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.
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| Gene | Phenotype | Experiment Type | Mutant Information | Strain Background | Chemical | Details |
|---|---|---|---|---|---|---|
| SEN1 | cold sensitivity: increased | classical genetics | conditional Allele: sen1-Δ4-1003 N terminal residues 4-1003 deleted | Other | Temperature: reduced temperature, 16 °C Details: mild cold sensitivity; also observed for sen1-delta4-964 | |
| SEN1 | cold sensitivity: increased | classical genetics | conditional Allele: sen1-Δ1859-2231 C terminal residues 1859 to end deleted | Other | Temperature: reduced temperature, 16 °C Details: severe sensitivity; also observed at 23 deg C | |
| SEN1 | heat sensitivity: increased | classical genetics | conditional Allele: sen1-Δ4-1003 N terminal residues 4-1003 deleted | Other | Temperature: elevated temperature, 37 °C Details: strong heat sensitivity; also observed for sen1-delta4-964 | |
| SEN1 | heat sensitivity: increased | classical genetics | conditional Allele: sen1-Δ1859-2231 C terminal residues 1859 to end deleted | Other | Temperature: elevated temperature, 37 °C Details: severe growth defect at 37 deg C, but also grows somewhat slowly at 30 deg C | |
| SEN1 | heat sensitivity: increased | classical genetics | conditional Allele: sen1-E1597K predicted salt bridge residue with R1641 | Other | Temperature: semi-permissive temperature, 33.5 °C Details: heat sensitivity somewhat suppressed as double mutant with sen1-R1641E | |
| SEN1 | heat sensitivity: increased | classical genetics | conditional Allele: sen1-R1641E predicted salt bridge residue with sen1-E1597K | Other | Temperature: semi-permissive temperature, 33.5 °C Details: heat sensitivity somewhat suppressed as double mutant with sen1-E1597K | |
| SEN1 | heat sensitivity: increased | classical genetics | conditional Allele: sen1-W1166S analogous to mutation in SETX implicated in ataxia oculomotor apraxia type 2 | Other | Temperature: elevated temperature, 37 °C | |
| SEN1 | heat sensitivity: increased | classical genetics | conditional Allele: sen1-T1779P analogous to mutation in SETX implicated in ataxia oculomotor apraxia type 2 | Other | Temperature: elevated temperature, 37 °C | |
| SEN1 | heat sensitivity: increased | classical genetics | conditional Allele: sen1-K1788E analogous to mutation in SETX implicated in ataxia oculomotor apraxia type 2 | Other | Temperature: elevated temperature, 37 °C | |
| SEN1 | heat sensitivity: increased | classical genetics | conditional Allele: sen1-N1413S analogous to mutation in SETX implicated in ataxia oculomotor apraxia type 2 | Other | Temperature: elevated temperature, 37 °C |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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