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.