Senataxin homologue Sen1 is required for efficient termination of RNA polymerase III transcription

Abstract

R‐loop disassembly by the human helicase Senataxin contributes to genome integrity and to proper transcription termination at a subset of RNA polymerase II genes. Whether Senataxin also contributes to transcription termination at other classes of genes has remained unclear. Here, we show that Sen1, one of two fission yeast homologues of Senataxin, promotes efficient termination of RNA polymerase III (RNAP3) transcription in vivo. In the absence of Sen1, RNAP3 accumulates downstream of RNAP3‐transcribed genes and produces long exosome‐sensitive 3′‐extended transcripts. Importantly, neither of these defects was affected by the removal of R‐loops. The finding that Sen1 acts as an ancillary factor for RNAP3 transcription termination in vivo challenges the pre‐existing view that RNAP3 terminates transcription autonomously. We propose that Sen1 is a cofactor for transcription termination that has been co‐opted by different RNA polymerases in the course of evolution.

RNA polymerase backtracking results in the accumulation of fission yeast condensin at active genes

“…In the absence of Sen1, condensin did not accumulate either at COC sites (Fig 2A), which recruit TFIIIC but not RNAP3 (Noma et al , 2006), consistent with a transcription-mediated effect. To further determine whether the accumulation of condensin was mechanistically linked to the transcription termination defects observed in the absence of Sen1, we corrected those defects by strengthening the terminator sequences at two tRNA genes by inserting long polyT sequences, as described previously (Rivosecchi et al , 2019). As expected, this strategy was sufficient to correct the accumulation of RNAP3 downstream of the terminator sequences in mitotic cells lacking Sen1 (Fig 2CD, lower panels).…”

“…CD. Distribution of condensin (cnd2-GFP, top) and RNAP3 (rpc37-flag, bottom) around SPCTRNATHR.10 ( C ) and SPCTRNAARG.10 ( D ) in mitotic cells, in the presence or not of super-terminator sequences ( thr10-20T and arg10-23T respectively) which correct the transcription termination defects in the absence of Sen1 (Rivosecchi et al , 2019) (compare the yellow and red curves). Results are presented as (mean ± std) of 3 ( C ) or 4 ( D ) biological replicates.…”

“…Because backtracking is a prominent feature in the 3’ end of genes (Sheridan et al , 2019; Lemay et al , 2014), this might explain why condensin accumulates particularly over the 3’ of transcriptionally active genes in fission yeast (Toselli-Mollereau et al , 2016; Sutani et al , 2015). In the absence of Sen1, RNAP3 accumulates strongly over and downstream of class III genes (Rivosecchi et al , 2019). We speculate that those accumulated RNAP3 molecules are also often backtracked and that the increased levels of condensin downstream of class III genes in the absence of Sen1 depend on the size and the density of the domain occupied by these read-through polymerases.…”

“…Chromatin Immunoprecipitation (ChIP). ChIP was carried out as described previously (Rivosecchi et al, 2019), using the primers listed in Table EV2. GFP-tagged proteins were immuno-precipitated with the A11122 antibody (Thermo Fisher Scientific); Myc-tagged proteins were immuno-precipitated with the 9E10 antibody (Merck); Rpb1 was immunoprecipitated using the 8WG16 antibody (Merck).…”

A pan-plant protein complex map reveals deep conservation and novel assemblies

“…Some of these eukaryotic complexes, such as the Conserved Oligomeric Golgi (COG) complex, the SRP68/72 heterodimer, and the TRAPP and BRISC complexes have to our knowledge only previously been inferred in plants by gene content. Similarly, we ind orthologs of complex members that have only been reported in non-plant species, such as a MAA3 (a plant ortholog of the yeast protein Sen1 and human Senataxin) interacting with RNA polymerase III, an interaction recently found in yeast to regulate RNA polymerase III termination (Rivosecchi et al, 2019) .…”