Spliced Leader RNA Gene Research Articles

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.

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.

Diplonema spp. possess spliced leader RNA genes similar to the Kinetoplastida.

The phylogenetic placement of the genus Diplonema in relation to fellow phylum members Euglena and Trypanosoma has been uncertain. The spliced leader RNA gene, present in the euglenids and kinetoplastids in distinct forms, was a potential target for resolving this question. The first indication supporting a closer relationship to the kinetoplastids was the recognition of potential spliced leader RNA exon sequences in the genomic DNA of two Diplonema isolates. Examination of total cell RNA revealed transcripts in the anticipated size range at approximately 120 and 130 nt. Specific PCR amplification of a spliced leader RNA gene repeat was performed. The hallmark features of the kinetoplastid-type spliced leader RNA, specifically the 39-nt exon, splice-donor site, Sm-binding site and poly-T tract and the potential to form the requisite stem-loop structures, were found. Diplonema spp. are different from the kinetoplastids by virtue of C residues at positions 4 and 18 in the exon. While the intergenic spacer regions varied in size, each contained the complete sequence or remnants of a 5S ribosomal RNA gene. Possession of a functional spliced leader RNA gene of the kinetoplastid variety in Diplonema supports a closer evolutionary relationship with this group than with the euglenids.

Trans-spliced leader RNA, 5S-rRNA genes and novel variant orphan spliced-leader of the lymphatic filarial nematode Wuchereriabancrofti, and a sensitive polymerase chain reaction based detection assay

A genomic library of Wuchereriabancrofti was examined for the presence of the 22 nucleotide spliced leader (SL) which plays a vital role in the maturation of the 5′ end of certain mRNAs through the addition of a small spliced leader (SL) exon and also in the generation of monocistronic mRNA from initial polycistronic transcripts in nematodes. Here, we report the characterization of three SL RNA genes (SLG1, SLG2 and SLG3), an internal copy of a novel variant SL1 sequence (SL1v) with 23 nucleotides within an open reading frame of 75 amino acid residues of an unknown gene and two 5S-rRNA genes (5SR2 and 5SR3) from two genomic clones (TZP/11, TZP/91) of W.bancrofti. Our results revealed that the genes for the spliced leader RNA of W.bancrofti (SL RNA) is reiterated within the 5S-rRNA gene cluster and are in the same orientation. The genes SLG1, SLG2 and SLG3 were identical in nucleotide sequence except for an additional nucleotide at position 43 on SLG2. Sequence analysis of the three genes indicated that the 22-nt sequence is invariably adjacent to the dinucleotide GT, characteristic of a potential spliced donor site. The Sm-binding sequence AATTTTGG was conserved in SLG1, SLG2 and SLG3. Further, both 5′ and 3′ flanking regions of genes SLG1, SLG2 and SLG3 shared considerable sequence similarity. Two 5S-rRNA genes characterized from the genomic clone TZP 11 were shown to have sequence heterogeneity. Genomic southern showed that the spliced leader sequence is multicopy within the W.bancrofti genome and is also encoded in the region of DNA unlinked to the 5S rRNA gene cluster. Primers designed to amplify intergenic regions between 5S-rRNA and SL RNA genes in a PCR assay were found to be specific for W.bancrofti and was sensitive enough to detect 1 pg of W.bancrofti DNA or 1/8th of a microfilariae in infected blood samples. The high specificity and sensitivity of the optimised PCR assay makes it an ideal diagnostic tool for the identification of W.bancrofti in both the host and the vector.

Trypanosome spliced leader RNA genes contain the first identified RNA polymerase II gene promoter in these organisms.

Typical general transcription factors, such as TATA binding protein and TFII B, have not yet been identified in any member of the Trypanosomatidae family of parasitic protozoa. Interestingly, mRNA coding genes do not appear to have discrete transcriptional start sites, although in most cases they require an RNA polymerase that has the biochemical properties of eukaryotic RNA polymerase II. A discrete transcription initiation site may not be necessary for mRNA synthesis since the sequences upstream of each transcribed coding region are trimmed from the nascent transcript when a short m(7)G-capped RNA is added during mRNA maturation. This short 39 nt m(7)G-capped RNA, the spliced leader (SL) sequence, is expressed as an approximately 100 nt long RNA from a set of reiterated, though independently transcribed, genes in the trypanosome genome. Punctuation of the 5' end of mRNAs by a m(7)G cap-containing spliced leader is a developing theme in the lower eukaryotic world; organisms as diverse as EUGLENA: and nematode worms, including Caenorhabditis elegans, utilize SL RNA in their mRNA maturation programs. Towards understanding the coordination of SL RNA and mRNA expression in trypanosomes, we have begun by characterizing SL RNA gene expression in the model trypanosome Leptomonas seymouri. Using a homologous in vitro transcription system, we demonstrate in this study that the SL RNA is transcribed by RNA polymerase II. During SL RNA transcription, accurate initiation is determined by an initiator element with a loose consensus of CYAC/AYR(+1). This element, as well as two additional basal promoter elements, is divergent in sequence from the basal transcription elements seen in other eukaryotic gene promoters. We show here that the in vitro transcription extract contains a binding activity that is specific for the initiator element and thus may participate in recruiting RNA polymerase II to the SL RNA gene promoter.

The spliced leader RNA gene array in phloem-restricted plant trypanosomatids (Phytomonas) partitions into two major groupings: epidemiological implications.

The arbitrary genus Phytomonas includes a biologically diverse group of kinetoplastids that live in a wide variety of plant environments. To understand better the subdivisions within the phytomonads and the variability within groups, the exon, intron and non-transcribed spacer sequences of the spliced leader RNA gene were compared among isolates of the phloem-restricted members. A total of 29 isolates associated with disease in coconut, oil palm and red ginger (Alpinia purpurata, Zingibreaceae) were examined, all originating from plantations in South America and the Caribbean over a 12-year period. Analysis of non-transcribed spacer sequences revealed 2 main groups, I and II; group II could be further subdivided into 2 subgroups, IIa and Ilb. Three classes of spliced leader (SL) RNA gene were seen, with SLI corresponding to group I, SLIIa to group lIa, and SLIIb to group IIb. Two isolates showed some characteristics of both major groups. Group-specific oligonucleotide probes for hybridization studies were tested, and a multiplex amplification scheme was devised to allow direct differentiation between the 2 major groups of phloem-restricted Phytomonas. These results provide tools for diagnostic and molecular epidemiology of plant trypanosomes that are pathogenic for commercially important flowers and palms.

In vitro transcription of mutated Leishmania tarentolae spliced leader RNA genes approximates in vivo patterns

To elucidate the process of transcription in the kinetoplastids and to aid in the purification of transcription factors, we have developed a transcriptionally-competent nuclear extract from Leishmania tarentolae for the study of the spliced leader (SL) RNA gene. The extract was competent to transcribe a tagged SL RNA gene. The in vitro SL RNA transcripts initiated accurately and their synthesis was dependent on the presence of the promoter defined in vivo. The nuclear extract was then challenged rigorously using an exhaustive set of mutated SL RNA gene templates previously tested for transcriptional activity in vivo. Mutation of four nucleotides (CCGG) at positions −34 to −31 had a detrimental effect on transcription in vitro; the CC dinucleotide overlaps one element necessary in vivo. Similarly, four nucleotides (TGAC; positions −67 to −64) important for transcription in vitro overlapped the other core promoter element defined in vivo, but were generally not effective as point mutations. The promoter-binding ability of the transcriptionally-competent extract for the −60 region mutations mirrored the in vitro transcription pattern. Although it does not reflect precisely the in vivo results, this in vitro system provides us with an important tool for monitoring the purification of potential transcription factors, as well as the basis for future reconstitution experiments.

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 ∼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.

Transcription Initiation at the TATA-less Spliced Leader RNA Gene Promoter Requires at Least Two DNA-binding Proteins and a Tripartite Architecture That Includes an Initiator Element

Eukaryotic transcriptional regulatory signals, defined as core and activator promoter elements, have yet to be identified in the earliest diverging group of eukaryotes, the primitive protozoans, which include the Trypanosomatidae family of parasites. The divergence within this family is highlighted by the apparent absence of the "universal" transcription factor TATA-binding protein. To understand gene expression in these protists, we have investigated spliced leader RNA gene transcription. The RNA product of this gene provides an m(7)G cap and a 39-nucleotide leader sequence to all cellular mRNAs via a trans-splicing reaction. Regulation of spliced leader RNA synthesis is controlled by a tripartite promoter located exclusively upstream from the transcription start site. Proteins PBP-1 and PBP-2 bind to two of the three promoter elements in the trypanosomatid Leptomonas seymouri. They represent the first trypanosome transcription factors with typical double-stranded DNA binding site recognition. These proteins ensure efficient transcription. However, accurate initiation is determined an initiator element with a a loose consensus of CYAC/AYR (+1), which differs from that found in metazoan initiator elements as well as from that identified in one of the earliest diverging protozoans, Trichomonas vaginalis. Trypanosomes may utilize initiator element-protein interactions, and not TATA sequence-TATA-binding protein interactions, to direct proper transcription initiation by RNA polymerase II.

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.

Developmental Regulation of Spliced Leader RNA Gene inLeishmania donovani Amastigotes Is Mediated by Specific Polyadenylation

Leishmania cycles between the insect vector and its mammalian host undergoing several important changes mediated by the stage-specific expression of a number of genes. Using a genomic differential screening approach, we isolated differentially expressed cosmid clones carrying several copies of the mini-exon gene. We report that the spliced leader (SL) RNA, essential for the maturation of all pre-mRNAs by trans-splicing, is developmentally regulated in Leishmania donovani amastigotes and that this regulation is rapidly induced upon parasite growth under acidic conditions. Stage-specific regulation of the SL RNA is associated with the expression of a larger approximately 170-nucleotide transcript that bears an additional 15-nucleotide sequence at its 3'-end and is polyadenylated in contrast to the mature SL RNA. The poly(A)+ SL RNA represents 12-16% of the total SL transcript synthesized in amastigotes and is 2.5-3-fold more stable than the poly(A)- transcript. The poly(A)+ SL transcript is synthesized specifically from one class of the genomic mini-exon copies. Polyadenylation of the SL RNA may control the levels of the SL mature transcript under amastigote growth and may represent an additional step in the gene regulation process during parasite differentiation.

Transcription termination and 3'-End processing of the spliced leader RNA in kinetoplastids.

Addition of a 39-nucleotide (nt) spliced leader (SL) by trans splicing is a basic requirement for all trypanosome nuclear mRNAs. The SL RNA in Leishmania tarentolae is a 96-nt precursor transcript synthesized by a polymerase that resembles polymerase II most closely. To analyze SL RNA genesis, we mutated SL RNA intron structures and sequence elements: stem-loops II and III, the Sm-binding site, and the downstream T tract. Using an exon-tagged SL RNA gene, we examined the phenotypes produced by a second-site 10-bp linker scan mutagenic series and directed mutagenesis. Here we report that transcription is terminated by the T tract, which is common to the 3' end of all kinetoplastid SL RNA genes, and that more than six T's are required for efficient termination in vivo. We describe mutants whose SL RNAs end in the T tract or appear to lack efficient termination but can generate wild-type 3' ends. Transcriptionally active nuclear extracts show staggered products in the T tract, directed by eight or more T's. The in vivo and in vitro data suggest that SL RNA transcription termination is staggered in the T tract and is followed by nucleolytic processing to generate the mature 3' end. We show that the Sm-binding site and stem-loop III structures are necessary for correct 3'-end formation. Thus, we have defined the transcription termination element for the SL RNA gene. The termination mechanism differs from that of vertebrate small nuclear RNA genes and the SL RNA homologue in Ascaris.

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 GN 3CCC element at −39 to −33 and a GACN 5G 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 GN 3CCC and GACN 5G 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 GACN 5G 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 GACN 5G fit the consensus PSE promoter structure of pol II-transcribed snRNA genes in metazoa.

In vitro transcription of the Leptomonas seymouri SL RNA and U2 snRNA genes using homologous cell extracts

A cell-free transcription system for the spliced leader (SL) RNA gene of the trypanosomatid Leptomonas seymouri has been developed. Accurately initiated transcription was achieved using cell extracts and a template in which the transcribed region of the SL RNA was replaced with a guanosine-less sequence (G-less cassette). The extract was also able to direct accurate initiation of RNA from an L. seymouri tagged U2 snRNA gene, which may be expressed via a transcriptional apparatus shared by the SL RNA gene. In vivo transcription analysis was used previously to define essential sequence components of the SL RNA gene promoter (Hartree D, Bellofatto V. Mol Biochem Parasitol 1995;71:27–39). A substitution mutation in the upstream promoter element (bp −50 to −70) markedly reduced transcription in vitro as did deletion of this and the middle promoter element (bp −30 to −40). Thus, the in vitro transcription system correctly responds to promoter mutations and is useful for investigating SL RNA and snRNA gene expression.

Characterization of Two Protein Activities That Interact at the Promoter of the Trypanosomatid Spliced Leader RNA

All trypanosome mRNAs have a spliced leader (SL). The SL RNA gene in Leptomonas seymouri is a member of the small nuclear RNA gene family. However, the SL RNA is required in stoichiometric amounts for trans-splicing during mRNA formation. Expression of the SL RNA gene requires sequence elements at bp -60 to -70 and bp -30 to -40 upstream from the transcription initiation site. Using conventional and affinity chromatography, we have identified and characterized an approximately 122-kDa protein, promoter-binding protein (PBP) -1, that binds to double-strand DNA. The PBP-1-binding site is within the bp -60 to -70 element determined by DNase I footprinting. Therefore, PBP-1 is the first characterized double-strand DNA binding activity that interacts with a trypanosome gene promoter. A second protein, PBP-2, interacts with the PBP-1:DNA complex and its DNase I footprint extends to include the second promoter element (bp -30 to -40). An alteration of the spacing between the two promoter elements or mutation of the second element decreases PBP-2/PBP-1:DNA stability. Taken together, these data suggest that PBP-1 and PBP-2 are components of a transcription initiation complex that assembles within the SL RNA gene promoter.

Trypanosoma cruzi strains partition into two groups based on the structure and function of the spliced leader RNA and rRNA gene promoters

We have previously identified a major proximal sequence element (PSE) responsible for transcription of the spliced leader (SL) gene from Trypanosama cruzi strain CL, and showed that the sequence encompassing this PSE exhibits ∼30% divergence between two major groups of T. cruzi isolates, but strong conservation within the groups. In this report, we show that the SL RNA gene promoter from the CL strain (group I) is efficiently expressed only in T. cruzi isolates from group I. Similarly, the sequence of the ∼643 bp promoter region of the T. cruzi rRNA is strongly conserved within, but diverged ∼20% between, the two groups. Reporter constructs driven by the rRNA promoter sequences from group I strains are strongly expressed after electroporation into other group I strains, but not expressed in group II strains. In contrast, constructs bearing rRNA promoter sequences from group II strains are active in strains from both groups. Phylogenetic analyses performed with both the rRNA and the SL RNA gene promoter sequences yielded similar trees, and these trees strongly reinforce the partitioning of known T. cruzi into two major groups that parallel the observed functional specificity of the promoters. Given the well-documented species specific pattern of both rRNA promoters and PSEs in higher eukaryotes, these results suggest an ancient evolutionary divergence among organisms currently classified as T. cruzi.

Transcription of the Trypanosoma brucei spliced leader RNA gene is dependent only on the presence of upstream regulatory elements

The spliced leader (SL) RNA plays a key role in mRNA maturation in trypanosomatid protozoa by providing the SL sequence, which is joined to the 5′ end of every mRNA. As a first step towards a better understanding of the biogenesis and function of the SL RNA, we expressed a tagged SL RNA gene in a cell-free system of procyclic Trypanosoma brucei cells. Transcription initiates at +1 and can be detected as early as 1 min after addition of extract. Transcription of the SL RNA gene in vitro, as well as in permeable cells, is mediated by an α-amanitin/tagetitoxin resistant complex, suggesting a promoter that is intermediate between a classical RNA polymerase II and RNA polymerase III promoter. An analysis of the promoter architecture of the SL RNA gene revealed that regulatory elements are located upstream of the coding region and that the SL sequence, in contrast to the nematode SL sequence, is not required for T. brucei SL RNA gene transcription.

Structure and Expression of Novel Spliced Leader RNA Genes in Caenorhabditis elegans

Approximately 25% of Caenorhabditis elegans genes are organized as operons. Polycistronic transcripts are converted to monocistronic mRNAs by 3' cleavage/polyadenylation and 5' trans-splicing with untranslated, 5' termini of mRNAs encoded by downstream genes in operons are acceptors for > or = 7 recently discovered "novel" SLs and a classical SL (SL2). Diversity in SL exons is now partly explained by the discovery and characterization of five novel genes that encode C. elegans SL RNAs. These novel SL RNAs contain a 22- or 23-nucleotide SL followed by conserved splice donor and downstream sequences that are essential for catalysis of trans-splicing reactions. The SL3 alpha, SL4, and SL5 RNA genes are tightly clustered on chromosome III; their 114-nucleotide transcripts deliver three distinct SLs to mRNAs. The SL3 beta and SL3 gamma RNA genes are on chromosome I, but are not tightly linked. SL RNAs 3 alpha, 3 beta, and 3 gamma provide identical 5' leader exons, although their 3' sequences diverge. Transcription of SL 3-5 RNA genes appears to be driven by flanking DNA elements that are homologous with segments of promoters for the C. elegans SL2 RNA and small nuclear RNA genes. RNase protection assays demonstrated that novel SL RNAs are transcribed in vivo and accumulate in the poly(A-) RNA pool. SL3 exons are transferred to mRNAs as frequently as SL2 exons. In contrast, SL4 is appended to mRNAs 10% as frequently as SL3. The abundance of SL4 RNA increased 6-fold during postembryonic development, and the SL4 RNA gene promoter is active principally in hypodermal cells.

The Spliced Leader RNA Gene of Leptomonas collosoma

The Spliced Leader RNA Gene of Leptomonas collosoma

RNA trans-splicing in Fasciola hepatica. Identification of a spliced leader (SL) RNA and SL sequences on mRNAs.

We have identified a new spliced leader (SL) in Fasciola hepatica by characterizing the 5'-terminal sequences of its enolase mRNA, an mRNA also trans-spliced in the flatworm Schistosoma mansoni (Rajkovic, A., Davis, R.E., Simonsen, J.N., and Rottman, F.M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8879-8883). This 37-nucleotide spliced leader is at the 5' ends of multiple Fasciola mRNAs and is likely to be derived from the 5' terminus of a nonpolyadenylated, 108-nucleotide RNA with a trimethylguanosine cap. The SL RNA gene is present in approximately 100 copies within a 1.1-kilobase genomic tandem repeat. Secondary structure predictions indicate that the Fasciola SL RNA contains three stem loops in contrast to two previously observed in S. mansoni. Fasciola and S. mansoni SLs are likely to be evolutionarily related although their sequence identity is only 65%. In contrast with nematodes, absolute conservation of SL sequences and secondary structure does not occur in trematodes. A spliced leader in Fasciola indicates that trans-splicing is likely to be a common feature in other trematodes and perhaps other flatworms.