[Abstract unavailable

Trypanosomes use trans splicing to place a common 39-nucleotide spliced-leader sequence on the 5' ends of all of their mRNAs. To identify likely participants in this reaction, we used antiserum directed against the characteristic U RNA 2,2,7-trimethylguanosine (TMG) cap to immunoprecipitate six candidate U RNAs from total trypanosome RNA. Genomic Southern analysis using oligonucleotide probes constructed from partial RNA sequence indicated that the four largest RNAs (A through D) are encoded by single-copy genes that are not closely linked to one another. We have cloned and sequenced these genes, mapped the 5' ends of the encoded RNAs, and identffied three of the RNAs as the trypanosome U2, U4, and U6 analogs by virtue of their sequences and structural homologies with the corresponding metazoan U RNAs. The fourth RNA, RNA B (144 nucleotides), was not sufficiently similar to known U RNAs to allow us to propose an identity. Surprisingly, none of these U RNAs contained the consensus Sm antigen-binding site, a feature totally conserved among several classes of U RNAs, including U2 and U4. Similarly, the sequence of the U2 RNA region shown to be involved in pre-mRNA branchpoint recognition in yeast, and exactly conserved in metazoan U2 RNAs, was totally divergent in trypanosomes. Like all other U6 RNAs, trypanosome U6 did not contain a TMG cap and was immunoprecipitated from deproteinized RNA by anti-TMG antibody because of its association with the TMG-capped U4 RNA. These two RNAs contained extensive regions of sequence complementarity which phylogeneticaily support the secondary-structure model proposed by D. A. Brow and C. Guthrie (Nature [London] 334:213-218, 1988) for the organization of the analogous yeast U4-U6 complex.Eucaryotic cells contain many small nuclear (sn) U RNAs complexed in vivo with proteins to form ribonucleoprotein (RNP) particles termed snRNPs (3, 9). Individual snRNPs are distinguished by the U snRNA species they contain and by their complement of unique and shared protein antigens (2,6,40). Eleven U snRNAs have been detected in mammals; eight of these (Ul through U8) have been isolated and sequenced (21,41,42). They are abundant RNAs (104 to 106 copies per cell) ranging in size from 57 to 216 nucleotides (nt) and are usually encoded by multicopy gene families. With the exception of U6, the U snRNAs are characteristically capped at their 5' termini by 2,2,7-trimethylguanosine (TMG). In Saccharomyces cerevisiae, 24 U snRNA species have been identified; these are approximately 100-fold less abundant than their metazoan counterparts, range in size from 70 to 1,000 nt, and, except for the U3 analog, are encoded by single-copy genes (1,36,43,59). Several of the yeast U RNA genes have been shown genetically to be necessary for cellular viability (3,8,22,38,50,51 ble by autoimmune antisera having Sm specificity (44). These Sm snRNPs play essential roles in the processing of nuclear mRNA precursors in both metazoa and yeasts; in vitro, each is required for formation of the 40-to 60S pre-mRNA splicin...

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.

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Highlights d Loss of the repair protein kinase ATR in Trypanosoma brucei is lethal d Loss of T. brucei ATR alters VSG coat expression needed for immune evasion d Monoallelic RNA polymerase I VSG expression is undermined by ATR loss d ATR loss leads to expression of subtelomeric VSGs, indicative of recombination