Department of Genetics, Washington University School of Medicine, 4523 Clayton Avenue, St Louis, MO 63110, USA.
Department of Ophthalmology, Mount Sinai School of Medicine, New York, NY, USA.
Open Biol. 2018 Mar;8(3). doi: 10.1098/rsob.170211.
Intraflagellar transport moves proteins in and out of flagella/cilia and it is essential for the assembly of these organelles. Using whole-genome sequencing, we identified splice site mutations in two genes, () and (), which lead to flagellar assembly defects in the unicellular green alga The splicing defects in these mutants are partially corrected by mutations in two conserved spliceosome proteins, DGR14 and FRA10. We identified a deletion mutant, which suppresses the 3' splice site mutation in , and a frameshift mutant of , which suppresses the 5' splice site mutation in Surprisingly, we found and mutations suppress both splice site mutations. We suggest these two proteins are involved in facilitating splice site recognition/interaction; in their absence some splice site mutations are tolerated. Nonsense mutations in , which is involved in nonsense-mediated decay, lead to accumulation of aberrant transcripts and partial restoration of flagellar assembly in the mutants. The high density of introns and the conservation of noncore splicing factors, together with the ease of scoring the mutant phenotype, make an attractive organism to identify new proteins involved in splicing through suppressor screening.
鞭毛/纤毛内运输将蛋白运进和运出鞭毛/纤毛，这对于这些细胞器的组装是必不可少的。通过全基因组测序，我们在两个基因（ 和 ）中鉴定出剪接位点突变，这些突变导致单细胞绿藻 的鞭毛组装缺陷。在这些 突变体中，两个保守的剪接体蛋白 DGR14 和 FRA10 的突变部分纠正了剪接缺陷。我们鉴定出一个 缺失突变体，它抑制了 在 3'剪接位点的突变，以及一个 在 5'剪接位点的移码突变体，它抑制了 在 5'剪接位点的突变。令人惊讶的是，我们发现 和 突变都能抑制这两个剪接位点的突变。我们认为这两种蛋白参与促进剪接位点的识别/相互作用；在它们缺失的情况下，一些剪接位点突变是可以容忍的。参与无意义介导衰变的 的无意义突变导致异常转录本的积累，并在 突变体中部分恢复鞭毛组装。内含子的高密度和非核心剪接因子的保守性，加上易于评分 突变体表型，使 成为一个有吸引力的生物体，可以通过抑制筛选来鉴定新的参与剪接的蛋白。
