October 15, 2002;
A ubiquitous and conserved signal for RNA localization in chordates.
During oogenesis in Xenopus laevis, several RNAs that localize to the vegetal cortex via one of three temporally defined pathways have been identified. Although individual mRNAs utilize only one pathway, there is functional overlap and apparent continuity between them, suggesting that common cis-acting sequences may exist. Because previous work with the Vg1
mRNA revealed that short nontandem repeats are important for localization, we developed a new computer program, called REPFIND, to expedite the identification of repeated motifs in other localized RNAs. Here we show that clusters of short CAC
-containing motifs characterize the localization elements (LEs) of virtually all mRNAs localized to the vegetal cortex of Xenopus oocytes. A search for this signal in GenBank  resulted in the identification of new localized mRNAs, demonstrating the applicability of REPFIND to predict localized RNAs. CAC
-rich LEs are also found in ascidians and other vertebrates, indicating that these cis regulatory elements are conserved in chordates. Interestingly, biochemical evidence shows that distinct CAC
-containing motifs have different functions in the localization process. Thus, clusters of CAC
-containing motifs are a ubiquitous
signal for RNA localization and can signal localization in a variety of pathways through slight variations in sequence composition.
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Figure 2. In Situ Hybridization of Newly Discovered Localized mRNAsIn situ hybridization toXcat-2 mRNA and Xpat mRNAs demonstrates examples of RNAs that localize via the early pathway. Note that most of the RNA is associated with the mitochondrial cloud in stage I oocytes, with little background in the rest of the cytoplasm. By stage II, these RNAs have migrated with the cloud to the vegetal cortex, and there is no detectable RNA in the remaining cytoplasm. Xlerk, which was identified by computational screening of Xenopus 3′ UTRs for highly significant clusters of CAC-containing motifs, follows the intermediate pathway in which localization to the cloud of stage I oocytes is clearly visible but there is also a high level of signal in the remaining cytoplasm. During stage II, labeling is observed both in the cloud at the cortex and in the remaining cytoplasm. However, by stage III, no cytoplasmic labeling is detected, and all Xlerk RNA is localized to the vegetal pole. By stage IV, this labeling pattern is broader than that of either Xcat-2 or Xpat. C3H-3, also determined computationally to contain CAC-rich clusters in its 3′ UTR, utilizes the late, or Vg1, pathway. In this case, localization does not begin until stage II, and background labeling is observed through stage III. By stage IV of oogenesis, however, this late-pathway RNA shows no labeling in the animal hemisphere. Vg1 is shown as a known example of late-pathway localization, and histone mRNA (Hist), which is expressed throughout the cytoplasm of stage I–IV oocytes, is shown as a negative control for localization. The scale bar represents 100 μm in all panels.
nanos1 ( nanos homolog 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF oocyte stage iV, vegetal view.
Figure 1. Microinjection Analysis of the CAC-Rich Regions of 3′ UTRs from Six Localized mRNAs
(A) Various fragments from the 3′ UTRs of six different localized RNAs were injected into stage I oocytes and assayed for localization to the mitochondrial cloud. White arrows indicate where RNA (blue) has localized to the mitochondrial cloud in all panels except for XβG (Xenopus β-globin), which was used as a negative control for localization and shows no labeling of the mitochondrial cloud. The numbers in the lower right corner of each panel indicate the number of oocytes that showed localization over the number of oocytes tested from a minimum of three independent experiments.
(B) A schematic representation of all six 3′ UTRs is shown, with the name of each construct that was assayed for localization shown at the right and preceded by a letter indicating its corresponding panel in (A). Regions containing the CAC-rich clusters are indicated by open lines with coordinates from the experimental LE column in Table 1. A scale bar in nucleotides is included at the bottom of the figure. Note that all CAC-rich clusters show localization and that removal of the CAC-rich region from the Xpat 3′ UTR (c) or deletion of the UGCAC motifs from Xcat-2 LE (e) abolishes localization.
(C) A schematic diagram of the Macho-1 mRNA is shown at the left, with the sequence of the CAC-rich region of its 3′ UTR from Table 1 shown in the box. All CAC and ACAC motifs are shown in red text. The CA of each motif was mutated to GU, indicated below the sequence of each CAC or ACAC motif. It can be seen that the wild-type (wt), but not the mutant (mut) version of the Macho-1 CAC-rich region localizes in Xenopus stage I oocytes.