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

The derepression of ESAG 6 and 7 genes adjacent to the BES promoter along with the VSG ~40 566 kb downstream (S2 Table) , suggests that PJW/PP1 may function throughout the telomeric PTU. To 567 examine this more closely, we analyzed the RNA-seq reads mapping to the 14 telomeric BES sequences 568 (80). RNA-seq reads mapping to BES were counted in 200bp windows with a 100bp steps. Read counts 569 were converted into reads per million (RPM) and compared between +/-Tet to estimate log2 fold change 570 and plotted in Fig 7C and S11 Fig. The location of BES promoters is indicated by an arrow. The 571 transcription of the active BES1 was not affected by the loss of TbPNUTS. However, when the remaining 572 13 silent BESs are analyzed we see derepression of ESAGs as well as the terminal VSG. In some cases, 573 it seems that derepression extends 10-20 kb from the promoter to express ESAG 6 and 7, with no . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 574 significant effect on the remaining genes (ESAGs) within the silent BES, and derepression of the VSG at 575 the 3' end. In other cases there is selective upregulation of the VSG gene, including the pseudo VSGs 576 that are present upstream of some telomeric VSGs. The apparent selective VSG upregulation may be 577 due to the combined effect of low level transcription of the entire BES and enhanced stability of VSG 578 mRNAs. For example, the increased VSG mRNA half-life (4.5 hrs) compared with ESAG 6 and 7 (1.8-2.8 579 hrs) (81, 82). Transcripts of genes located approximately 5 kb upstream of the VSG have also been 580 shown to be selectively rapidly degraded, presumably by nonsense-mediated decay (83). We also 581 noticed that increased levels of RNA close to the promoter are significantly higher when there is an 582 additional BES promoter upstream for ESAG10. In fact, the most significant gene expression changes are 583 in BESs that have dual promoters (S2 Table) . These data would suggest derepression of telomere-584 proximal VSG genes after PNUTS depletion is due to transcriptional activation of silent Pol I promoters. 585 However, these results are also consistent with increased Pol I elongation along the BES. Repression of 586 silent ESs is mediated at least in part by the inhibition of Pol I elongation within the BES preventing the 587 production of VSG mRNA from the silent BESs (84-86). Similar to its inhibition of Pol II transcription 588 elongation at termination sites at the 5' and 3' end of PTUs genome-wide, PNUTS may also function at 589 telomeric regions to attenuate Pol I transcription elongation within the silent ESs. The data here suggest 590 PJW/PP1 controls VSG silencing at BESs by regulating Pol I elongation (termination) and/or regulating 591 access of the polymerase to silent promoter regions.