CEM-A cell cultures by trans-splicing group I introns targeting PAS/PBS sequences and conditionally expressing ΔN-Bax.

Anti-HIV group I introns containing antisense guide sequences directed against the HIV-1 primer activation signal and primer-binding site (PAS/PBS) were designed and evaluated. Because PAS/PBS sequences are present in the viral RNA species examined, these RNAs can serve as trans-splicing substrates. The introns were active against both artificial target RNAs and viral RNA generated during infection. Cleavage and degradation of targeted viral RNA may have contributed to suppression, whereas inclusion of a 3' exon encoding the proapoptotic protein ΔN-Bax was associated with increased programmed cell death and may have augmented suppression of viral replication. In cultured CEM-A cells, transgene expression of these introns markedly suppressed HIV-1 replication, with p24 levels falling below the assay detection limit in selected clones. RESULTS: RT-PCR and sequence analysis detected splice products containing the expected PAS/PBS junctions. In the dual-luciferase assay, intron expression reduced normalized Gaussia luciferase signal by approximately 70% relative to the negative control. Qualitative Annexin V imaging and caspase-3 assays were consistent with infection-dependent apoptosis after ΔN-Bax splice-product formation. Transient expression of each intron in HEK293T cells followed by infection with VSV-G-pseudotyped HIV-1NL4-3 at an MOI of 2 reduced p24 levels by approximately 50% at 4 days post-infection. Construct 128L produced the strongest RT-PCR band under the tested conditions and was selected for subsequent experiments. A canonical splice product and a low-abundance noncanonical splice product were detected; both involved the intended HIV-derived target RNA, although transcriptome-wide off-target splicing was not assessed. Heterogeneous transformed HEK293T populations showed an approximately 2-log10 reduction in p24. In selected clonal HEK293T and CEM-A lines, p24 was below the assay detection limit at the measured endpoints, including up to 90 days after infection in some CEM-A clones. CONCLUSIONS: PAS/PBS-targeting group I introns suppressed HIV-1-associated p24 production in the tested cell-culture models. Linking the introns to a ΔN-Bax 3' exon was associated with infection-dependent apoptosis and may further limit viral replication and spread. The use of highly conserved, functionally constrained target sequences may reduce the likelihood of escape, but viral evolution and transcriptome-wide off-target effects were not assessed. This conditional death-upon-infection strategy warrants further evaluation in primary-cell and in vivo models.

Humans↗
PubMed2024

Split intein-mediated protein trans-splicing to express large dystrophins.

Gene replacement using adeno-associated virus (AAV) vectors is a promising therapeutic approach for many diseases1,2. However, this therapeutic modality is challenged by the packaging capacity of AAVs (approximately 4.7 kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase messenger RNA. Here we developed a new method for expressing large dystrophins by utilizing the protein trans-splicing mechanism mediated by split inteins. We identified several split intein pairs that efficiently join two or three fragments to generate a large midi-dystrophin or the full-length protein. We show that delivery of two or three AAVs into dystrophic mice results in robust expression of large dystrophins and significant physiological improvements compared with micro-dystrophins. Moreover, using the potent myotropic AAVMYO4, we demonstrate that low total doses (2 × 1013 viral genomes per kg) are sufficient to express large dystrophins in striated muscles body-wide with significant physiological corrections in dystrophic mice. Our data show a clear functional superiority of large dystrophins over micro-dystrophins that are being tested in clinical trials. This method could benefit many patients with Duchenne or Becker muscular dystrophy, regardless of genotype, and could be adapted to numerous other disorders caused by mutations in large genes that exceed the AAV capacity.

Animals↗
PubMed2026

Natural variation in Miniature5 determines mitochondrial nad1 splicing and seed development in maize.

Seed size is a key determinant of cereal grain yield, but natural variations in defective-kernel genes have rarely been applied in maize breeding. Here, we report the positional cloning of maize Miniature5 (Mn5), which encodes a mitochondrial-targeted P-class pentatricopeptide repeat (PPR) protein. Further analysis shows that a missense mutation of Mn5, Mn5Val109, presents in maize populations and correlates with reduced seed size. The Mn5Val109 variant exhibits compromised function in the miniature5 (mn5-ref) mutant, failing to trans-splice mitochondrial nad1 intron1, drastically reducing the abundance and activity of respiratory complex I, accompanied by disorganized mitochondrial cristae. Mn5 directly binds to domain IV of the pre-nad1.1 transcript. Notably, this binding site is located downstream of the previously presumed 3'-terminus bound by MITOCHONDRIA STABILITY/PROCESSING PPR FACTOR1 (MSP1), thus redefining the 3'-end of the nad1.1 pre-RNA. Furthermore, Mn5 physically interacts with the maturases ZmnMAT1 and ZmnMAT3, as well as the PPR proteins PPR-SMR1 and SPR2, which are broadly involved in organellar group II intron splicing. Together, our results suggest that Mn5 recruits maturases and PPR proteins to form spliceosomal complexes responsible for the trans-splicing of nad1 intron1. Importantly, natural variations in Mn5 confer differences in seed size control, offering potential for breeding high-yield maize varieties.

Zea mays↗
PubMed1992

Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides.

Synthesis of mRNA in kinetoplastid protozoa involves the process of trans-splicing, in which an identical 39-41-nucleotide (depending on the species) mini-exon is placed at the 5' end of mature mRNAs. The mini-exon sequence is highly conserved among all members of the Kinetoplastida, nucleotides 1-6 being identical in the four genera so far examined. Prior to trans-splicing, the mini-exon donor RNA is capped by the addition of a (5'-5') triphosphate-linked 7-methylguanosine, followed by modification of the first four transcribed nucleotides. Partial structures have been previously deduced for this cap 4 moiety from Trypanosoma brucei and Leptomonas collosoma. We have purified enough cap 4 from T. brucei and Crithidia fasciculata to allow definitive structural analysis by combined liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. The results, together with the known mini-exon sequence, show that cap 4 in both species has the structure m7G(5')ppp(5')m6(2)AmpAmpCmpm3Ump. The presence of N6,N6,2'-O-trimethyladenosine and 3,2'-O-dimethyluridine, nucleosides previously unknown in nature, were confirmed by rigorous comparison with synthetic standards. The conservation of cap 4 between these divergent genera suggests that this structure may be common to most if not all Kinetoplastida.

Animals↗
PubMed2010

Gene therapy of mdx mice with large truncated dystrophins generated by recombination using rAAV6.

Recombinant adeno-associated viral (rAAV) vector-mediated gene transfer represents a promising approach for many diseases. However, the applicability of rAAV vectors has long been hindered by the small (~4.8 kb) DNA packaging capacity. This limitation can hamper the packaging and delivery of critical regulatory elements and/or larger coding sequences, such as the ~14-kb dystrophin complementary DNA (cDNA) that is of interest for gene therapy of Duchenne muscular dystrophy (DMD). Here, we have demonstrated reconstitution of an expression cassette (7.3 kb) encoding a highly functional "minidystrophin" protein (ΔH2-R19, 222 kd) in vivo following intravascular co-delivery of two independent rAAV6 vectors sharing a central homologous recombinogenic region of 372 nucleotides. Similar to previously reported trans-splicing approaches, one rAAV vector provides the promoter with the ~1/2 initial portion of minidystrophin, while the second vector provides the remaining minidystrophin cDNA followed by the polyadenylation signal. Significantly, administering a modest dose [2 × 10(12) vector genomes (vg)] of the two minidystrophin-encoding rAAV vectors to dystrophic mice elicited an improvement of physiological performance indicative of prevention or amelioration of the disease state. These studies provide evidence that functional dystrophin transgenes larger than that typically carried by a single rAAV genome can be reconstituted in vivo by homologous recombination (HR) following intravascular co-delivery with rAAV6.

Animals↗
PubMed1992

Segments of Escherichia coli genome similar to the exons of human prothymosin alpha gene.

Identification of the putative prothymosin alpha homolog in Escherichia coli cells prompted the search for a prothymosin alpha-coding gene in the E. coli genome. A set of interspersed DNA segments was identified, which match various parts of the human prothymosin alpha molecule. Their location in the E. coli genome and high degree of similarity with the appropriate regions of the human prothymosin alpha gene suggest that some kind of trans-splicing should exist in E. coli, which could be responsible for bringing these putative bacterial prothymosin alpha-coding exons together.

Amino Acid Sequence↗
PubMed1992

The P-glycoprotein gene family of Caenorhabditis elegans. Cloning and characterization of genomic and complementary DNA sequences.

P-glycoproteins, encoded by families of evolutionarily conserved genes, can confer a multidrug-resistant phenotype to mammalian tumor cells. To obtain more information on their functions in normal cells we have cloned genomic and complementary DNA sequences of four P-glycoprotein gene homologs of the genetically well-characterized nematode Caenorhabditis elegans, termed pgp-1, pgp-2, pgp-3 and pgp-4, respectively. The genes were physically mapped on chromosome IV (pgp-1), I (pgp-2) and X (pgp-3 and pgp-4). Phenotypic mutants corresponding to these loci have not yet been described. Two of the genes, pgp-1 and pgp-3, were analyzed in detail. They are predicted to encode ATP-binding membrane-spanning proteins of 1321 and 1254 amino acid residues, respectively, with the characteristic features shared by most P-glycoproteins described thus far. Intra-species divergence of P-glycoprotein genes is more pronounced in C. elegans than in mammals. Only 40% of the amino acids of pgp-1 and pgp-3 are identical, in contrast to 77% identity between human MDR1 and MDR3. pgp-1 consists of 14 exons, pgp-3 of 13. The two genes share only one intron position, whereas they share four (pgp-1) and five (pgp-3) intron positions with mammalian P-glycoprotein genes. pgp-1, pgp-2, and pgp-3 are transcribed into low abundance mRNAs in wild-type nematodes. pgp-1 and pgp-3 mRNAs have the trans-spliced leader SL1 at their 5' ends. Arsenite, emetine and actinomycin D drugs did not increase the steady state levels of pgp mRNA, unlike in some mammalian cell types. Heat shock disturbed trans as well as cis-splicing of pgp-1 and led to the accumulation of partially processed pgp-1 RNA. Thus, in C. elegans these genes are not induced in the context of a general stress response, as has been proposed for mammalian P-glycoprotein genes in certain tissues.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

The actin genes of Onchocerca volvulus.

The genome of Onchocerca volvulus was found to contain 2 actin gene classes (called 1 and 2) of 2 genes each. The 4 genes are located in 2 clusters (called A and B), each containing a gene class member. Five short introns of 122-207 bp occur within each gene. The sequences of the fourth intron and the 5' and 3' untranslated regions of all the 2 gene classes are completely different even though their coding regions share 95% identity. Mature transcripts from the actin genes have the nematode spliced leader (SL) at their 5' ends. One actin cDNA was found to be derived from an actin pre-mRNA which locks both the 5 introns and the 5' SL, suggesting that in at least some transcripts cis-splicing is completed before trans-splicing occurs.

Actins↗
PubMed1992

Expression of the polyubiquitin-encoding gene (ubq-1) in transgenic Caenorhabditis elegans.

The expression of the polyubiquitin-encoding gene (ubq-1) of Caenorhabditis elegans was analysed using transgenic nematode lines carrying translational ubq-1::lacZ fusions. Animals carrying a construct consisting of 938 bp of ubq-1 upstream sequences fused to lacZ (ubq938::lacZ) expressed beta Gal in embryos and in a tissue-general manner in 20% of L1 larvae. Somatic expression in later stages was usually confined to body muscle. Progressively larger deletions extending from the 5' end of ubq938::lacZ did not significantly alter the pattern of expression until 827 bp of sequence had been removed. Thus, sequences upstream from the transcription start point, including a G+C-rich block and a sequence resembling a TATA box (GAATAA), are not required to generate the expression pattern seen with ubq938::lacZ. Moreover, a basal level of expression was maintained in embryos when 903 bp were deleted. These results suggest that the promoter elements required for efficient expression of ubq-1 may reside within the transcribed region of the gene; alternatively, they must lie more than 1.7 kb upstream or 0.8 kb downstream from this region. Polymerase chain reaction analysis indicates that RNA molecules transcribed from the ubq938::lacZ and ubq delta 827::lacZ transgenes are trans-spliced to SL1, as is ubq-1 RNA.

Animals↗
PubMed2026

Functional chimeric mRNAs encode proteins in mammalian immunity.

Individual mammalian mRNAs and proteins are typically believed to originate from single genomic loci, with isoform diversity arising through cis-splicing of pre-mRNA. Whether mRNA from distant genes can undergo trans-splicing to generate functionally relevant chimeric transcripts has remained unclear. Here we develop a pipeline combining long-read direct RNA sequencing with non-targeted and targeted validation to identify chimeric transcripts in macrophages. Chromatin conformation capture studies reveal that inflammation induces interchromosomal DNA interactions, positioning parent genes proximally to facilitate the formation of chimeric mRNA. Notably, we identify a protein-coding chimeric mRNA representing a fusion between the pore-forming protein gasdermin D (GSDMD)1,2 and a C-terminal domain translated out of frame from Tmem106a (Gsdmd-Tmem106a) in mice. We show that inflammasome priming upregulates Gsdmd-Tmem106a, with the protein localizing to the plasma membrane. After activation of the inflammasome, GSDMD-TMEM106A directly interacts with canonical GSDMD N termini to accelerate and enhance pore formation and IL-1β release. Finally, we show that GSDMD-TMEM106A balances host defence and immunopathology in vivo: its loss protects against lethal sepsis but compromises antibacterial defence, whereas overexpression enhances host protection while increasing sepsis lethality. We establish that protein-coding chimeric mRNAs formed by regulated transcript fusion events are operative during inflammation and immunity.

Journal Article↗
PubMed1992

A ribosomal RNA gene promoter at the telomere of a mini-chromosome in Trypanosoma brucei.

The parasitic protozoan Trypanosoma brucei has some hundred mini-chromosomes of 50-150 kb, which mainly consist of telomeric repeats, sub-telomeric repeats and internal 177-bp repeats. Their primary function seems to be to expand the repertoire of non-transcribed sub-telomeric variant surface glycoprotein (VSG) genes. Here we report that two of the smaller mini-chromosomes (55 and 60 kb) contain sequences homologous to the ribosomal RNA gene promoter region. We have targeted by homologous recombination the neomycin phosphotransferase (neo(r)) gene behind the promoter on the 55 kb chromosome and show that this promoter mediates the efficient synthesis of properly trans-spliced and polyadenylated neo mRNA. The resulting high resistance to G418 (a neo analogue) is stable in the absence of drug showing that mitotic segregation of this mini-chromosome is precise. Downstream of the transcription start the wild-type version of the ribosomal promoter is flanked by telomeric repeats. The absence of the sub-telomeric repeats found in other T.brucei chromosome ends suggests that the rDNA-telomeric junction has been formed by de novo addition of telomeric repeats to a broken chromosome end (healing). Our results provide a plausible explanation for the alpha-amanitin-resistant transcription of telomeric repeats in T.brucei reported by Rudenko and Van der Ploeg and they show that trypanosomes can efficiently use RNA polymerase I for the expression of sub-telomeric genes, supporting the notion that the alpha-amanitin-resistant transcription of sub-telomeric VSG genes may also be catalyzed by this enzyme.

Animals↗
PubMed1992

Genomic organisation and expression of a differentially-regulated gene family from Leishmania major.

We have isolated and characterised a differentially-regulated gene family in the protozoan parasite Leishmania major. The family contains 5 genes linked within a 10Kb region of the genome: three of the genes are closely related in DNA sequence, the other two have only limited homology. Post-transcriptional control of the differential expression pattern is suggested by detection of precursor RNA molecules containing intergenic sequences and evidence that mature mRNA molecules contain a 35nt spliced leader sequence at their 5' ends. These features support a model of polycistronic transcription in which the stability and differential processing of precursor RNA molecules determine the steady state levels of mature mRNA. We have identified several DNA sequence motifs within the gene family that have potential roles in differential processing and/or RNA stability: an alternative 5' splice acceptor site for trans-splicing; a putative polyadenylation site; and a region of potential secondary structure within 3' flanking sequences. The 3' sequence elements are conserved in those genes that share the same pattern of differential regulation. To our knowledge, this is the first example of coordinated differential-regulation of a non-identical gene cluster in Leishmania.

Animals↗
PubMed1992

Domain structure of U2 and U4/U6 small nuclear ribonucleoprotein particles from Trypanosoma brucei: identification of trans-spliceosomal specific RNA-protein interactions.

Maturation of mRNAs in trypanosomes involves trans splicing of the 5' end of the spliced leader RNA and the exons of polycistronic pre-mRNAs, requiring small nuclear ribonucleoproteins (snRNPs) as cofactors. We have mapped protein-binding sites in the U2 and U4/U6 snRNPs by a combination of RNase H protection analysis, native gel electrophoresis, and CsCl density gradient centrifugation. In the U2 snRNP, protein binding occurs primarily in the 3'-terminal domain; through U2 snRNP reconstitution and chemical modification-interference assays, we have identified discrete positions within stem-loop IV of Trypanosoma brucei U2 RNA that are essential for protein binding; significantly, some of these positions differ from the consensus sequence derived from cis-spliceosomal U2 RNAs. In the U4/U6 snRNP, the major protein-binding region is contained within the 3'-terminal half of U4 RNA. In sum, while the overall domain structure of the U2 and U4/U6 snRNPs is conserved between cis- and trans-splicing systems, our data suggest that there are also trans-spliceosomal specific determinants of RNA-protein binding.

Animals↗
PubMed2025

Proteomics-based evaluation of AAV dystrophin gene therapy outcomes in mdx skeletal muscle.

Duchenne muscular dystrophy (DMD) is a fatal genetic muscle-wasting disease characterized by loss of dystrophin protein. Therapeutic attempts to restore a functional copy of dystrophin to striated muscle are under active development, and many utilize adeno-associated viral (AAV) vectors. However, the limited cargo capacity of AAVs precludes delivery of full-length dystrophin, a 427 kDa protein, to target tissues. Recently, we developed a method to express large dystrophin constructs using the protein trans-splicing mechanism mediated by split inteins and myotropic AAV vectors. The efficacy of this approach to restore muscle function in mdx4cv mice was previously assessed using histology, dystrophin immunolabeling, and Western blotting. Here, we expand our molecular characterization of dystrophin constructs with variable lengths using a mass spectrometry-based proteomics approach, providing insight into unique protein expression profiles in skeletal muscles of wild-type, dystrophic mdx4cv, and AAV-treated mdx4cv mice. Our data reveal several affected cellular processes in mdx4cv skeletal muscles with changes in the expression profiles of key proteins to muscle homeostasis, whereas successful expression of dystrophin constructs results in an intermediate to complete restoration. This study highlights several biomarkers that could be used in future preclinical or clinical studies to evaluate the effectiveness of therapeutic strategies.

Animals↗