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

Rudo Kieft; Yang Zhang; Alexandre P. Marand; Jose Dagoberto Moran; Robert Bridger; Lance Wells; Robert J. Schmitz; Robert Sabatini

297 This is consistent with one of three replicates of LtJGT purification that resulted in all components of the 298 complex except PP1 (S1 Table) , suggesting PP1 is the least stable component of the Leishmania 299 complex. It has been demonstrated that the phosphorylation of Ser residue(s) within or close to the RVxF 300 motif of PP1 regulatory subunits, including PNUTS, disrupts the binding of this motif to PP1 (56-59). The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/753004 doi: bioRxiv preprint 323 PJW/PP1 complex, we analyzed inducible RNAi ablation of Wdr82, JBP3, and PNUTS in BSF T. brucei. 324 As shown in Fig 3, induction of RNAi against Wdr82, JBP3 and PNUTS and ablation of mRNA levels from 325 30-60% leads to reduced parasite growth, indicating the essential nature of these genes and thus the 326 PJW/PP1 complex in BSF T. brucei. We also detect evidence of read-through transcription at the 327 representative PTU internal termination site on chromosome 5 upon ablation of the three factors. RT-PCR 328 using oligos flanking the termination site (see diagram, Fig 3A) detects increased RNA upon ablation of 329 PNUTS, Wdr82 and JBP3 ( Fig 3C) . As a control a separate RT-PCR utilized the same 5' primer and a 3' 330 primer immediately upstream of the termination site. We have previously shown that an RNA species 331 spanning the termination site is indicative of read-through transcription and is only detected following the 332 loss of base J or H3.V, due to continued transcription elongation at termination sites (21, 24, 25).