A Consensus RNA Signal That Directs Germ Layer Determinants to the Vegetal Cortex of Xenopus Oocytes

Abstract

RNA localization is an important mechanism for generating cellular diversity and polarity in the early embryo. In Xenopus, the correct localization of the RNA encoding the T-box transcription factor VegT is essential for the correct spatial organization and identity of endoderm and mesoderm. Although localization signals in the 3' UTR have been identified for many localized RNAs, insight into what constitutes an RNA localization signal remains elusive. To investigate possible common features between signals that direct different RNAs to the same subcellular region, we carried out a detailed analysis of the uncharacterized VegT RNA localization signal and compared it with the well-studied Vg1 localization signal. Both RNAs localize to the vegetal cortex during the same period of oogenesis. Our results suggest a common RNA localization signal at the level of clustered redundant protein-binding motifs and trans-acting factors. We propose that what characterizes RNA localization signals in general is not the nucleotide sequence or secondary structure per se, but the critical clustering of specific redundant protein-binding motifs.

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

“…to previous observations, 16 mutational analysis revealed no correlation between the presence of E2/E2-like-elements and Vg1RBP protein interaction in the context of the XGrip2.1-LE, since we were able to identify 5'-as well as 3'-LE-mutants that still contain multiple such elements but do not bind to Vg1RBP. The extended region required for Vg1RBP binding could indicate that binding of more than one copy of Vg1RBP protein to the XGrip2.1-LE is required for the formation of more robust RNA-protein complexes.…”

“…[10][11][12][13][14][15] Comparative analysis of Vg1 and VegT-LE defined Vg1RBP and VgRBP60 as core localization components that assemble on the E2-and VM1-element containing LEs and are required for late pathway localization. 16 Although a similarly detailed characterization of early pathway localization elements is still missing, they were also found to be enriched in CAC-containing motifs. 17 In addition to Vg1RBP and VgRBP60, a number of other proteins also critical for vegetal localization has been identified via their affinity for the localization element of the late pathway RNA Vg1; these include Prrp, VgRBP71, 40LoVe and ElrA/B.…”

“…Thus, localization of the early pathway XGrip2.1-LE might require recruitment of the same core localization components as the true late pathway RNAs Vg1 and VegT. 16 In a previous publication, we had provided experimental evidence for Elr-type proteins as being required for the localization of the XDead end mRNA via the late localization pathway in Xenopus oocytes. 21 Conversely, in the context of this study, mutational interference with Elr-type protein recruitment had no detectable effect on late pathway localization mediated by the XGrip2.1-LE.…”

40LoVe interacts with Vg1RBP/Vera and hnRNP I in binding the Vg1-Localization Element

“…Some unidentified characteristic (a sequence or structure) of the Vg1LE RNA is likely required for stable 40LoVe binding, and by immunoprecipitation subsequent to UV cross-linking we have demonstrated cross-linking of 40LoVe to the Vg1LE (Czaplinski et al 2005). It has been proposed that the unique configuration of E2 and VM1 elements in Vg1 and VegT LEs accounts for late localization (Bubunenko et al 2002). Therefore, an explanation for 40LoVe's E2 and VM1 motif dependence is that 40LoVe can be recruited to RNAs by interactions with Vg1RBP/Vera and hnRNP I uniquely in situations where binding sites for the two proteins are appropriately organized.…”

Cis-acting determinants of asymmetric, cytoplasmic RNA transport

“…In Xenopus, both the VegT and Vg1 RNAs are vegetally localized by the late pathway. The 39 UTRs of both transcripts reveal clusters of E2 and VM1 sites (Bubunenko et al 2002) that bind Vera and hnRNP I, respectively. These clusters are similar in length and are located at similar positions in the 39 UTRs, although the exact numbers and locations of each type of site vary between the two UTRs.…”