Saccharomyces cerevisiae Sen1 as a Model for the Study of Mutations in Human Senataxin That Elicit Cerebellar Ataxia

Abstract

The nuclear RNA and DNA helicase Sen1 is essential in the yeast Saccharomyces cerevisiae and is required for efficient termination of RNA polymerase II transcription of many short noncoding RNA genes. However, the mechanism of Sen1 function is not understood. We created a plasmid-based genetic system to study yeast Sen1 in vivo. Using this system, we show that (1) the minimal essential region of Sen1 corresponds to the helicase domain and one of two flanking nuclear localization sequences; (2) a previously isolated terminator readthrough mutation in the Sen1 helicase domain, E1597K, is rescued by a second mutation designed to restore a salt bridge within the first RecA domain; and (3) the human ortholog of yeast Sen1, Senataxin, cannot functionally replace Sen1 in yeast. Guided by sequence homology between the conserved helicase domains of Sen1 and Senataxin, we tested the effects of 13 missense mutations that cosegregate with the inherited disorder ataxia with oculomotor apraxia type 2 on Sen1 function. Ten of the disease mutations resulted in transcription readthrough of at least one of three Sen1-dependent termination elements tested. Our genetic system will facilitate the further investigation of structure-function relationships in yeast Sen1 and its orthologs.T RANSCRIPTION termination by eukaryotic RNA polymerase II (Pol II) uses at least two pathways, one that is coupled to cleavage and polyadenylation of the nascent transcript [the poly(A)-dependent pathway] and one that involves the activity of the RNA/DNA helicase Sen1 (the Sen1-dependent pathway) (Kuehner et al. 2011). The Sen1-dependent pathway was first identified in the budding yeast Saccharomyces cerevisiae and is responsible for transcription termination of many short, noncoding RNA genes, including small nuclear (sn) and small nucleolar (sno) RNAs (Winey and Culbertson 1988;Steinmetz and Brow 1996;Rasmussen and Culbertson 1998;Steinmetz et al. 2001). It also restricts the elongation and accumulation of pervasive cryptic unstable transcripts (Arigo et al. 2006b;Thiebaut et al. 2006) and regulates transcription of some protein-coding genes by premature termination, i.e., attenuation (Steinmetz et al. 2001;Arigo et al. 2006a;Steinmetz et al. 2006b;Jenks et al. 2008;Kuehner and Brow 2008). A set of core factors distinguish the Sen1-dependent pathway from the poly(A)-dependent pathway, including Sen1 and the RNA-binding proteins Nrd1 and Nab3 Brow 1996, 1998;Conrad et al. 2000;Steinmetz et al. 2001;Carroll et al. 2007). However, some short messenger RNA (mRNA) genes, such as CYC1, may have hybrid terminators that require factors from both pathways (Steinmetz et al. 2006b).S. cerevisiae Sen1 is a 252-kDa superfamily 1 helicase encoded by the essential SEN1 gene. Its helicase domain is located in the C-terminal half of the protein ( Figure 1A), and the N-terminal 975 amino acids are dispensable for viability (DeMarini et al. 1992). The ortholog of Sen1 in the fission yeast Schizosaccharomyces pombe has ATP-dependent, 59-to-39 DNA and RNA unw...

Sen1 has unique structural features grafted on the architecture of the Upf1‐like helicase family

“…Residues in the “brace” are important in vivo (Chen et al , 2014) and are highly conserved in Sen1 orthologues, including the human SETX, further supporting the importance of this region. From the structural and biochemical analysis, the “brace” appears to push the “barrel” in a position competent for RNA binding, similar to the effect exerted by the presence of the CH domain in Upf1.…”

“…ND, not done; HS, heat sensitive.aGrowth of yeast expressing the indicated version of Sen1 according to Chen et al (2014), except for ΔLP (see Fig EV4). …”

Saccharomyces cerevisiae Sen1 Helicase Domain Exhibits 5′- to 3′-Helicase Activity with a Preference for Translocation on DNA Rather than RNA

“…1A) (24). Based on alignment of the sequence of S. cerevisiae Sen1 with senataxin and its paralogs Upf1 and IGHMBP2 (Fig.…”

“…2). The sen1-K1363R mutation is recessive lethal in yeast, whereas the remaining alleles result in heatsensitive growth and defects in pol II termination (24).…”

“…In the absence of ATP, Sen1-HD bound strongly to all three trimers 3 X. Chen and D. A. Brow, unpublished data. (24). Overlapping arrowheads mark the two AOA2 substitutions that were tested for expression in E. coli (see text).…”