Discussion

The mechanism governing gene expression regulation along the proliferative cycle of Trypanosoma cruzi are still poorly understood. In view of the biological and therapeutic relevance of the parasite replication we sought to apply RNA-seq approaches to identify global gene expression patterns during the progression of the parasite cell cycle. For that purpose, we deep sequenced the polyA-RNAs (transcriptome) and the ribosome footprints (translatome) of different cell cycle phases. We analyzed epimastigotes of a TcI strain synchronized with hydroxyurea, obtaining cell populations in G1, S and G2/M phases (at 70% enrichment). We extracted RNA and prepared RNA-seq libraries for NGS. We found 305 differentially expressed mRNAs (DEGs) (fold change>1.5, p-value <0.01) in the total RNA fraction. These transcriptomic changes involve proteins dedicated to carbohydrates metabolism and energy production at G1-phase (70 genes), DNA and chromatin replication at S-phase (97 genes) and microtubules-based movement at G2/M-phase (138 genes). For the ribosome profiling, we only studied the G1-S transition. Interestingly, translational regulation affects more than 1150 genes at >2.0-fold change, 20% and 80% of which were up-regulated in G1 and S-phase respectively. Enriched molecular functions in the latter dataset include ribosome synthesis, nucleotide metabolism and microtubule dynamics. We found specific sequence and structural RNA motifs in the UTRs, including the known CS sequence responsible for the periodical expression of some mRNAs. Several known as well as novel RNA binding proteins are found to periodically modify the abundance of their mRNA; indeed, some are predicted to bind the enriched RNA sequence motifs.