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Discovery of a splicing regulator required for cell cycle progression

ABSTRACT: Toxoplasma gondii temperature sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. We describe transcriptome of the temperature sensitive mutant 12-109C6. Transcriptome studies demonstrated that gene expression is largely downregulated in the mutant at the restrictive temperature 40oC and is accompanied by a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs.

ORGANISM(S): Toxoplasma gondii

PROVIDER: GSE43315 | GEO | 2013/01/08

SECONDARY ACCESSION(S): PRJNA185412

REPOSITORIES: GEO

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Project description:Toxoplasma gondii is an obligate intracellular protozoan parasite whose rapid lytic replication cycles define its pathogenicity. We identified a temperature sensitive growth mutant, FV-P6, which irreversibly arrests before the middle of the G1 stage of the tachyzoite cell cycle. This arrest is caused by a point mutation in a gene conserved across eukaryotes, Cactin, whose product localizes to the nucleus. To elucidate the role of TgCactin we performed genome-wide expression profiling of FV-P6 mutant parasites at 35C and 40C, as well as FV-P6 complemented with a wild-type Cactin encoding cosmid (TOXO93 or comp) and as a pseudo-diploid wild-type line encoding both the wild-type and mutant Cactin locus (S4.2), also at both temperatures. Besides the expected G1 expression profile, many genes associated with the extracellular state as well as with the bradyzoite cyst stage were identified. Consistent with these profiles were the expression of AP2 transcription factors typically associated with extracellular and bradyzoite stage parasites. This suggests a role for TgCactin in control of gene expression. Since TgCactin does not contain any functionally defined domains we reasoned TgCactin exerts its function through interactions with other proteins. In support of this model we demonstrated that TgCactin is present in a protein complex and can oligomerize. Taken together, these results suggest that TgCactin acts as a pivotal protein potentially regulating gene expression at several transition points in parasite development. The role of Cactin in G1 progression of Toxoplasma gondii tachyzoites was assessed by expression profiling a temperature senstive mutant of Cactin at both the permissive (35C) as well as the restrictive (40C) temperature. Controls under the same temperature conditions inlcude the mutant line complemented with a wild-type Cactin allele encoded on a cosmid and a wild-type parasite line expressing an additional, mutant allele of Cactin (pseudo-diploid line).

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Discovery of a splicing regulator required for cell cycle progression

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