NIH grant: Molecular analysis of transcription termination by RNA polymerase II in trypanosomatids (1R01AI195646-01A1)

retained text

NIH grant 1R01AI195646-01A1: Molecular analysis of transcription termination by RNA polymerase II in trypanosomatids
Institute: {'code': 'AI', 'abbreviation': 'NIAID', 'name': 'National Institute of Allergy and Infectious Diseases', 'admin_org_id': '12500003', 'admin_funding_url': ''}. PI: ROBERT S SABATINI. Award: $675790.
Start: 2026-08-07T00:00:00.
Project Summary
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.

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