Sunday, April 27, 2025
4/27/2025
Alternative polyadenylation regulation in Trypanosoma brucei - Human African Trypanosomiasis (HAT), and the related livestock disease, nagana, are caused by infection of the kinetoplastid parasite, Trypanosoma brucei. These maladies cause devastating health and economic impacts in sub-Saharan Africa. Kinetoplastids, including T. brucei, T. cruzi and Leishmania spp., exhibit many novel biological features, such as U insertion/deletion editing and universal trans-splicing of mRNAs. Thus, understanding the basic biology of these parasites is a cornerstone on the path to discovery of unique biological processes that could potentially serve as much-needed new drug targets. One novel kinetoplastid feature is the almost complete lack of transcriptional control of gene expression. Instead, these parasites constitutively generate long polycistronic transcripts that are resolved into monocistrons by 5’ trans-splicing and 3’ cleavage and polyadenylation (PA). The resulting obligate posttranscriptional gene regulation relies on RNA binding proteins (RBPs), and RBP regulation of transcript fate is mediated by their binding to cis-acting sequence elements typically located in mRNA 3’ untranslated regions (UTRs). Transcriptome-wide studies in T. brucei revealed widespread alternative PA (APA) sites on many transcripts. APA is significant because use of different PA sites can lead to inclusion/exclusion of critical 3’UTR elements that regulate transcript stability or translation efficiency. Despite the potential for APA to profoundly affect gene expression, nothing is known regarding the mechanisms that control APA in kinetoplastids, constituting a major gap in our knowledge. We discovered that knockdown of the T. brucei RBP, DRBD18, leads to substantial changes in APA, marking DRBD18 as the first factor in trypanosomes known to modulate APA, and highlighting its potential to control inclusion/exclusion of distinct cis-regulatory 3’UTR elements. Single cell RNAseq in DRBD18-replete vs. -depleted cells analyzed using different bioinformatic platforms revealed that distinct 3’ UTR variants, generated by DRBD18-mediated APA, are more important drivers of cell identity than is overall transcript abundance. These findings underpin our central hypothesis that DRBD18 regulates APA by controlling access of the cleavage/PA machinery to distinct 3’ UTR sites and, in this manner, dramatically influences cell identity. To test this hypothesis, we will (1) Identify DRBD18-regulated 3’UTR variants associated with alterations in cell identity; (2) Define transcriptome-wide whether DRBD18-mediated APA results in altered trans-splice sites on downstream open reading frames or whether it leads to generation of new trans-spliced and polyadenylated intergenic fragments, thereby informing its mechanism of action; (3) Determine the transcriptome-wide effect of DRBD18 on cleavage/PA machinery RNA binding. The proposed studies are significant because they will fill major gaps in our knowledge regarding fundamental aspects of mRNA processing, RBP regulation of APA, and the functional consequences of APA in controlling cell-to-cell heterogeneity in this medically and economically important parasite.
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