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

Abstract

Xenopus Vg1 mRNA is localized to the vegetal cortex during oogenesis in a process involving microtubules and microfilaments and proteins that specifically recognize the vegetal localization element (VLE) within the 39 untranslated region. One of the best characterized VLE-binding proteins is Vg1RBP or Vera. Primary sequence analysis of Vg1RBP and its homologs suggests that most of its open reading frame is occupied by RNA-binding modules, including two RRMs and four KH domains, arranged as three pairs of didomains. In the first detailed domain analysis of Vg1RBP, we show that the interaction of Vg1RBP with the VLE requires both KH didomains, but not the RRM didomain, and moreover that the KH didomains contribute cooperatively to RNA binding. In the full-length protein, individual KH domains display significant redundancy, and their relative importance appears to vary with the RNA target. We also demonstrate that the KH34 didomain mediates Vg1RBP self-association, which is stabilized by RNA, and occurs in vivo as well as in vitro. Altogether, our findings highlight the importance of multiple KH domains in mediating RNA-protein and protein-protein interactions in the formation of a stable complex of Vg1RBP and Vg1 mRNA.

Participation of Xenopus Elr-type Proteins in Vegetal mRNA Localization during Oogenesis

“…The Vg1RBP expression construct was as described (22). His-tagged ElrB and Vg1RBP proteins were purified as described previously (48).…”

Multimerization of poly(rC) binding protein 2 is required for translation initiation mediated by a viral IRES

“…KH domain-containing proteins have been shown to multimerize and interact with other proteins. The Xenopus Vg1RBP protein contains four KH domains that contribute cooperatively to RNA binding and also mediate self-association (Git and Standart 2002). hnRNP K interacts with proteins containing SH domains, including src and vav, through a proline-rich domain found between KH2 and KH3.…”

Functional dissection of the RNA signal sequence responsible for vegetal localization ofXGrip2.1mRNA in Xenopus oocytes

“…As Vg1RBP has been described to self-associate in the presence of RNA, such protein-protein interactions could contribute to stabilizing such RNA-protein complexes. 44 Alternatively, individual KH-domains of Vg1RBP might interact with spatially separated target sequences thereby inducing the looping of the interjacent RNA stretch, as has recently been proposed for IMP1, the human ortholog of Vg1RBP. 45 Binding of 40LoVe to the XGrip2.1-LE in vitro turned out to be relatively weak and might require binding of additional factors such as Vg1RBP and VgRBP60 to stabilize the RNA/protein interactions.…”