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 mouse quaking lethality (E¦G) occurs due to the absence of Qk1 self-association mediated by the GSG domain.

The KH domains of Xenopus Vg1RBP mediate RNA binding and self-association

“…Members of the GSG/STAR subfamily of RNA-binding proteins, which contain a single KH domain embedded in a larger conserved domain of ;200 amino acids called the GSG domain, also oligomerize and bind RNA+ Oligomerization of Qk1 is mediated by a coiled-coil in the N-terminal extension to the KH domain, whereas RNA binding requires the entire GSG domain+ Importantly, a lethal mutation in mouse, quaking, was shown to be due to absence of self-association mediated by the GSG domain (Chen & Richard, 1998)+ Similarly, FMR-1, which targets G quartet mRNAs important for neuronal function (Darnell et al+, 2001), associates with its close homologs FXR-1 and 2 family members through a coiled-coil motif located outside of the KH domains (Siomi et al+, 1996)+ In the case of Vg1RBP, however, self-association is mediated principally by the K34 didomain, and to a lesser extent by K12, and the glutamine-rich stretch present C-terminal to KH4 is not required+ Although the ability to oligomerize presumably has important implications for the function of Vg1RBP, we do not know whether self-association is required for RNA binding because no dimerization mutant is available in the context of the full-length protein+ An attractive model relating dimerization and high affinity RNA binding has recently been proposed (Harvey et al+, 2002), in which the forced engagement of a RNA/ protein complex by a chemical inducer of dimerization is shown to modulate gene expression+ On presentation of the chemical inducer of dimerization to an mRNA template by the protein, cooperative binding ensued, resulting in higher-affinity complexes, and inhibition of mRNA translation (Harvey et al+, 2002 In summary, we propose that Vg1RBP self-associates initially through interactions mediated by the K34 didomain, which is subsequently stabilized by RNA binding, and furthermore, that self-association appears to be the first step in the formation of very much larger complexes+ An important future avenue of investigation will be to address the functional importance of Vg1RBP self-association in the formation of a Vg1 mRNA localization complex+ …”

A novel glioblastoma cancer gene therapy using AAV-mediated long-term expression of human TERT C-terminal polypeptide

“…Among all these genes, apoptosis was found to be the category, which composed of the highest number of differentially expressed genes (Figure 6a). The expression levels of four representative apoptosis-related genes, including mitogen-activated protein kinase kinase kinase 7 interacting protein (MAP3K7IP2), 24 lysyl oxidase-like 2 (LOXL2), 25 immunoglobulin heavy constant gamma 1 (IGHG1) 26 and Qk1, 27 were further validated by semiquantitative RT-PCR (Figure 6b and c). 28,29 Using this model, we showed that intratumoral injection of rAAV-hTERTC27 dramatically reduces the growth and tumorigenicity of the human glioblastoma U87-MG cells.…”

Human QKI, a potential regulator of mRNA expression of human oligodendrocyte-related genes involved in schizophrenia

“…The gene is highly conserved over different species and is important for normal development in both vertebrates and invertebrates (4,5). The function of the Qki gene has been well studied in mouse by using an autosomal recessive mutant (qk v ), characterized by body tremor and severe dysmyelination of the CNS (6).…”