Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes


Autoři: Rudo Kieft aff001;  Yang Zhang aff001;  Alexandre P. Marand aff002;  Jose Dagoberto Moran aff001;  Robert Bridger aff001;  Lance Wells aff001;  Robert J. Schmitz aff002;  Robert Sabatini aff001
Působiště autorů: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, United States of America aff001;  Department of Genetics, University of Georgia, Athens, Georgia, United States of America aff002
Vyšlo v časopise: Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes. PLoS Genet 16(2): e32767. doi:10.1371/journal.pgen.1008390
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008390

Souhrn

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.

Klíčová slova:

DNA transcription – DNA-binding proteins – Gene expression – Messenger RNA – RNA interference – Transcriptional control – Transcriptional termination – Trypanosoma brucei gambiense


Zdroje

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