Defective importin β recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations

Abstract

The architectural transcription factor SRY (sex-determining region of the Y chromosome) plays a key role in sex determination as indicated by the fact that mutations in SRY are responsible for XY gonadal dysgenesis in humans. Although many SRY mutations reduce DNA-binding͞bending activity, it is not clear how SRY mutations that do not affect interaction with DNA contribute to disease. The SRY high-mobility group domain harbors two nuclear localization signals (NLSs), and here we examine SRY from four XY females with missense mutations in these signals. In all cases, mutant SRY protein is partly localized to the cytoplasm, whereas wild-type SRY is strictly nuclear. Each NLS can independently direct nuclear transport of a carrier protein in vitro and in vivo, with mutations in either affecting the rate and extent of nuclear accumulation. The N-terminal NLS function is independent of the conventional NLS-binding importins (IMPs) and requires unidentified cytoplasmic transport factors, whereas the C-terminal NLS is recognized by IMP␤. The SRY-R133W mutant shows reduced IMP␤ binding as a direct consequence of the sex-reversing C-terminal NLS mutation. Of the N-terminal NLS mutants examined, SRY-R62G unexpectedly shows a marked reduction in IMP␤ binding, whereas SRY-R75N and SRY-R76P show normal IMP␤ binding, suggesting defects in the IMP-independent pathway. We conclude that SRY normally requires the two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for sex determination. This study documents cases of human disease being explained, at a molecular level, by the impaired ability of a protein to accumulate in the nucleus.

Dynein Light Chain Association Sequences Can Facilitate Nuclear Protein Import

“…Fixed cells were viewed on a Bio-Rad MRC-600 CLSM with a 60ϫ oil immersion objective (Harley et al, 2003). Analysis of digitized confocal files used Image J 1.62 public domain software (NIH) as previously described Xiao et al, 1997).…”

Human SRY inhibits β-catenin-mediated transcription

“…DNA encoding wild-type FLAG-tagged human SRY and clinical mutants R75N, R76P and R133W were previously described (Harley et al, 2003). SRY clinical mutants S18N, R30I, I90M and L163X were produced using the Site-directed Quick change mutagenesis kit (Qiagen) according to the manufacturer's instructions.…”

“…Interestingly, R133W and R76P, which have similar nuclear import defects, 52% and 50% of wild-type activity respectively (Harley et al, 2003, Sim et al, 2005, displayed differing abilities to inhibit TOPFLASH activity induced by β-catenin. R133W, which retains almost wild-type DNA binding and bending activities (Harley et al, 2003, Li et al, 2001, was less potent in inhibiting β-catenin-induced TOPFLASH activity than R76P which retains only 33% of DNA binding activity (Harley et al, 2003). We conclude that SRY does not seem to require DNA-binding properties to inhibit β-catenin signaling.…”

“…Error bars indicate standard errors of the means for triplicate samples from two independent experiments. (Mitchell and Harley, 2002); 2 (Assumpcao et al, 2002); 3 (Pontiggia et al, 1994); 4 (Harley et al, 2003); 5 (Li et al, 2001)). (C).…”

Swyer’s Syndrome: In a Fifty-Year-Old Female

“…High-mobility group domain of SRY harbors two nuclear localization signals (NLSs). Harley and collaborators [9] examined SRY from four XY females with missense mutations in these signals. They concluded that SRY normally requires the two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for sex determination.…”