Molecular analysis of transcription termination by RNA polymerase II in trypanosomatids

The National Institute of Allergy and Infectious Diseases awarded the University of Georgia Research Foundation, Inc. $675,790 on August 7, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to conduct molecular analysis of transcription termination by RNA polymerase II in trypanosomatids.

The research investigates epigenetic mechanisms of gene expression control in Trypanosoma brucei and Leishmania species, protozoan parasites that cause human African sleeping sickness and leishmaniasis. The principal objective is to determine the mechanism by which base J and histone H3 variant regulate RNA polymerase II transcription termination through a torpedo-exonuclease pathway involving protein phosphatase dephosphorylation of the polymerase C-terminal domain and the SPT5 elongation factor. The work centers on characterizing the PJW/PP1 protein phosphatase complex, specifically the roles of regulatory subunits PNUTS, WDR82, and the J-binding protein JBP3 in coordinating termination events. Understanding these regulatory mechanisms has implications for parasite viability and virulence, advancing knowledge toward long-term control of trypanosomatid-caused diseases.

Performance occurs in Athens, Georgia. The period of performance runs from August 7, 2026, through July 31, 2031.

Abstract

Trypanosoma brucei is a single cell protozoan parasite that causes human african sleeping sickness, that is fatal if untreated. Related trypanosomatid pathogens, including numerous leishmania species, cause even more morbidity and mortality worldwide. The regulation of gene expression is essential for parasite development and survival in their sequential host environments. Exactly how trypanosomatid gene expression is regulated is currently not well understood, but the relative contribution of transcriptional control was thought to be absent. We have demonstrated a role for a modified DNA base, called base j, and histone H3 variant (H3V) in the regulation of RNA polymerase II (POL II) transcription termination in t. Brucei and l. Major. Furthermore, defects in POL II termination lead to gene expression changes impacting parasite viability and virulence. Our long-term goal is to dissect epigenetic mechanisms of gene expression control in trypanosomatids, and consequences on parasite biology. The objective of this grant is to determine the mechanism of POL II transcription termination in t. Brucei. The central hypothesis is that base j/H3V regulates termination via controlling specific stages of a "torpedo-PP1" termination mechanism. The torpedo component involves 5'-3' exonuclease (xrnd), and an RNA binding protein (RBP33); enhanced by POL II pausing involving dephosphorylation of the c-terminal domain (CTD) of POL II and elongation factor SPT5 by a PP1 protein phosphatase. We recently identified a PP1 complex (PJW/PP1) composed of regulatory subunits pnuts, WDR82 and a j-binding protein (JBP3) and illustrated a link between dephosphorylation of POL II by the PP1-pnuts complex and termination in t. Brucei and leishmania. We also identified p-sites in TBSPT5 involved in termination. These data led to the current model where base j (via JBP3) recruits the PJW/PP1 complex at termination sites and PP1 dephosphorylates POL II CTD and SPT5, pausing elongation allowing the torpedo mechanism to efficiently dissociate POL II. Our specific aims will test the following hypothesis: aim 1 will identify the phosphorylated sites in SPT5 and the POL II-CTD that are targeted by PP1-pnuts using a combination of in vitro and in vivo analyses. We will also identify other substrates of PP1-pnuts involved in termination. Aim 2 will directly test our model by assessing the role of these protein factors in transcription termination in t. Brucei. This will include elucidating the role of PP1-medited dephosphorylation of specific sites in POL II-CTD and SPT5 in the transcription cycle. Aim 3 will characterize the PJW/PP1 complex, including the roles of pnuts and JBP3 as the assembly/PP1 regulatory and chromatin recruitment factor, respectively. The proposed work will provide a fundamental understanding trypanosomatid gene expression, yielding a comprehensive view of the role of transcriptional control and clues to IT mechanisms. These studies may prove useful in identifying novel approaches to prevention, treatment and diagnosis of the debilitating and deadly diseases caused by these parasites.