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At least 19 recordsLinked to original sources
PubMed2004

Spliced-leader RNA trans splicing in a chordate, Oikopleura dioica, with a compact genome.

trans splicing of a spliced-leader RNA (SL RNA) to the 5' ends of mRNAs has been shown to have a limited and sporadic distribution among eukaryotes. Within metazoans, only nematodes are known to process polycistronic pre-mRNAs, produced from operon units of transcription, into mature monocistronic mRNAs via an SL RNA trans-splicing mechanism. Here we demonstrate that a chordate with a highly compact genome, Oikopleura dioica, now joins Caenorhabditis elegans in coupling trans splicing with processing of polycistronic transcipts. We identified a single SL RNA which associates with Sm proteins and has a trimethyl guanosine cap structure reminiscent of spliceosomal snRNPs. The same SL RNA, estimated to be trans-spliced to at least 25% of O. dioica mRNAs, is used for the processing of both isolated or first cistrons and downstream cistrons in a polycistronic precursor. Remarkably, intercistronic regions in O. dioica are far more reduced than those in either nematodes or kinetoplastids, implying minimal cis-regulatory elements for coupling of 3'-end formation and trans splicing.

Animals↗
PubMed2005

The Trypanosoma brucei La protein is a candidate poly(U) shield that impacts spliced leader RNA maturation and tRNA intron removal.

By virtue of its preferential binding to poly(U) tails on small RNA precursors and nuclear localisation motif, the La protein has been implicated for a role in the stabilisation and nuclear retention of processing intermediates for a variety of small RNAs in eukaryotic cells. As the universal substrate for trans-splicing, the spliced leader RNA is transcribed as a precursor with just such a tail. La protein was targeted for selective knockdown by inducible RNA interference in Trypanosoma brucei. Of three RNA interference strategies employed, a p2T7-177 vector was the most effective in reducing both the La mRNA as well as the protein itself from induced cells. In the relative absence of La protein T. brucei cells were not viable, in contrast to La gene knockouts in yeast. A variety of potential small RNA substrates were examined under induction, including spliced leader RNA, spliced leader associated RNA, the U1, U2, U4, and U6 small nuclear RNAs, 5S ribosomal RNA, U3 small nucleolar RNA, and tRNATyr. None of these molecules showed significant variance in size or abundance in their mature forms, although a discrete subset of intermediates appear for spliced leader RNA and tRNATyr intron splicing under La depletion conditions. 5'-end methylation in the spliced leader RNA and U1 small nuclear RNA was unaffected. The immediate cause of lethality in T. brucei was not apparent, but may represent a cumulative effect of multiple defects including processing of spliced leader RNA, tRNATyr and other unidentified RNA substrates. This study indicates that La protein binding is not essential for maturation of the spliced leader RNA, but does not rule out the presence of an alternative processing pathway that could compensate for the absence of normally-associated La protein.

Animals↗
PubMed2001

Two sequence classes of kinetoplastid 5S ribosomal RNA gene revealed among bodonid spliced leader RNA gene arrays.

The spliced leader RNA genes of Bodo saltans, Cryptobia helicis and Dimastigella trypaniformis were analyzed as molecular markers for additional taxa within the suborder Bodonina. The non-transcribed spacer regions were distinctive for each organism, and 5S rRNA genes were present in Bodo and Dimastigella but not in C. helicis. Two sequence classes of 5S rRNA were evident from analysis of the bodonid genes. The two classes of 5S rRNA genes were found in other Kinetoplastids independent of co-localization with the spliced leader RNA gene.

Animals↗
PubMed1999

The role of intron structures in trans-splicing and cap 4 formation for the Leishmania spliced leader RNA.

A 39-nucleotide leader is trans-spliced onto all trypanosome nuclear mRNAs. The precursor spliced leader RNA was tested for trans-splicing function in vivo by mutating the intron. We report that in Leishmania tarentolae spliced leader RNA 5' modification is influenced by the primary sequence of stem-loop II, the Sm-binding site, and the secondary structure of stem-loop III. The sequence of stem-loop II was found to be important for cap 4 formation and splicing. As in Ascaris, mutagenesis of the bulge nucleotide in stem-loop II was detrimental to trans-splicing. Because restoration of the L. tarentolae stem-loop II structure was not sufficient to restore splicing, this result contrasts the findings in the kinetoplastid Leptomonas, where mutations that restored stem-loop II structure supported splicing. Methylation of the cap 4 structure and splicing was also dependent on both the Sm-binding site and the structure of stem-loop III and was inhibited by incomplete 3' end processing. The critical nature of the L. tarentolae Sm-binding site is consistent with its essential role in the Ascaris spliced leader RNA, whereas in Leptomonas mutation of the Sm-binding site and deletion of stem-loop III did not affect trans-splicing. A pathway for Leishmania spliced leader RNA processing and maturation is proposed.

Animals↗
PubMed1996

Spliced leader RNA trans-splicing in metazoa.

Spliced leader trans-splicing is a form of RNA processing originally described and studied in parasitic kinetoplastida. This mechanism of gene expression also occurs in parasitic and free-living metazoa. In this review, Dick Davis describes current knowledge of the distribution, substrates, specificity and functional significance of trans-splicing in metazoa.

Journal Article↗
PubMed1991

Trans splicing in trypanosomes requires methylation of the 5' end of the spliced leader RNA.

Trypanosoma brucei spliced leader (SL) RNA contains an unusual cap 4 structure consisting of 7-methylguanosine linked to four modified nucleosides. During RNA maturation, trans splicing transfers the first 39 nucleotides of the SL RNA including the cap structure to the 5' end of all mRNAs. Here we show that exposure of permeable trypanosome cells to S-adenosyl-L-homocysteine inhibits methylation of the nucleosides adjacent to 7-methylguanosine of newly synthesized SL RNA and prevents utilization of the SL RNA in trans splicing. However, trans splicing of the SL RNA preexisting in the cells is not inhibited by S-adenosyl-L-homocysteine as shown by the observation that newly synthesized alpha-tubulin RNA is trans spliced at the same level as in control cells. Therefore, it appears that the newly synthesized SL RNA is the only known component of the trans-splicing machinery that is impaired in its function by inhibition of methylation. Undermethylation does not alter either the stability of the SL RNA or the electrophoretic mobility and chromatographic behavior of the core SL ribonucleoprotein particle. Taken together, our data suggest that the cap 4 structure of the SL RNA plays an essential role in the trans-splicing process.

Animals↗
PubMed1990

The nematode spliced leader RNA participates in trans-splicing as an Sm snRNP.

The trans-spliced leader RNA (SL RNA) of nematodes resembles U snRNAs both in cap structure and in the presence of a consensus Sm binding site. We show here that synthetic SL RNA, synthesized by in vitro transcription, is efficiently used as a spliced leader donor in trans-splicing reactions catalyzed by a cell free extract prepared from developing embryos of the parasitic nematode, Ascaris lumbricoides. Efficient utilization of synthetic SL RNA requires a functional Sm binding site. Mutations within the Sm binding sequence that prevent immunoprecipitation by Sm antisera and prevent cap trimethylation abolish trans-splicing. The effect on trans-splicing is not due to undermethylation of the cap structure.

Animals↗
PubMed2003

Exportin 1 mediates nuclear export of the kinetoplastid spliced leader RNA.

The kinetoplastid protozoan spliced leader (SL) RNA is the common substrate pre-mRNA utilized in all trans-splicing reactions. Here we show by fluorescence in situ hybridization that the SL RNA is present in the cytoplasm of Leishmania tarentolae and Trypanosoma brucei. Treatment with the karyopherin-specific inhibitor leptomycin B was toxic to T. brucei and eliminated the cytoplasmic SL RNA, suggesting that cytoplasmic SL RNA was dependent on the nuclear exporter exportin 1 (XPO1). Ectopic expression of xpo1 with a C506S mutation in T. brucei conferred resistance to leptomycin B. A reduction in SL RNA 3' extension removal and 5' methylation of nucleotide U(4) was observed in wild-type T. brucei treated with leptomycin B, suggesting that the cytoplasmic stage is necessary for SL RNA biogenesis. This study demonstrates spatial and mechanistic similarities between the posttranscriptional trafficking of the kinetoplastid protozoan SL RNA and the metazoan cis-spliceosomal small nuclear RNAs.

Active Transport, Cell Nucleus↗
PubMed1990

Transcription and cap trimethylation of a nematode spliced leader RNA in a cell-free system.

Maturation of a fraction of mRNAs in nematodes involves the acquisition of a common 5' terminal spliced leader sequence derived from a nonpolyadenylylated spliced leader RNA by trans splicing. We have developed a cell-free system prepared from Ascaris lumbricoides embryos that accurately and efficiently synthesized the spliced leader RNA of A. lumbricoides. Transcription of the spliced leader RNA was catalyzed by RNA polymerase II, and the majority of the spliced leader RNAs synthesized in vitro possessed a trimethylguanosine cap structure identical to that found on in vivo-synthesized spliced leader RNA.

Animals↗
PubMed2004

3'-End polishing of the kinetoplastid spliced leader RNA is performed by SNIP, a 3'-->5' exonuclease with a Motley assortment of small RNA substrates.

In all trypanosomatids, trans splicing of the spliced leader (SL) RNA is a required step in the maturation of all nucleus-derived mRNAs. The SL RNA is transcribed with an oligo-U 3' extension that is removed prior to trans splicing. Here we report the identification and characterization of a nonexosomal, 3'-->5' exonuclease required for SL RNA 3'-end formation in Trypanosoma brucei. We named this enzyme SNIP (for snRNA incomplete 3' processing). The central 158-amino-acid domain of SNIP is related to the exonuclease III (ExoIII) domain of the 3'-->5' proofreading epsilon subunit of Escherichia coli DNA polymerase III holoenzyme. SNIP had a preference for oligo(U) 3' extensions in vitro. RNA interference-mediated knockdown of SNIP resulted in a growth defect and correlated with the accumulation of one- to two- nucleotide 3' extensions of SL RNA, U2 and U4 snRNAs, a five-nucleotide extension of 5S rRNA, and the destabilization of U3 snoRNA and U2 snRNA. SNIP-green fluorescent protein localized to the nucleoplasm, and substrate SL RNA derived from SNIP knockdown cells showed wild-type cap 4 modification, indicating that SNIP acts on SL RNA after cytosolic trafficking. Since the primary SL RNA transcript was not the accumulating species in SNIP knockdown cells, SL RNA 3'-end formation is a multistep process in which SNIP provides the ultimate 3'-end polishing. We speculate that SNIP is part of an organized nucleoplasmic machinery responsible for processing of SL RNA.

Animals↗
PubMed2004

SmD1 is required for spliced leader RNA biogenesis.

The Sm-binding site of the kinetoplastid spliced leader RNA has been implicated in accurate spliced leader RNA maturation and trans-splicing competence. In Trypanosoma brucei, RNA interference-mediated knockdown of SmD1 caused defects in spliced leader RNA maturation, displaying aberrant 3'-end formation, partial formation of cap 4, and overaccumulation in the cytoplasm; U28 pseudouridylation was unaffected.

Alternative Splicing↗
PubMed1998

Single nucleotide resolution of promoter activity and protein binding for the Leishmania tarentolae spliced leader RNA gene.

In Kinetoplastid protozoa, trans-splicing is a central step in the maturation of nuclear mRNAs. In Leishmania, a common 39 nt spliced-leader (SL) is transferred via trans-splicing from the precursor 96 nt SL RNA to the 5' terminus of all known protein-encoding RNAs. In this study, promoter elements of the L. tarentolae SL RNA gene have been identified with respect to transcriptional activity and putative transcription factor binding. We have mapped the essential regions in the SL RNA gene promoter at single nucleotide resolution using both in vivo transcription and in vitro protein/DNA binding approaches. Two regions located upstream of the SL RNA gene were identified: a GN3CCC element at -39 to -33 and a GACN5G element at -66 to -58 were essential for SL RNA gene transcription in stably transfected cells. Consistent with other known bipartite promoter elements, the spacing between the GN3CCC and GACN5G elements was found to be critical for proper promoter function and correct transcription start point selection, as was the distance between the two elements and the wild-type transcription start point. The GACN5G element interacts specifically and in a double-stranded form with a protein(s) in Leishmania nuclear extracts. The degree of this protein DNA interaction in vitro correlated with SL RNA gene transcription efficiency in vivo, consistent with a role of the protein as a transcription factor. The core nucleotides GACN5G fit the consensus PSE promoter structure of pol II-transcribed snRNA genes in metazoa.

Animals↗
PubMed2001

The Leptomonas seymouri spliced leader RNA promoter requires a novel transcription factor.

The spliced leader RNA gene promoter in Leptomonas seymouri requires three promoter elements for efficient and accurate transcription of the spliced leader RNA. The upstream most element appears to have a functional homolog in Leishmania species and in the African trypanosomes. The protein factor, promoter binding protein-1, interacts with the upstream element and appears to function as a basal transcription factor. Promoter binding protein-1 has three subunits; 36, 41 and 57 kDa. Using microsequencing techniques, we have obtained peptide sequence from each subunit. These data have enabled us to recently identify the Leptomonas gene that encodes the 41 kDa subunit. The 41 kDa subunit, comprised of 381 amino acids, is a founding member of a new class of transcription factors since extensive database searches revealed no homology to any known protein. This subunit, encoded by a single copy gene, has a potential nuclear localisation signal at amino acid positions 71-76. There are also multiple dileucine repeats with unknown function. Anti-41 kDa protein polyclonal antibodies are being employed to test the function of the 41 kDa subunit in PBP-1 activities.

Amino Acid Sequence↗
PubMed1999

In vivo transcriptional analysis of the spliced leader RNA gene in the trypanosomatid Leptomonas seymouri.

Gene expression in all organisms requires the direct and indirect interaction of multiple proteins with specific DNA sequence elements. Using the monogenetic trypanosomatid, Leptomonas seymouri, we investigated the cis- and trans-acting components that determine expression of a central trypanosomatid RNA, the spliced leader (SL) RNA. Using base substitution mutagenesis and DNA transfection assays, we determined that the SL RNA gene promoter lies exclusively up-stream from the transcription initiation site. Accordingly, the SL RNA gene can be used as a gene cassette to express short heterologous RNAs of interest. We utilized two pharmacological agents, alpha-amanitin and tagetitoxin, and the detergent sarkosyl to assess components of the trans-acting machinery involved in transcription. The SL RNA inhibition pattern was distinct from that of alpha-tubulin, tRNA or ribosomal RNA. Taken together, these data suggest that the upstream SL RNA gene promoter serves to nucleate a transcriptional complex that is distinct, in either its initiation and/or elongation abilities, from other genes. A comparison of trypanosomatid SL RNA gene promoter structures with that found in the nematode Ascaris lumbricoides underscores a taxonomic difference in promoter architectures which may reflect differential requirements for the SL RNA in these organisms.

Animals↗
PubMed2002

Two distinct functional spliced leader RNA gene arrays in Leishmania tarentolae are found in several lizard Leishmania species.

A second distinct array of spliced leader RNA genes has been found in several Leishmania species particular to lizards. This is the first report of two non-allelic arrays of spliced leader RNA genes within a species cell line. The arrays are identical to each other in their transcribed spliced leader RNA gene sequences, but variable in their non-transcribed spacer sequences. In the two arrays from Leishmania tarentolae UC strain the promoter regions are similar, but not identical, at positions shown previously to be critical for spliced leader RNA transcription. These arrays contain similar numbers of genes and are both transcribed in L. tarentolae in vitro transcription extract as well as in vivo. The -66/-58 regions of both genes, which contain an element of the spliced leader RNA gene promoter, bind proteins likely to be transcription factors in a specific manner. A survey of lizard Leishmania spp. revealed a second spliced leader RNA gene array in three of four species. Phylogenetic analyses of these sequences with each other and with the spliced leader RNA gene sequences of non-lizard Leishmania spp. and their near-relatives showed that the lizard groups are more closely related to each other than to arrays from other Leishmania spp. As the transcripts of the two arrays are identical, they may co-exist to fulfil the substantial requirement for spliced leader RNA production; however, they have the potential for differential usage modulated by their distinct promoter elements. The presence of two distinct spliced leader RNA gene arrays within a single cell type may represent dissociated evolution of two redundant loci, or a previously unsuspected level of control in the post-transcriptional gene expression within some kinetoplastids.

Alleles↗
PubMed2000

Cotranscriptional cap 4 formation on the Trypanosoma brucei spliced leader RNA.

mRNA cap formation in trypanosomatid protozoa is mediated through trans-splicing of the capped spliced leader (SL) sequence of the SL RNA onto the 5' end of all mRNAs. The SL RNA cap structure in Trypanosoma brucei is unique among eukaryotes and consists of 7-methylguanosine (m(7)G) followed by four methylated nucleotides (cap 4): m(7)Gpppm(2)(6)AmpAmpCmpm(3)Um. Using transcriptional arrest in permeable T. brucei cells, we have analyzed the temporal progression of cap 4 formation on the 140-nucleotide-long SL RNA. m(7)G capping of the SL RNA could be detected on prematurely terminated SL RNA transcripts of 56 nucleotides in length and longer. Subsequent modifications characteristic of the SL RNA cap 4 were added successively in a 5' to 3' direction and appeared to be independent of core ribonucleoprotein formation. Transcripts between 56 and 67 nucleotides in length were partially modified and carried methyl groups on the first two adenosine residues, whereas a fully modified cap 4 structure was present on transcripts arrested at position 117 and beyond. Taken together, our results are consistent with a cotranscriptional mechanism for generating the cap 4 structure on the SL RNA.

Animals↗
PubMed1994

Identification of a small RNA that interacts with the 5' splice site of the Trypanosoma brucei spliced leader RNA in vivo.

In vivo psoralen cross-linking of the trypanosome spliced leader (SL) RNA has led to the discovery of a small RNA that we provisionally call the spliced leader-associated (SLA) RNA. The 72 nt SLA RNA is unlike any known small RNA except for a small region that resembles U5 snRNA. The SL/SLA RNA cross-links map to two regions, the predominant interactions occurring between the 5' splice site region of the SL RNA and a CUUUUA sequence in the SLA RNA. The resemblance between these cross-links and interactions of U5 snRNA with cis-spliced pre-mRNAs suggests that the SLA RNA may be the trans-splicing analog of U5 snRNA in trypanosomes.

Animals↗
PubMed2000

PPB1, a putative spliced leader RNA gene transcription factor in Trypanosoma cruzi.

In trypanosomatids, the spliced leader RNA, or SL RNA, donates its 5' 39 nucleotides to mature nuclear mRNAs in a process termed trans-splicing. We have previously characterized the SL RNA gene from Trypanosoma cruzi and identified its transcription promoter, including a 14 nt proximal sequence element, or PSE, that binds a putative transcription factor and activates transcription of the gene. Herein, we describe establishment of a yeast one-hybrid system using the 14 nt PSE as bait, and use this system to select T. cruzi cDNAs encoding a putative transcription factor that activates transcription of the SL RNA gene. The cDNA was selected from a normalized library and encodes an approximately 45 kDa putative PSE promoter-binding protein, PPB1. PPB1 in vitro translated or overexpressed in and isolated from transformed E. coli, showed PSE-specific binding activity by electrophoretic mobility shift assays. Finally, overexpression of PPB1 in T. cruzi led to increased expression of the SL RNA gene as well as reporter genes in episomal constructs under the control of the SL RNA gene promoter. These observations suggest that PPB1 is a transcription factor that plays an important role in SL RNA gene expression.

Amino Acid Sequence↗