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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↗
PubMed2001

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

A genomic library of Wuchereria bancrofti 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.

Amino Acid Sequence↗
PubMedDate not supplied

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.

Animals↗
PubMed1992

Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo.

The Trypanosoma brucei spliced leader (SL) RNA donates its 5' leader sequence to all nuclear pre-mRNAs via trans RNA splicing. The SL RNA is a small-nuclear U RNA-like molecule which is present in the cell as part of a small ribonucleoprotein particle. However, unlike the trimethylguanosine-capped small nuclear U RNAs, the SL RNA has a highly modified 5' terminus containing an m7G cap and methylations on the first four transcribed nucleotides. Here, we show that incubation of procyclic-form T. brucei in the presence of the S-adenosylmethionine analog, sinefungin, leads to a rapid inhibition of SL RNA methylation. A concomitant inhibition of trans splicing and an accumulation of high-molecular-weight tubulin transcripts were also observed. The effects of sinefungin on SL RNA methylation and on trans splicing were correlated by labeling of cells incubated in the presence of the antibiotic. The results indicate that 5' modifications of the SL RNA are necessary for it to participate in trans splicing. SL RNA modification is not required for assembly of the core SL ribonucleoprotein, as these Cs2SO4-resistant particles can be formed with either methylated or undermethylated SL RNA.

Adenosine↗
PubMed2001

Identification of a spliced leader RNA binding protein from Trypanosoma cruzi.

Nuclear mRNAs in trypanosomatids are generated by trans-splicing. Although trans-splicing resembles cis-splicing in many ways and most of the U RNA participants have been characterized, relatively few involved proteins have been identified. Herein, we employed a yeast three-hybrid system to identify a protein, XB1, which binds to the Trypanosoma cruzi SL RNA. XB1 is a approximately 45 kDa protein which is homologous to the essential pre-mRNA-splicing factor PRP31p from Saccharomyces cerevisiae. Gel shift assays and UV cross-linking experiments with recombinant XB1 confirmed that this T. cruzi protein binds the SL RNA in vitro. The binding site of XB1 on the SL RNA was mapped to stem-loop II by deletion of the SL RNA 'bait' in the three-hybrid system. Finally, UV cross-linking SL RNA with S100 extract indicated native XB1 protein and SL RNA interaction in T. cruzi extract.

Amino Acid Sequence↗
PubMed2003

Silencing of Sm proteins in Trypanosoma brucei by RNA interference captured a novel cytoplasmic intermediate in spliced leader RNA biogenesis.

In Trypanosoma brucei the small nuclear (sn) RNAs U1, U2, U4, and U5, as well as the spliced leader (SL) RNA, bind the seven Sm canonical proteins carrying the consensus Sm motif. To determine the function of these proteins in snRNA and SL RNA biogenesis, two of the Sm core proteins, SmE and SmD1, were silenced by RNAi. Surprisingly, whereas the level of all snRNAs, including U1, U2, U4, and U5 was reduced during silencing, the level of SL RNA was dramatically elevated, but the levels of U6 and spliced leader-associated RNA (SLA1) remained unchanged. The SL RNA that had accumulated in silenced cells lacked modification at the cap4 nucleotide but harbored modifications at the cap1 and cap2 nucleotides and carried the characteristic psi. This SL RNA possessed a longer tail and had accumulated in the cytoplasm in 10 and 50 S particles that were found by in situ hybridization to be present in "speckles." We propose a model for SL RNA biogenesis involving a cytoplasmic phase and suggest that the trypanosome-specific "cap4" nucleotides function as a signal for export and import of SL RNA out and into the nucleus. The SL RNA biogenesis pathway differs from that of U sn ribonucleoproteins (RNPs) in that it is the only RNA that binds Sm proteins that were stabilized under Sm depletion in a novel RNP, which we termed SL RNP-C.

Active Transport, Cell Nucleus↗
PubMed2001

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.

Amino Acid Sequence↗
PubMedDate not supplied

[Analysis of the dynamics of transitions in Leptomonas collosoma spliced leader RNA and a possible role of gaps upon splicing].

In [1], the existence of two competing secondary structures at the 5'-end of Leptomonas collosoma spliced leader RNA was shown. On going from one steady-state secondary structure to the other, and vice versa, RNA passes through several states in which the chain gets tied up into nodes. If the transitions are reversible, the nodes are figurative both in the forth and back directions although the secondary structures that arise in this process are quite different, and the forth and back routs do not coincide. It is assumed that under certain conditions, the chain can get tied into the next node of series 5(2), which most likely will not get untied. This node is tightened around GG nucleotides. In this case, the migration of the gap is possible, as a result of which the chain can break. Independent experiments indicate that the RNA chain breaks just at this site upon splicing. Based on these results, a possible role of nodes in the enzymatic activity is discussed.

Animals↗
PubMed2006

RNAi interference of XPO1 and Sm genes and their effect on the spliced leader RNA in Trypanosoma brucei.

In trypanosomes, trans-splicing is a major essential RNA-processing mechanism that involves the addition of a spliced leader sequence to all mRNAs from a small RNA species, known as the spliced leader RNA (SL RNA). SL RNA maturation is poorly understood and it is not clear where assembly with Sm proteins takes place. In this study, we followed the localization of the SL RNA during knockdown of Sm proteins and XPO1, which in metazoa functions in transport of mRNA and U snRNAs from the nucleus to the cytoplasm. We found that XPO1 has no role in SL RNA biogenesis in wild-type cells, or when the cells are depleted of Sm proteins. During Sm depletion, 'defective' SL RNA lacking cap modification at position +4 first accumulates in the nucleus, suggesting that Sm assembly on SL RNA most probably takes place in this compartment. Only after massive nuclear accumulation is the 'defective' SL RNA exported to the cytoplasm to form SL RNP-C, which may be a route to dispose of SL RNA when its normal biogenesis is blocked.

Active Transport, Cell Nucleus↗
PubMed2005

Intragenomic spliced leader RNA array analysis of kinetoplastids reveals unexpected transcribed region diversity in Trypanosoma cruzi.

The spliced leader RNA gene (SL RNA) repeat is present in large multicopy arrays and has been used as a marker for the diversity of kinetoplastid protozoans. Intra-array variation could affect conclusions made using a randomly isolated repeat as a marker. We examined the Leishmania major (Friedlin) and Trypanosoma cruzi (CL Brener) genome projects for SL RNA repeat sequences in order to assess their homogeneity and the possible effects of sequence variation on taxonomic interpretation. Of the dozens of distinct sequence classes examined, no single copy would bias clustering analyses with regard to other closely related species or isolates. Six dimorphic sites within the T. cruzi transcribed region were found to be linked and are predicted to yield a heterogeneous SL RNA population. The variation that exists among the repeats paints a picture of the broad mechanisms of array maintenance and evolution where site-specific mutations in a single repeat may be spread throughout the array and recombined with existing repeats to create new sequence classes, all occurring under selective pressure to maintain or increase the fitness of the cell line in which these events occur.

Animals↗
PubMed1997

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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1998

Structure-function analysis of the trypanosomatid spliced leader RNA.

In trypanosomes, all mRNAs possess a spliced leader (SL) at their 5' end. SL is added to pre-mRNA via trans -splicing from a small RNA, the SL RNA. To examine structure-function aspects of the trypanosomatid SL RNA, an in vivo system was developed in the monogenetic trypanosomatid Leptomonas collosoma to analyze the function of chimeric and site-directed SL RNA mutants in trans -splicing. Stable cell lines expressing chimeric and mutated SL RNA from the authentic SL RNA regulatory unit were obtained. The chimeric RNA was expressed and assembled into an SL RNP particle, but could not serve as a substrate in splicing. Mutations in loop II and III of L.collosoma SL RNA formed the Y structure intermediate. In addition, a double SL RNA mutant in loop II, and positions 7 and 8 of the intron, also formed the Y structure intermediate, suggesting that these intron positions, although proposed to participate in the interaction of SL RNA with U5, may not be crucial for the first step of the trans -splicing reaction. A mutation in the exon located in loop I was not utilized in splicing, suggesting the importance of exon sequences for trans -splicing in trypanosomes. However, a double SL RNA mutant in loop II and exon position 31 was utilized in both steps of splicing; the mutant thus provides a model molecule for further analysis of positions essential for the function of the SL RNA.

Animals↗
PubMed2000

Transcription of the kinetoplastid spliced leader RNA gene.

In recent years, much has been learned about the cis-elements controlling transcription of the kinetoplastid spliced leader (SL) RNA gene. The SL RNA gene contains the first 39 nucleotides that are trans-spliced on to all nuclear-derived mRNAs in these organisms. Transcription initiation is determined by two precisely spaced upstream elements and transcription termination is directed by the downstream poly-T tract, although the RNA polymerase responsible for SL RNA synthesis is still questioned. In this article, David Campbell, Nancy Sturm and Michael Yu review the field of kinetoplastid SL RNA gene transcription, address past proposals in light of current data and discuss some of the differences that appear in the literature.

Amanitins↗