Genetic mapping of nuclear suppressors of splicing-deficient chloroplast introns, and a novel rhodanese-domain protein required for chloroplast translation in Chlamydomonas
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Although many group I (GI) introns can self-splice in vitro, their splicing is promoted by proteins in vivo. Only a few splicing factors that specifically promote GI intron splicing have been identified, however, none are from chloroplasts, which is the subject of this study. In previous work from our lab, a strategy was developed to identify splicing factors for chloroplast GI introns of Chlamydomonas by using suppressor genetics. A mutant with reduced splicing of the chloroplast 23S rRNA intron (Cr.LSU) was generated. Then, 3 nuclear suppressors (7120, 71N1 and 7151) with substantially restored splicing of Cr.LSU were isolated and partially characterized. However, the suppressor gene(s) were not identified. In this study, I have used genetic mapping to make a renewed attempt to isolate these genes. Using polymorphisms between the 137C strain that was used for suppressor isolation, and a new strain of C.reinhardtii (S1D2), the nuclear suppressor mutations in 7120 and 71N1 were mapped to a region on chromosome III that is essentially devoid of recombination. Based on the recombination maps, the suppressor gene in 7120 is located within a ~418-kb region from bp 2,473,064 to 2,891,232, whereas the suppressor in 71N1 is likely located within a ~236-kb subregion from bp 2,473,064 to 2,709,377. It is possible that these mutations are in the same gene; however, the maps could not be refined further due to the lack of recombination in this 418-kb region. I also attempted to compare the genomic sequence of the 7120 suppressor, which was obtained by next-generation sequencing, with the Chlamydomonas reference genome (JGI, v.4). Next-generation sequencing of 7120 revealed the existence of abundant repetitive sequences and transposable elements clustered in a ~40-kb subregion of the recombinationally suppressed 418-kb region on chromosome III. I suggest that the high frequency of repetitive sequences and transposable elements in this region may be the reason for the suppressed recombination. Searching for candidate genes in the mapped region led me to examine a novel protein that was predicted to have a putative chloroplast transit-peptide, and an RNA binding domain. Further bioinformatic analysis revealed a single rhodanese domain with an active-site cysteine. The protein was expressed in E.coli as the full-length and predicted mature forms, plus a small His-tag. The purified mature protein had rhodanese catalytic activity, based on the fact that it was able to transfer sulfur from thiosulfate to cyanide. Also, western blot analysis with a polyclonal antibody produced in rabbits showed that the cellular protein migrated on SDS gels close to the mature protein and faster than the full-length protein, indicative of an organelle-targeted protein. The antibody also showed that the cellular protein co-fractionated with chloroplasts. To gain insight into its in vivo function, the gene was knocked down using the tandem RNAi system (Rohr et al., 2004), which produced strains (5) with reductions of 31% to 76% in the mRNA level, and ~30% to ~60% in the protein level. These strains were sensitive to bright light, and had reduced rates of growth under all conditions, which are characteristics of chloroplast translation mutants. Thus, chloroplast protein synthesis was examined by radioisotope pulse-labeling in the presence of cycloheximide, which showed that the RNAi strains were broadly and negatively affected, and RT-PCR and northern blot revealed only normal chloroplast mRNA levels. These data have identified a new rhodanese-family enzyme that is required for chloroplast translation, which I have designated “CRLT”, for chloroplast rhodanese-like translation.