Structure-Function Analysis of Qk1: a Lethal Point Mutation in Mouse quaking Prevents Homodimerization

ABSTRACT

Qk1 is a member of the KH domain family of proteins that includes Sam68, GRP33, GLD-1, SF1, and Who/How. These family members are RNA binding proteins that contain an extended KH domain embedded in a larger domain called the GSG (for GRP33–Sam68–GLD-1) domain. An ethylnitrosourea-induced point mutation in the Qk1 GSG domain alters glutamic acid 48 to a glycine and is known to be embryonically lethal in mice. The function of Qk1 and the GSG domain as well as the reason for the lethality are unknown. Here we demonstrate that the Qk1 GSG domain mediates RNA binding and Qk1 self-association. By using in situ chemical cross-linking studies, we showed that the Qk1 proteins exist as homodimers in vivo. The Qk1 self-association region was mapped to amino acids 18 to 57, a region predicted to form coiled coils. Alteration of glutamic acid 48 to glycine (E➤G) in the Qk1 GSG domain (producing protein Qk1:E➤G) abolishes self-association but has no effect on the RNA binding activity. The expression of Qk1 or Qk1:E➤G in NIH 3T3 cells induces cell death by apoptosis. Approximately 90% of the remaining transfected cells are apoptotic 48 h after transfection. Qk1:E➤G was consistently more potent at inducing apoptosis than was wild-type Qk1. These results suggest that the mousequaking lethality (E➤G) occurs due to the absence of Qk1 self-association mediated by the GSG domain.

ACKNOWLEDGMENTS

We thank Guillermina Almazan for purified extracts of rat astrocytes and oligodendrocytes. We thank Janet Henderson and Antonis Koromilas for critically reading the manuscript and helpful comments. We are grateful to Rongtuan Lin, John Th’ng, and Hans Zingg for providing reagents and to Bassam Damaj for technical assistance with the rabbit polyclonal antibody.

T.C. is supported by a studentship from the Cancer Research Society of Canada and funds from Canderel. This work was supported by grants from the Medical Research Council of Canada, the Cancer Research Society of Canada, Fonds de la Recherche en Santé du Québec, and the Multiple Sclerosis Society of Canada. S.R. is a Scholar of the Medical Research Council of Canada.