Discovery, identification and sequence analysis of RNAs selected for very short or long poly a tail in immature bovine oocytes

Abstract

A major challenge in applying genomics to oocyte physiology is that many RNAs are present but will not be translated into proteins, making it difficult to drawconclusions fromRNAseqand array data. Oocyte maturation and early embryo development rely on maternal storage of specific RNAs with a short poly(A) tail, which must be elongated for translation. To resolve the role of key genes during that period, we aimed to characterize both extremes of mRNA: deadenylated RNA and long polyA tails mRNA population in immature bovine oocytes. Using magnetic beads coupled to oligodT,we isolated deadenylated (A2, 20-50 adenosines) frompolyadenylated (A+, up to 200 adenosines) RNAs. After transcriptomic analysis, we observed that A+ candidates are associated with short-term processes required for immediate cell survival (translation or protein transport) or meiotic resumption, while several A2 candidates are involved in processes (chromatin modification, gene transcription and post-transcriptional modifications) that will be extremely important in the development of the early embryo. In addition to a list of candidates probably translated early or late, sequence analysis revealed that cytoplasmic polyadenylation element (CPE) and U3GU3 were enriched in A2 sequences. Moreover, a motif associated with polyadenylation signals (MAPS, U5CU2) appeared to be enriched in 3'untranslated regions (UTR) with CPE or U3GU3 sequences in bovine but also in zebrafish and Xenopus tropicalis. To further validate our methodology, we measured specific tail length of known candidates (AURKA, PTTG1, H2A1) but also determined the poly(A) tail length of other candidate RNAs (H3F3A, H1FOO, DAZAP2, ATF1, ATF2, KAT5, DAZL, ELAVL2). In conclusion, we have reported a methodology to isolate deadenylated from polyadenylated RNAs in samples with small total RNA quantities such as mammals. Moreover, we identified deadenylated RNAs in bovine oocytes that may be stored for the long-term process of early embryo development and described a conserved motif enriched in the 3'UTR of deadenylated RNAs. © The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.