Nucleic Acids Res
January 1, 2006;
Identification of post-transcriptionally regulated Xenopus tropicalis maternal mRNAs by microarray.
Cytoplasmic control of the adenylation state of mRNAs is a critical post-transcriptional process involved in the regulation of mRNAs stability and translational efficiency. The early development of Xenopus laevis has been a major model for the study of such regulations. We describe here a microarray analysis to identify mRNAs that are regulated by changes in their adenylation state during oogenesis and early development of the diploid frog Xenopus tropicalis. The microarray data were validated using qRT-PCR and direct analysis of the adenylation state of endogenous maternal mRNAs during the period studied. We identified more than 500 mRNAs regulated at the post-transcriptional level among the 3000 mRNAs potentially detected by the microarray. The mRNAs were classified into nine different adenylation behavior categories. The various adenylation profiles observed during oocyte
maturation and early development and the analyses of 3''-untranslated region sequences suggest that previously uncharacterized sequence elements control the adenylation behavior of the newly identified mRNAs. These data should prove useful in identifying mRNAs with important functions during oocyte
maturation and early development.
Nucleic Acids Res
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Figure 1. Comparison of microarray and qRT–PCR results. Relative amounts of poly(A)+ mRNA of the indicated gene products in stage VI oocytes (black bars), UFE (gray bars) and 64-cell embryos (open bars), as measured by (A) microarrays and (B) qRT–PCR. Relative levels for microarray data are given with the maximal normalized intensity as 100%, and for qRT–PCR, the maximal poly(A)+ mRNA amount is considered as 100%.
Figure 2. Sequence analysis of mRNAs classified according to their adenylation behaviors. Regulatory sequences were searched for in putative 3′-UTR of mRNAs in each category. For each category, the percentage of sequences containing the regulatory element is indicated. The number of sequences analyzed and the total length of the sequence in each category are indicated. The percentage of each regulatory sequence elements in the totality of the 3′-UTR sequences of categorized mRNAs (cat UTR) and of all the UTR's recovered from the genome annotation (whole 3′-UTR) is indicated.
Figure 3. RL-PAT specificity. (A) Schematic of the RNA ligation mediated poly(A) test, as described by Rassa et al. (35). P1 denotes the primer ligated to the 3′-UTR of the RNA, the 3′ end of P1 is blocked by an amino group. cDNA synthesis is achieved from primer P′1 and PCR products are radiolabeled by the 32P phosphorylated gene-specific primer (GSP). X indicates a restriction site, radiolabeled fragments are in gray. (B) Specificity of RL-PAT was tested by restriction digest (D) of PCR products obtained from StVI oocyte or UFE RNA as indicated (St., stage). The identity of the tested mRNAs is indicated above each lane. When indicated, RNase H (RH) digestion in the presence of oligo(dT) was performed prior to the RL-PAT. M, radiolabeled DNA size markers as indicated (base pairs).
Figure 4. Adenylation behaviors of selected X.tropicalis endogenous mRNAs. Total RNAs from StVI oocytes, UFE and 64C embryos were analyzed by RL-PAT. The identity of the tested mRNAs is indicated above each panel. Oligo(dT)/RNase H digestion (RH) was performed on StVI oocyte total RNA. M, DNA size markers. In RT- control lanes, ligated RNAs were not reverse transcribed before PCR. (A) RL-PAT results for mRNAs with known adenylation behaviors in X.laevis. (B) RL-PAT results for mRNAs with unknown behaviors.
Zygotic regulation of maternal cyclin A1 and B2 mRNAs.