Identification of a Novel Base J Binding Protein Complex Involved in RNA Polymerase II Transcription Termination in Trypanosomes

PLoS Genet. 2020 Feb 21;16(2):e1008390. doi: 10.1371/journal.pgen.1008390. eCollection 2020 Feb.

Abstract

Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • DNA, Kinetoplast / genetics
  • DNA, Kinetoplast / metabolism*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Genes, Protozoan
  • Glucosides / metabolism
  • Glucosyltransferases / genetics
  • Glucosyltransferases / metabolism
  • Histones / genetics
  • Histones / metabolism
  • Leishmania / physiology
  • Mutation
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • RNA Interference
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism*
  • Thymine / metabolism
  • Transcription Termination, Genetic*
  • Trypanosoma brucei brucei / physiology*
  • Uracil / analogs & derivatives
  • Uracil / metabolism

Substances

  • DNA, Kinetoplast
  • DNA-Binding Proteins
  • Glucosides
  • Histones
  • Protozoan Proteins
  • 5-((glucopyranosyloxy)methyl)uracil
  • Uracil
  • Glucosyltransferases
  • RNA Polymerase II
  • Thymine

Grant support

This work was supported by the National Institutes of Health [grant number AI109108](to R.S). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.