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PubMed2003

An archaeal endoribonuclease catalyzes cis- and trans- nonspliceosomal splicing in mouse cells.

The tRNA endonuclease from the archaebacterium Methanococcus jannaschii (MJ endonuclease) can cleave RNAs forming specific bulge-helix-bulge (BHB) structures recognized by the enzyme. The resulting cleavage products are subsequently joined together by an endogenous ligase. We demonstrate the potential of using this strategy for repairing RNA in higher organisms by expressing the enzyme in mouse cells. Reporter target mRNAs modified with 17-nucleotide introns, flanked by sequences capable of forming BHB structures in cis, were expressed in mouse cells. RNA molecules that can form BHB substrates in trans with targeted mRNAs were also designed. Co-transfection of mouse cells with plasmids expressing these RNAs and the MJ endonuclease led to formation of RNA chimeras in which the target and exogenous RNA were recombined across the BHB. This technology is not limited to mRNA, but could in principle be used to destroy, modify or restore the function of a vast repertoire of RNA species or to join selectable tags to target RNAs.

3T3 Cells↗
PubMed2006

MATN and LAPTM are parts of larger transcription units produced by intergenic splicing: intergenic splicing may be a common phenomenon.

Intergenic splicing, the joining of exons from separate genes, has been observed only rarely in mammals. While the matrilin (MATN) and lysosomal-associated protein transmembrane (LAPTM) genes comprise distinct gene families, we have demonstrated intergenic splicing between two sets of family genes, the matrilin-3 (MATN3) and lysosomal-associated protein transmembrane 4alpha (LAPTM4A), and the matrilin-2 (MATN2) and lysosomal-associated protein transmembrane 4beta (LAPTM4B). The expression pattern and sub-cellular localization of the MATN-LAPTM hybrid transcripts differ from those of the original genes, suggesting unique functions for the products. Our observations indicate that intergenic splicing is a common and well-regulated phenomenon and underscore the fundamental challenges in defining the gene (transcriptional unit). Given these findings, the number of gene in the human genome may be smaller than present estimates suggest.

Cell Line↗
PubMed1999

Caenorhabditis elegans mRNAs that encode a protein similar to ADARs derive from an operon containing six genes.

The Caenorhabditis elegans T20H4.4 open reading frame (GenBank accession no. U00037) predicted by Genefinder encodes a 367 amino acid protein that is 32-35% identical to the C-terminal domain of adenosine deaminases that act on RNA. We show that T20H4.4 cDNAs (GenBank accession no. AF051275) encode a larger 495 amino acid protein that is extended at its N-terminus to include a single double-stranded RNA-binding motif, and that T20H4.4 occupies the second position in a six-gene operon (5'-T20H4.5, T20H4.4, R151.8A, R151.8B, R151.7, R151.6-3'). Ten different spliced-leader (SL) sequences were found attached to T20H4.4 mRNAs, including SL1, SL2 and eight SL2-like leaders that include two new variants. Characterization of cDNAs derived from all six genes confirmed the essential features of C.elegans operons: intercistronic distances in the range of 104-257 nt between the upstream polyadenylation sites and the downstream trans -splice sites; SL2, or SL2-like leaders, attached to the downstream mRNAs. Polycistronic mRNA fragments revealed a 5'-untranslated region (5'-UTR) >705 nt. The 5'-UTR is removed in mature mRNAs from the first gene (T20H4.5) and replaced primarily by SL1, and to a lesser extent by SL2. Our study provides new information regarding operons and how they are processed.

5' Untranslated Regions↗
PubMed1999

Delta ribozyme has the ability to cleave in transan mRNA.

We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy.

Base Sequence↗
PubMed2006

A TFIIB-like protein is indispensable for spliced leader RNA gene transcription in Trypanosoma brucei.

The lack of general class II transcription factors was a hallmark of the genomic sequences of the human parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. However, the recent identification of TFIIA as part of a protein complex essential for RNA polymerase II-mediated transcription of SLRNA genes, which encode the trans splicing-specific spliced leader RNA, suggests that trypanosomatids assemble a highly divergent set of these factors at the SLRNA promoter. Here we report the identification of a trypanosomatid TFIIB-like (TFIIB(like)) protein which has limited overall sequence homology to eukaryotic TFIIB and archaeal TFB but harbors conserved residues within the N-terminal zinc ribbon domain, the B finger and cyclin repeat I. In accordance with the function of TFIIB, T.brucei TFIIB(like) is encoded by an essential gene, localizes to the nucleus, specifically binds to the SLRNA promoter, interacts with RNA polymerase II, and is absolutely required for SLRNA transcription.

Amino Acid Sequence↗
PubMed2002

Alternative splicing of LYT1 transcripts in Trypanosoma cruzi.

As a result of alternative trans splicing, three distinct LYT1 mRNAs are produced in Trypanosoma cruzi, two encoding the full-length LYT1 protein and the third encoding a truncated LYT1 protein lacking a possible signal sequence. Analysis of the three mRNAs in different developmental forms of the parasite revealed that the alternative processing events were regulated differently during the parasite life cycle.

Alternative Splicing↗
PubMed2006

Cotranscription and intergenic splicing of the PPARG and TSEN2 genes in cattle.

BACKGROUND: Intergenic splicing resulting in the combination of mRNAs sequences from distinct genes is a newly identified mechanism likely to contribute to protein diversity. Few cases have been described, most of them involving neighboring genes and thus suggesting a cotranscription event presumably due to transcriptional termination bypass. RESULTS: We identified bovine chimeric transcripts resulting from cotranscription and intergenic splicing of two neighboring genes, PPARG and TSEN2. These two genes encode the Peroxisome Proliferator Activated Receptors gamma1 and gamma2 and the tRNA Splicing Endonuclease 2 homolog and are situated in the same orientation about 50 kb apart on bovine chromosome 22q24. Their relative position is conserved in human and mouse. We identified two types of chimeric transcripts containing all but the last exon of the PPARG gene followed by all but the first exon of the TSEN2 gene. The two chimers differ by the presence/absence of an intermediate exon resulting from transcription of a LINE L2 sequence situated between the two genes. Both transcripts use canonical splice sites for all exons coming from both genes, as well as for the LINE L2 sequence. One of these transcripts harbors a premature STOP codon and the other encodes a putative chimeric protein combining most of the PPARgamma protein and the entire TSEN2 protein, but we could not establish the existence of this protein. CONCLUSION: By showing that both individual and chimeric transcripts are transcribed from PPARG and TSEN2, we demonstrated regulation of transcription termination. Further, the existence and functionality of a chimeric protein harboring active motifs that are a priori unrelated is hypothesized.

Animals↗
PubMed2004

Signal sequence analysis of expressed sequence tags from the nematode Nippostrongylus brasiliensis and the evolution of secreted proteins in parasites.

BACKGROUND: Parasitism is a highly successful mode of life and one that requires suites of gene adaptations to permit survival within a potentially hostile host. Among such adaptations is the secretion of proteins capable of modifying or manipulating the host environment. Nippostrongylus brasiliensis is a well-studied model nematode parasite of rodents, which secretes products known to modulate host immunity. RESULTS: Taking a genomic approach to characterize potential secreted products, we analyzed expressed sequence tag (EST) sequences for putative amino-terminal secretory signals. We sequenced ESTs from a cDNA library constructed by oligo-capping to select full-length cDNAs, as well as from conventional cDNA libraries. SignalP analysis was applied to predicted open reading frames, to identify potential signal peptides and anchors. Among 1,234 ESTs, 197 (~16%) contain predicted 5' signal sequences, with 176 classified as conventional signal peptides and 21 as signal anchors. ESTs cluster into 742 distinct genes, of which 135 (18%) bear predicted signal-sequence coding regions. Comparisons of clusters with homologs from Caenorhabditis elegans and more distantly related organisms reveal that the majority (65% at P < e-10) of signal peptide-bearing sequences from N. brasiliensis show no similarity to previously reported genes, and less than 10% align to conserved genes recorded outside the phylum Nematoda. Of all novel sequences identified, 32% contained predicted signal peptides, whereas this was the case for only 3.4% of conserved genes with sequence homologies beyond the Nematoda. CONCLUSIONS: These results indicate that secreted proteins may be undergoing accelerated evolution, either because of relaxed functional constraints, or in response to stronger selective pressure from host immunity.

Animals↗
PubMed2005

In vivo excision of a single targeted nucleotide from an mRNA by a trans excision-splicing ribozyme.

We have previously reported the development of a group I intron-derived ribozyme that can bind an exogenous RNA substrate and excise from that substrate an internal segment in vitro, which allows for sequence-specific modification of RNA molecules. In this report, the activity of this trans excision-splicing ribozyme in a cellular environment, specifically Escherichia coli, was investigated. The ribozyme was re-engineered to target for excision a single-base insertion in the transcript of a green fluorescent protein, and fluorescence was exploited as a reporter for trans excision-splicing. We show that the ribozyme is able to catalyze the trans excision-splicing reaction in vivo and can repair the mutant transcripts. On average, 12% correction is observed as measured by fluorescence and at least 0.6% correction as confirmed through sequence analysis. This represents the first report of a biomolecule (in this case a ribozyme) that can selectively excise a targeted nucleotide from within an mRNA transcript in vivo. This new class of biochemical tools makes possible a wide variety of new experimental strategies, perhaps including a new approach to molecular-based therapeutics.

Escherichia coli↗
PubMed2005

A novel strategy to identify the location of necessary and sufficient cis-acting regulatory mRNA elements in trypanosomes.

Expression of nearly all protein coding genes in trypanosomes is regulated post-transcriptionally, predominantly at the level of mRNA half-life. The identification of cis-acting elements involved in mRNA stability has been hindered by a lack of ability to screen for loss-of-regulation mutants. The method described in this article allows the region containing the necessary and sufficient elements within a mRNA to be identified and uses antibiotic resistance genes as both selectable markers and reporters. In the case of unstable mRNAs, the strategy can be extended by performing a screen for spontaneous loss-of-function mutants in regulatory parts of a mRNA. The method was validated by using the GPI-PLC mRNA, which is unstable in procyclic form trypanosomes and showed that the 3'UTR of the GPI-PLC mRNA contains all elements required for developmentally regulated instability. Loss-of-instability mutants all contained deletions within the 2300-nucleotide-long 3'UTR, and their analysis showed that a deletion including the last 800 nt of the gene stabilized the mRNA. The method is nonpresumptive, allows far more rapid screening for cis-elements than existing procedures, and has the advantage of identifying functional mutants. It is applicable to all eukaryotes using polycistronic transcription.

3' Untranslated Regions↗
PubMed2004

Multiple links between transcription and splicing.

Transcription and pre-mRNA splicing are extremely complex multimolecular processes that involve protein-DNA, protein-RNA, and protein-protein interactions. Splicing occurs in the close vicinity of genes and is frequently cotranscriptional. This is consistent with evidence that both processes are coordinated and, in some cases, functionally coupled. This review focuses on the roles of cis- and trans-acting factors that regulate transcription, on constitutive and alternative splicing. We also discuss possible functions in splicing of the C-terminal domain (CTD) of the RNA polymerase II (pol II) largest subunit, whose participation in other key pre-mRNA processing reactions (capping and cleavage/polyadenylation) is well documented. Recent evidence indicates that transcriptional elongation and splicing can be influenced reciprocally: Elongation rates control alternative splicing and splicing factors can, in turn, modulate pol II elongation. The presence of transcription factors in the spliceosome and the existence of proteins, such as the coactivator PGC-1, with dual activities in splicing and transcription can explain the links between both processes and add a new level of complexity to the regulation of gene expression in eukaryotes.

Alternative Splicing↗
PubMed1999

Prenatal diagnosis of sporadic neurofibromatosis type 1 (NF1) by RNA and DNA analysis of a splicing mutation.

Neurofibromatosis type 1 (NF1) is one of the most common genetic disorders in humans with an incidence of 1 in 3500. Most of the NF1 mutations reported so far (over 240 mutations) are unique. Specific prenatal diagnosis can only be provided to familial cases by an indirect linkage analysis or to families with a previously identified mutation. Here we report the first prenatal diagnosis in sporadic NF1 by direct characterization of the mutation. We first identified the skipping of exon 10b of NF1 in the mRNA from a woman affected by NF1 and without familial history of the disease. The analysis of genomic DNA identified mutation IVS10b+1G-->A as the cause of the skipping of exon 10b. Chorionic villus sampling (CVS) was performed at 10 weeks of gestation and total RNA was directly extracted from the sample. After reverse transcription (RT) and polymerase chain reaction (PCR) of the cDNA, the skipping of exon 10b was not identified in the CVS upon agarose gel electrophoresis. The fetal origin of the CVS was confirmed via polymorphic markers and the absence of the IVS10b+1G-->A mutation was confirmed by genomic analysis.

Adult↗