mRNA Polyadenylation Machineries in Intestinal Protozoan Parasites.

In humans, mRNA polyadenylation involves the participation of about 20 factors in four main complexes that recognize specific RNA sequences. Notably, CFIm25, CPSF73, and PAP have essential roles for poly(A) site selection, mRNA cleavage, and adenosine residues polymerization. Besides the relevance of polyadenylation for gene expression, information is scarce in intestinal protozoan parasites that threaten human health. To better understand polyadenylation in Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum, which represent leading causes of diarrhea worldwide, genomes were screened for orthologs of human factors. Results showed that Entamoeba histolytica and C. parvum have 16 and 12 proteins out of the 19 human proteins used as queries, respectively, while G. lamblia seems to have the smallest polyadenylation machinery with only six factors. Remarkably, CPSF30, CPSF73, CstF77, PABP2, and PAP, which were found in all parasites, could represent the core polyadenylation machinery. Multiple genes were detected for several proteins in Entamoeba, while gene redundancy is lower in Giardia and Cryptosporidium. Congruently with their relevance in the polyadenylation process, CPSF73 and PAP are present in all parasites, and CFIm25 is only missing in Giardia. They conserve the functional domains and predicted folding of human proteins, suggesting they may have the same roles in polyadenylation.

The FIP-1 like polyadenylation factor in trypanosomes and the structural basis for its interaction with CPSF30.

In trypanosomes transcription is polycistronic and individual mRNAs are generated by a trans-splicing/polyadenylation coupled reaction. We identified a divergent trypanosome FIP1-like, a factor required for mRNA 3' end formation from yeasts to human. Here we showed that it is a nuclear protein with a speckled distribution essential for trypanosome viability. A strong interaction was found between TcFIP1-like and TcCPSF30, a component of the polyadenylation complex. We determined the specific amino acids in each protein involved in the interaction. Significant differences were found between the trypanosome interaction surface and its human counterpart. Although CPSF30/FIP1 interaction is known in other organisms, this is the first report mapping the interaction surface at the amino acid level.

Entamoeba histolytica: comparative genomics of the pre-mRNA 3' end processing machinery.

We report here the pre-mRNA 3' end processing machinery in Entamoeba histolytica. Comparative analysis of the putative sequences participating in the pre-mRNA 3' end processing of E. histolytica genes shows similitude and differences to those described for yeast and human transcripts. By a genomic survey, we identified 16 putative genes encoding for cleavage/polyadenylation factors in this parasite. E. histolytica pre-mRNA 3' end processing machinery does not seem to contain homologous genes coding for human Symplekin, CFIm59, and CFIm68 proteins, neither sequences related to yeast Pta1p and Hrp1p. Protein sequence comparisons among E. histolytica, yeast, and human showed little variation in their functional domains through evolutive scale. E. histolytica pre-mRNA 3' end processing machinery appears to be in an intermediate evolutionary position between mammals and yeast. From these analyses, we propose a hypothetical working model for the pre-mRNA 3' end processing machinery in E. histolytica.