Nuclear RanGAP is required for the heterochromatin assembly and is reciprocally regulated by histone H3 and Clr4 histone methyltransferase in Schizosaccharomyces pombe.

Although the Ran GTPase-activating protein RanGAP mainly functions in the cytoplasm, several lines of evidence indicate a nuclear function of RanGAP. We found that Schizosaccharomyces pombe RanGAP, SpRna1, bound the core of histone H3 (H3) and enhanced Clr4-mediated H3-lysine 9 (K9) methylation. This enhancement was not observed for methylation of the H3-tail containing K9 and was independent of SpRna1-RanGAP activity, suggesting that SpRna1 itself enhances Clr4-mediated H3-K9 methylation via H3. Although most SpRna1 is in the cytoplasm, some cofractionated with H3. Sprna1(ts) mutations caused decreases in Swi6 localization and H3-K9 methylation at all three heterochromatic regions of S. pombe. Thus, nuclear SpRna1 seems to be involved in heterochromatin assembly. All core histones bound SpRna1 and inhibited SpRna1-RanGAP activity. In contrast, Clr4 abolished the inhibitory effect of H3 on the RanGAP activity of SpRna1 but partially affected the other histones. SpRna1 formed a trimeric complex with H3 and Clr4, suggesting that nuclear SpRna1 is reciprocally regulated by histones, especially H3, and Clr4 on the chromatin to function for higher order chromatin assembly. We also found that SpRna1 formed a stable complex with Xpo1/Crm1 plus Ran-GTP, in the presence of H3.

Schizosaccharomyces pombe RanGAP homolog, SpRna1, is required for centromeric silencing and chromosome segregation.

We isolated 11 independent temperature-sensitive (ts) mutants of Schizosaccharomyces pombe RanGAP, SpRna1 that have several amino acid changes in the conserved domains of RanGAP. Resulting Sprna1ts showed a strong defect in mitotic chromosome segregation, but did not in nucleocytoplasmic transport and microtubule formation. In addition to Sprna1+ and Spksp1+, the clr4+ (histone H3-K9 methyltransferase), the S. pombe gene, SPAC25A8.01c, designated snf2SR+ (a member of the chromatin remodeling factors, Snf2 family with DNA-dependent ATPase activity), but not the spi1+ (S. pombe Ran homolog), rescued a lethality of Sprna1ts. Both Clr4 and Snf2 were reported to be involved in heterochromatin formation essential for building the centromeres. Consistently, Sprna1ts was defective in gene-silencing at the centromeres. But a silencing at the telomere, another heterochromatic region, was normal in all of Sprna1ts strains, indicating SpRna1 in general did not function for a heterochromatin formation. snf2SR+ rescued a centromeric silencing defect and Deltaclr4+ was synthetic lethal with Sprna1ts. Taken together, SpRna1 was suggested to function for constructing the centromeres, by cooperating with Clr4 and Snf2SR. Loss of SpRna1 activity, therefore, caused chromosome missegregation.

Closing the (Ran)GAP on segregation distortion in Drosophila.

Segregation Distorter (SD) is a meiotic drive system in Drosophila that causes preferential transmission of the SD chromosome from SD/SD(+) males owing to induced dysfunction of SD(+) spermatids. Since its discovery in 1956, SD and its mode of action have baffled biologists. Recently, substantial progress has been made in elucidating this puzzle. Sd, the primary gene responsible for distortion encodes a mutant RanGAP, a key protein in the Ran signaling pathway required for nuclear transport and other nuclear functions. The mutant protein is enzymatically active but mislocalized to nuclei, which apparently disrupts Ran signaling by reducing intranuclear Ran-GTP levels. Some evidence suggests that a defect in nuclear transport may be the main cause of sperm dysfunction. Although important questions remain, the basic mechanism of distortion is now understood sufficiently well that specific hypotheses can be formulated and tested. This previously mysterious genetic system may now offer unique insights into novel aspects of regulation by Ran.

Segregation distortion induced by wild-type RanGAP in Drosophila.

Segregation Distorter (SD) is a meiotic drive system in Drosophila that causes preferential transmission of the SD chromosome from SD/SD(+) males owing to the induced dysfunction of SD(+) spermatids. The key distorter locus, Sd, is a dominant neomorphic allele encoding a truncated, but enzymatically active, RanGAP (RanGTPase-activating protein) whose nuclear mislocalization underlies distortion by disrupting the Ran signaling pathway. Here, we show that even wild-type RanGAP can cause segregation distortion when it is overexpressed in the male germ line or when the gene dosage of a particular modifier locus is increased. Both manipulations result in substantial nuclear accumulation of RanGAP. Distortion can be suppressed by overexpression of Ran or Ran guanine nucleotide exchange factor (RanGEF) in the male germ line, indicating that the primary consequence of nuclear mislocalization of RanGAP is reduction of intranuclear RanGTP levels. These results prove that segregation distortion does not depend on any unique properties of the mutant RanGAP encoded by Sd and provide a unifying explanation for the occurrence of distortion in a variety of experimental situations.