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PubMed1998

Trypanosome capping enzymes display a novel two-domain structure.

The ubiquitous m7G cap of eukaryotic mRNAs and of precursors to the spliceosomal small nuclear RNAs (snRNAs) is the result of an essential RNA modification acquired during transcript elongation. In trypanosomes, the m7G cap is restricted to the spliced leader (SL) RNA and the precursors of U2, U3, and U4 snRNAs. mRNA capping in these organisms occurs posttranscriptionally by trans splicing, which transfers the capped SL sequence to the 5' ends of all mRNAs. The SL cap is the most elaborate cap structure known in nature and has been shown to consist of an m7G residue followed by four methylated nucleotides. Using Crithidia fasciculata, we have characterized and purified the guanylyltransferase (capping enzyme), which transfers GMP from GTP to the diphosphate end of RNA. The corresponding gene codes for a protein of 697 amino acids, with the carboxy-terminal half of the C. fasciculata guanylyltransferase containing the six signature motifs previously identified in yeast capping enzymes. The amino-terminal half contains a domain that displays no resemblance to any other domain associated with capping enzymes. Intriguingly, this region harbors a consensus sequence for a phosphate-binding loop which is found in ATP- and GTP-binding proteins. This two-domain structure is also present in the Trypanosoma brucei capping enzyme, which shows 44% overall identity with the C. fasciculata capping enzyme. Thus, this structure appears to be common to all trypanosomatid protozoa and defines a novel class of capping enzymes.

Amino Acid Sequence↗
PubMed1999

Purification of the spliced leader ribonucleoprotein particle from Leptomonas collosoma revealed the existence of an Sm protein in trypanosomes. Cloning the SmE homologue.

Trans-splicing in trypanosomes involves the addition of a common spliced leader (SL) sequence, which is derived from a small RNA, the SL RNA, to all mRNA precursors. The SL RNA is present in the cell in the form of a ribonucleoprotein, the SL RNP. Using conventional chromatography and affinity selection with 2'-O-methylated RNA oligonucleotides at high ionic strength, five proteins of 70, 16, 13, 12, and 8 kDa were co-selected with the SL RNA from Leptomonas collosoma, representing the SL RNP core particle. Under conditions of lower ionic strength, additional proteins of 28 and 20 kDa were revealed. On the basis of peptide sequences, the gene coding for a protein with a predicted molecular weight of 11.9 kDa was cloned and identified as homologue of the cis-spliceosomal SmE. The protein carries the Sm motifs 1 and 2 characteristic of Sm antigens that bind to all known cis-spliceosomal uridylic acid-rich small nuclear RNAs (U snRNAs), suggesting the existence of Sm proteins in trypanosomes. This finding is of special interest because trypanosome snRNPs are the only snRNPs examined to date that are not recognized by anti-Sm antibodies. Because of the early divergence of trypanosomes from the eukaryotic lineage, the trypanosome SmE protein represents one of the primordial Sm proteins in nature.

Amino Acid Sequence↗
PubMed1994

Functional analysis of cis-acting DNA elements required for expression of the SL RNA gene in the parasitic protozoan Leishmania amazonensis.

DNA sequences, that control expression of the spliced leader (SL) RNA gene in the parasitic protozoan Leishmania amazonensis, were mapped by block substitution mutagenesis. In the absence of a functional in vitro system for transcription, no promoter elements have yet been identified in this organism. We therefore developed an alternative in vivo approach, in which the SL RNA gene was tagged and then subjected to a series of linker scanning mutations. Each tagged and mutated SL RNA construct was introduced into parasite cells via the pX transfection vector, and was examined for expression of the tagged SL RNA followed by characterization of its transcriptional start site. The replacement of a critical DNA element was expected to prevent expression of the tagged SL RNA. We found that the putative SL RNA promoter is complex and includes two elements: one is located upstream to the coding region, between positions -30 to -70; and the other is located between -10 to +10, and includes transcribed sequences. In addition to the functional relationship between the SL RNA and vertebrate U snRNAs, we found structural similarities in their regulatory elements, which may possibly indicate a common evolutionary ancestry for these molecules.

Animals↗
PubMed1995

Determination of the folding topology of the SL1 RNA from Caenorhabditis elegans by multidimensional heteronuclear NMR.

The process of trans-splicing involves the transfer of a short spliced leader (SL) RNA sequence to a consensus acceptor site on a separate pre-mRNA transcript. In this study, the first stem loop of the SL1 RNA from the nematode Caenorhabditis elegans was examined by homonuclear and heteronuclear NMR. Results of enzymatic cleavage patterns established that the first 36 nucleotides (which includes the splice site and a complementary base-paired region surrounding a nine-nucleotide hairpin loop) remain structurally independent of the rest of the 100-nucleotide full-length transcript. A comparison of exchangeable and non-exchangeable proton chemical shifts in the region of the splice site and loop between the native sequence and a modified 26-nucleotide fragment from which an asymmetric internal loop had been deleted was made. There was no significant difference between the resonance locations of the equivalent protons in the two molecules, establishing that there was no tertiary interaction between the hairpin and internal loops. Full chemical shift assignments of 1H, 13C, and 15N chemical shifts were obtained for the modified fragment by multidimensional homonuclear and heteronuclear NMR spectroscopy. The stem adopts an A-form helix typical of RNA. The A-type helical conformation of the stem appears to continue for the first three nucleotides of the 5' side of the loop, followed by a guanosine residue in a syn conformation about the glycosidic bond. Base stacking is not seen on the 3' side of the loop. There was no evidence for formation of Watson-Crick base-pairs within the loop, but several long distance NOEs indicated cross-loop contacts, indicative of a structured loop. The final loop residues, an adenine which is conserved among all known nematode SL RNA sequences, adopts an extrahelical conformation.

Animals↗
PubMed1997

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.

Animals↗
PubMed1988

Presence of the Caenorhabditis elegans spliced leader on different mRNAs and in different genera of nematodes.

Several different mRNAs from Caenorhabditis elegans contain the same 22-nucleotide leader sequence at their 5' ends that is acquired in a trans-splicing reaction. About 10 to 15% of the major proteins are translated from mRNAs that contain the spliced leader, among them two ribosomal proteins, ubiquitin, GAPDH, a heat shock protein (hsp70a), and three actins. The same spliced leader sequence is present in mRNAs isolated from nematodes from several different genera; but it is not present in mRNAs from other organisms. The spliced leader is encoded as a spliced leader (SL) RNA about 100 nucleotides long. The gene for the SL RNA is located in the 5S rDNA repeat in C. elegans; however, this association with the 5S repeat is not preserved in other genera. The 22-nucleotide spliced leader sequence is conserved in three genera of nematodes.

Actins↗
PubMed1990

Destruction of U2, U4, or U6 small nuclear RNA blocks trans splicing in trypanosome cells.

We have used permeable cells of Trypanosoma brucei to analyze the role of snRNAs in the trans splicing process. Degradation of U2, U4, or U6 snRNA by site-directed cleavage with complementary deoxyoligonucleotides and RNAase H inhibits trans splicing of the spliced leader (SL) RNA and newly synthesized alpha-tubulin pre-mRNAs. Cleavage of U snRNAs abolishes the appearance of putative trans splicing reaction intermediates and products, namely, linear branched molecules consisting of the SL intron joined to high molecular weight RNA (Y structures) and free SL intron. This indicates that U snRNAs are required for an early step in trans splicing. alpha-tubulin transcripts synthesized in the absence of trans splicing are unstable, suggesting that the addition of the SL sequence stabilizes pre-mRNAs against degradation. Our results provide direct evidence for the participation of U2 and U4/U6 snRNPs in trans splicing.

Animals↗
PubMed1996

Solution structure of the donor site of a trans-splicing RNA.

BACKGROUND: RNA splicing is both ubiquitous and essential for the maturation of precursor mRNA molecules in eukaryotes. The process of trans-splicing involves the transfer of a short spliced leader (SL) RNA sequence to a consensus acceptor site on a separate pre-mRNA transcript. In Caenorhabditis elegans, a majority of pre-mRNA transcripts receive the 22-nucleotide SL from the SL1 RNA. Very little is known about the various roles that RNA structures play in the complex conformational rearrangements and reactions involved in premRNA splicing. RESULTS: We have determined the solution structure of a domain of the first stem loop of the SL1 RNA of C. elegans, using homonuclear and heteronuclear NMR techniques; this domain contains the splice-donor site and a nine-nucleotide hairpin loop. In solution, the SL1 RNA fragment adopts a stem-loop structure: nucleotides in the stem region form a classical A-type helix while nucleotides in the hairpin loop specify a novel conformation that includes a helix, that extends for the first three residues; a syn guanosine nucleotide at the turn region; and an extrahelical adenine that defines a pocket with nucleotides at the base of the loop. CONCLUSION: The proximity of this pocket to the splice donor site, combined with the observation that the nucleotides in this motif are conserved among all nematode SL RNAs, suggests that this pocket may provide a recognition site for a protein or RNA molecule in the trans-splicing process.

Animals↗
PubMed1980

Characterisation of polyoma late mRNA leader sequences by molecular cloning and DNA sequence analysis.

The leader sequences of two of the three polyoma virus late mRNAs were characterised by molecular cloning and DNA sequence analysis. A short single-stranded DNA fragment complementary to the 5' end of the body of mVP1 was used to prime cDNA synthesis, and double-stranded cDNA was inserted into a derivative of pAT 153. Analysis of fourteen mVP1 cDNAs and two mVP3 cDNAs allowed the precise determination of the leader-body joints, and demonstrated that the majority of polyoma late leader sequences consist of exact tandem repeats of a 57 nucleotide sequence present only once in the genomic DNA at 66-67 m.u. The sequences in the genomic DNA borderline this unit are typical of those found at RNA splice points. Leader sequences contained on average three to four repeat units. Nuclease S1 mapping of total late mRNA demonstrated that most mRNA 5' ends map heterogeneously in the 50 nucleotides 5' to the repeated sequence unit. The structures of the leader sequences strongly suggest that they are generated by appropriate splicing events from a tandemly repeated transcript of the entire circular viral genome.

Base Sequence↗
PubMed2005

Quantifying optimal accuracy of local primary sequence bioinformatics methods.

MOTIVATION: Traditional bioinformatics methods scan primary sequences for local patterns. It is important to assess how accurate local primary sequence methods can be. RESULTS: We study the problem of donor pre-mRNA splice site recognition, where the sequence overlaps between real and decoy datasets can be quantified, exposing the intrinsic limitations of the performance of local primary sequence methods. We assess the accuracy of primary sequence methods generally by studying how they scale with dataset size and demonstrate that our new primary sequence ranking methods have superior performance.

Algorithms↗
PubMed1999

SR proteins are required for nematode trans-splicing in vitro.

SR (ser/arg) proteins have been shown to play roles in numerous aspects of pre-mRNA splicing, including modulation of alternative splicing, commitment of substrates to the splicing pathway, and splice site communication. The last of these, splice site communication, is particularly relevant to trans-splicing in which the 5' and 3' exons originate on separate molecules. The participation of SR proteins in naturally occurring, spliced leader RNA-dependent transsplicing has not been examined. Here, we have isolated SR proteins from an organism that performs both trans- and cis-splicing, the nematode Ascaris lumbricoides. To examine their activity in in vitro splicing reactions, we have also developed and characterized an SR protein-depleted whole-cell extract. When tested in this extract, the nematode SR proteins are required for both trans- and cis-splicing. In addition, the state of phosphorylation of the nematode SR proteins is critical to their activity in vitro. Interestingly, mammalian (HeLa) and A. lumbricoides SR proteins exhibit equivalent activities in cis-splicing, while the nematode SR proteins are much more active in trans-splicing. Thus, it appears that SR proteins purified from an organism that naturally trans-splices its pre-mRNAs promote this reaction to a greater extent than do their mammalian counterparts.

Animals↗
PubMed2006

Operon conservation and the evolution of trans-splicing in the phylum Nematoda.

The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five major clades of the phylum Nematoda, for the presence of operons and the use of trans-spliced leaders in resolution of polycistronic pre-mRNAs. Conserved operons were found in Pristionchus pacificus, Nippostrongylus brasiliensis, Strongyloides ratti, Brugia malayi, and Ascaris suum. In nematodes closely related to the rhabditine C. elegans, a related family of SL2-like spliced leaders is used for operonic transcript resolution. However, in the tylenchine S. ratti operonic transcripts are resolved using a family of spliced leaders related to SL1. Non-operonic genes in S. ratti may also receive these SL1 variants. In the spirurine nematodes B. malayi and A. suum operonic transcripts are resolved using SL1. Mapping these phenotypes onto the robust molecular phylogeny for the Nematoda suggests that operons evolved before SL2-like spliced leaders, which are an evolutionary invention of the rhabditine lineage.

Animals↗
PubMed2003

RNA polymerase I transcribes procyclin genes and variant surface glycoprotein gene expression sites in Trypanosoma brucei.

In eukaryotes, RNA polymerase (pol) I exclusively transcribes the large rRNA gene unit (rDNA) and mRNA is synthesized by RNA pol II. The African trypanosome, Trypanosoma brucei, represents an exception to this rule. In this organism, transcription of genes encoding the variant surface glycoprotein (VSG) and the procyclins is resistant to alpha-amanitin, indicating that it is mediated by RNA pol I, while other protein-coding genes are transcribed by RNA pol II. To obtain firm proof for this concept, we generated a T. brucei cell line which exclusively expresses protein C epitope-tagged RNA pol I. Using an anti-protein C immunoaffinity matrix, we specifically depleted RNA pol I from transcriptionally active cell extracts. The depletion of RNA pol I impaired in vitro transcription initiated at the rDNA promoter, the GPEET procyclin gene promoter, and a VSG gene expression site promoter but did not affect transcription from the spliced leader (SL) RNA gene promoter. Fittingly, induction of RNA interference against the RNA pol I largest subunit in insect-form trypanosomes significantly reduced the relative transcriptional efficiency of rDNA, procyclin genes, and VSG expression sites in vivo whereas that of SL RNA, alphabeta-tubulin, and heat shock protein 70 genes was not affected. Our studies unequivocally show that T. brucei harbors a multifunctional RNA pol I which, in addition to transcribing rDNA, transcribes procyclin genes and VSG gene expression sites.

Animals↗
PubMed2000

U4 small nuclear RNA genes of trypanosomes: cloning of the Leptomonas seymouri gene and mutational analysis of core snRNP assembly.

Trans mRNA splicing in trypanosomatids requires as cofactors small nuclear RNAs (snRNAs) U2, U4, U5, and U6, in addition to the spliced leader (SL) RNA. To allow a phylogenetic comparison and functional analysis of trypanosomatid U4 snRNAs, we have cloned the single-copy gene for the Leptomonas seymouri U4 snRNA. In addition, a putative U4 snRNA gene from Leishmania tarentolae was identified by database searching. Using an episomal expression system, we introduced mutations into the conserved Sm region of the L. seymouri U4, which is the putative binding site for the common proteins that are present in each of the trans-spliceosomal snRNPs. As demonstrated by CsCl density gradient centrifugation, Sm mutant U4 snRNAs are non-functional in core RNP assembly. Furthermore, we present evidence by cell fractionation that U4 snRNAs with Sm mutations are partially defective in nuclear-cytoplasmic translocation. Taken together this indicates that the Sm site of U4 snRNA is responsible for stable core RNP assembly and nuclear localization.

Animals↗
PubMed2005

Spliced-leader trans-splicing in freshwater planarians.

trans-Splicing, in which a spliced-leader (SL) RNA is appended to the most 5' exon of independently transcribed pre-mRNAs, has been described in a wide range of eukaryotes, from protozoans to chordates. Here we describe trans-splicing in the freshwater planarian Schmidtea mediterranea, a free-living member of the phylum Platyhelminthes. Analysis of an expressed sequence tag (EST) collection from this organism showed that over 300 transcripts shared one of two approximately 35-base sequences (Smed SL-1 and SL-2) at their 5' ends. Examination of genomic sequences encoding representatives of these transcripts revealed that these shared sequences were transcribed elsewhere in the genome. RNA blot analysis, 5' and 3' rapid amplification of cDNA ends, as well as genomic sequence data showed that 42-nt SL sequences were derived from small RNAs of approximately 110 nt. Similar sequences were also found at the 5' ends of ESTs from the planarian Dugesia japonica. trans-Splicing has already been described in numerous representatives of the phylum Platyhelminthes (trematodes, cestodes, and polyclads); its presence in two representatives of the triclads supports the hypothesis that this mode of RNA processing is ancestral within this group. The upcoming complete genome sequence of S. mediterranea, combined with this animal's experimental accessibility and susceptibility to RNAi, provide another model organism in which to study the function of the still-enigmatic trans-splicing.

Alternative Splicing↗