Authors

J Budzak¹; G Rudenko¹;  ¹ Imperial College London, UK

Discussion

A Variant Surface Glycoprotein (VSG) coat protects bloodstream form Trypanosoma brucei within the mammalian host. Prodigious amounts of VSG mRNA (~7-10% total) are generated by RNA polymerase I (Pol I), from a single VSG gene located within one of ~15 VSG expression sites (ES). Transcription of VSG-ESs occurs in an extra-nucleolar Pol I body called the Expression Site Body (ESB). Importantly, T. brucei must maintain very high levels of VSG expression, as VSG is essential for proliferation both in vitro and in vivo. Trypanosomes use Pol I to transcribe their ESs, presumably as Pol I has a higher rate of initiation than Pol II. It has been assumed that ESs are either fully transcribed or silent. However using a variety of different microscopy approaches, we have consistently observed that not all cells contain an ESB, indicating that transcription of the active ES may be heterogeneous. We therefore investigated this by generating cell lines where constructs containing MS2 sequences were inserted at less than 2, 6, 10, 20, 40 and 60 kb downstream of the active ES promoter.
Using single molecule RNA-FISH experiments, we now show that Pol I transcription elongation is not always fully processive at the active ES in individual cells. We find that transcription elongation is gradually attenuated as distance from the promoter increases. Interestingly, we find a significant reduction in transcription elongation after the first ~10kb of the active ES. In addition, across the length of the active ES, we find the highest pre-mRNA levels at the active ES promoter and the lowest pre-mRNA levels immediately upstream of the telomeric VSG. We have recently discovered that a number of nuclear splicing condensates are positioned in close proximity to the VSG at the active ES telomere. Possibly these boost VSG mRNA expression even in the absence of fully processive Pol I transcription. These unexpected observations challenge the paradigm that RNA Polymerase I provides continuously high levels of transcription throughout the active ES in T. brucei.