The KH domain protein encoded by quaking functions as a dimer and is essential for notochord development inXenopus embryos

Abstract

Mutations in the mouse indicate that quaking gene function is essential for both embryogenesis and for development of the nervous system. Recent isolation of the mouse quaking gene identified a putative RNA-binding protein containing a single KH domain. We have previously isolated the Xenopus homolog of quaking, Xqua, and shown that the sequence is highly conserved through evolution. Here, we report experimental data on the biochemical function of the quaking protein and its role during development. We demonstrate that the quaking protein expressed during early embryogenesis, pXqua 357 , can bind RNA in vitro, and we have mapped the regions of the protein that are essential for RNA binding. We present evidence that pXqua can form homodimers and that dimerization may be required for RNA binding. Oocyte injection experiments show that pXqua 357 is located in both the nucleus and cytoplasm. In the Xenopus embryo, Xqua is first expressed during gastrulation in the organizer region and its derivative, the notochord. In later stage embryos, Xqua is expressed in a number of mesodermal and neural tissues. We demonstrate that disruption of normal Xqua function, by overexpression of a dominant inhibitory form of the protein, blocks notochord differentiation. Xqua function appears to be required for the accumulation of important mRNAs such as Xnot, Xbra, and gsc. These results indicate an essential role for the quaking RNA-binding protein during early vertebrate embryogenesis.

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

“…This demonstrates that the entire Qk1 GSG domain is sufficient for RNA binding, as has been previously demonstrated for Sam68 (8,25). However, this is in contrast to what has been reported for Xqua by Zorn and Krieg; they demonstrated that the entire Xqua protein was required for optimal RNA binding (45). Deletion of the N-terminal GSG amino acids had only a slight reduction effect on RNA binding, whereas deletion of amino acids C terminal to the GSG domain (231 to 357) abolished RNA binding.…”

“…GSG proteins share several properties, including RNA binding (1,8,25,41,44) and self-association (8,45). With the exception of the human SF1 protein, which functions as a splicing factor (1), the roles of the GSG proteins in cellular processes are not known.…”

Expression of Hqk Encoding a KH RNA Binding Protein Is Altered in Human Glioma

“…Drosophila who/how gene has been shown to be critical for muscle development 18,19) and Xqua is involved in gastrulation, particularly notochord development. 20) In C. elegans, the GLD-1 protein is required for normal oocyte differentiation as well as sex determination. 13,16,17,21) In mouse, reduction of qkI expression results in neurological phenotypes such as body tremor and seizures due to hypomyelination in the CNS, 6,22) while disruption of qkI gene activity leads to embryonic lethality.…”

kep1 interacts genetically with dredd / Caspase-8 , and kep1 mutants alter the balance of dredd isoforms

“…5), Sam50, and QKI all belong to the STAR protein family that contains a GSG domain (3,6,7). Like other single KH domain-containing proteins (3,4,8,9), Kep1 can form homodimers and can bind homopolymeric RNA in vitro. In addition, lysates obtained from S2 cells transfected with Kep1 can induce isolated HeLa nuclei to show morphological characteristics indicative of apoptosis (3).…”