Multiple Functions of Aconitase-2 in Schizosaccharomyces pombe

Abstract

Aconitase functions as an enzyme in TCA cycle (Krebs cycle), converting citrate to isocitrate in bacteria and mitochondria of eukaryotes. In many organisms, aconitase serves additional roles, being a nucleic acids binding protein. In mammals and prokaryots, aconitases are known to bind RNA as well. DNA binding has been demonstrated in Saccharomyces cerevisiae. In fission yeast Schizosaccharomyces pombe, the aco2+ gene encodes a fusion protein between aconitase and a putative mitochondrial ribosomal protein bL21 (Mrpl49). In this study, the expression of the aco2+ gene to transcripts and protein products was analyzed. Two types of aco2+ transcripts were generated via alternative poly (A) site selection, producing both a single aconitase domain protein and the fusion form. The bL21-fused aconitase-2 Aco2 protein resides in mitochondria as well as in cytosol and the nucleus. In mitochondria, Aco2 is needed for mitochondrial translation. The viability defect of aco2 mutation was complemented not by the aconitase domain but by the bL21 domain, which enables mitochondrial translation. This suggests that essentiality of Aco2 protein is due to its role in mitochondrial translation. Based on the localization of Aco2 in the nucleus, novel nuclear functions of Aco2 were investigated. There has been a report from genome-wide genetic screenings that aconitase could be involved in RNAi pathway. Intrigued by this observation, involvement of Aco2 in heterochromatin formation in S. pombe was examined. Genetic and physical interaction of Aco2 with heterochromatin assembly factors and its effect on modulating transcription from the centromeric and subtelomeric regions were investigated. Loss of nuclear Aco2 (aco2ΔN) restored the defects of RNAi mutants, such as Δago1 and Δdcr1, in forming heterochromatin in the centromeric region. However, the aco2ΔN mutation did not restore the defect of Δchp1. Chp1, a component of RITS (RNA induced transcriptional silencing) complex, directly interacted with Aco2, especially through the chromodomain as monitored by GST pull-down assay. ChIP analysis demonstrated that Chp1 can recruit Aco2 to the centromeric region. RNA-IP assay showed that Aco2 can bind to the centromeric noncoding RNA from the repeat (dg/dh) region in a Chp1-dependent manner. These results support a model that Aco2 binds Chp1 through the chromodomain and deters Chp1 from being recruited to chromosome in an RITS complex-independent manner, and hence inhibits heterochromatin formation. Actually in the aco2ΔN mutant, the H3K9me2 level in the centromere core region that does not form heterochromatin is elevated compared to the wild-type cell. Therefore, it can be postulated that Aco2 inhibits Chp1 recruitment where RITS complex does not exist, so that heterochromatin may form in the right place. To modulate centromeric heterochromatin formation, the full-length Aco2 protein with both aconitase and bL21 domains are required as well as the three cysteine residues for [FeS] ligation. Aco2 (aco2ΔN) also restored the phenotype of elevated RNA level in Δswi6 mutant, one of the HP1 protein which can bind to H3K9me2. But unlike RNAi mutants, functional heterochromatin was not restored. Even though interaction between Aco2 and Swi6 was not detected by co-IP. It was monitored that Aco2 directly interacted with Swi6 hinge domain by GST pull-down assay. It is known that the hinge domain of Swi6 has RNA binding activity, so there is a possibility that RNA may interfere interaction between Aco2 and Swi6. Actually when RNase was treated in pull-down assay, interaction between two proteins was enhanced, as expected. Aco2 also interacted with Rrp6, a key component of a RNA exosome, that plays a role in supporting transcription of the dg/dh-like repeat-containing tlh1+ gene in the subtelomeric region. Involvement of Aco2 in the heterochromatin formation in the mating type locus has also been examined by monitoring mating type switch. Iodine staining of the homothallic h90 aco2ΔN cells resulted in pale color, suggesting that Aco2 may also function in mating type switching, possibly via the heterochromatin formation. Taken together, this study revealed novel functions of Aco2 in the nucleus, related with heterochromatin formation and transcriptional modulation.