Functional genomic characterization of mRNAs associated with TcPUF6, a pumilio-like protein from Trypanosoma cruzi.
Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. The PUF (Pumilio/FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypanosomatids. We used a ribonomic approach to identify the putative target mRNAs associated with TcPUF6, a member of the T. cruzi PUF family. TcPUF6 is expressed in discrete sites in the cytoplasm at various stages of the parasite life cycle and is not associated with the translation machinery. The overexpression of a tandem affinity purification-tagged TcPUF6 protein allowed the identification of associated mRNAs by affinity purification assays and microarray hybridization yielding nine putative target mRNAs. Whole expression analysis of transfected parasites showed that the mRNAs associated with TcPUF6 were down-regulated in populations overexpressing TcPUF6. The association of TcPUF6 with the TcDhh1 helicase in vivo and the cellular co-localization of these proteins in epimastigote forms suggest that TcPUF6 promotes degradation of its associated mRNAs through interaction with RNA degradation complexes. Analysis of the mRNA levels of the putative TcPUF6-regulated genes during the parasite life cycle showed that their transcripts were up-regulated in metacyclic trypomastigotes. In these infective forms no co-localization between TcPUF6 and TcDhh1 was observed. Our results suggest that TcPUF6 regulates the half-lives of its associated transcripts via differential association with mRNA degradation complexes throughout its life cycle.
Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader 3775, Curitiba, PR, Brazil.
Bruno
B
Dallagiovanna
Alejandro
A
Correa
Christian M
CM
Probst
Fabiola
F
Holetz
Pablo
P
Smircich
Alessandra Melo
AM
de Aguiar
Fernanda
F
Mansur
Claudio Vieira
CV
da Silva
Renato A
RA
Mortara
Beatriz
B
Garat
Gregory A
GA
Buck
Samuel
S
Goldenberg
Marco A
MA
Krieger
D000818:Animals; D003593:Cytoplasm / Q000378:metabolism; D015536:Down-Regulation; D018503:Genome, Protozoan / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D011485:Protein Binding; D020365:RNA Helicases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D015854:Up-Regulation
D012333:RNA, Messenger; D016053:RNA, Protozoan; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D020365:RNA Helicases