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

Abstract

19 Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA 20 Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how 21 specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription 22 termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-23 glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass 24 spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein 25 (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate 26 JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, 27 JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian 28 PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. 29 Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of 30 PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in 31 Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of promoters, 32 suggesting an additional role of the complex in regulating termination of bi-directional transcription. In 33 addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes important for 34 host immune evasion. Our results provide the first direct mechanistic link between base J and regulation 35 of Pol II termination and suggest a novel molecular model for the role of the CTD of Pol II in terminating 36 polycistronic transcription in trypanosomatids. 37 38 Author Summary 39 Trypanosoma brucei is an early-diverged parasitic protozoan that causes African sleeping 40 sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain 41 multiple genes that are co-transcribed from a single promoter. We have recently described the presence 42 of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene 43 clusters where the loss of base J and H3.V leads to read-through transcription and the expression of 44 downstream genes. We now identify a novel stable multimeric complex containing a J binding protein . CC-BY 4.0 International license is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/753004 doi: bioRxiv preprint 45 (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) 46 and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved 47 in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead 48 to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex reg...