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 draw conclusions from RNAseq and 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 (A-, 20-50 adenosines) from polyadenylated (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 A- 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 U(3)GU(3) were enriched in A- sequences. Moreover, a motif associated with polyadenylation signals (MAPS, U(5)CU(2)) appeared to be enriched in 3'untranslated regions (UTR) with CPE or U(3)GU(3) 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.

RNA‐Seq profiling of single bovine oocyte transcript abundance and its modulation by cytoplasmic polyadenylation

“…Anchored oligo(dT) primers were used in this study to create the sequencing libraries with the intent of determining the levels of polyadenylated transcripts, which could be considered active transcripts (available for immediate translation). Even though a short polyadenine tail (5À10 bp) has been shown to be sufficient for polyadenine-dependent reverse transcription (Graindorge et al, 2006), the length of the polyadenine tail influences the reverse transcriptase efficiency (Gohin et al, 2013), so changes in transcript abundance detected by anchored oligo(dT) priming may represent changes in transcript polyadenylation status.…”

Determinants and implications of mRNA poly(A) tail size – Does this protein make my tail look big?

“…In embryos, short poly(A) tails are necessary to repress translation until the appropriate stage of development is reached [9]. In addition, short poly(A) tails appear to denote mRNAs that are critical for early development, and may be a way to regulate translation in a dose and time-dependent manner [107]. Thus poly(A) tail length control and development go hand in hand.…”

Regulation of ATF1 and ATF2 transcripts by sequences in their 3′ untranslated region in cleavage‐stage cattle embryos

“…The late translation of ATF2LA − is an intriguing observation that suggests the existence of cis ‐elements in its 3′UTR that suppress polyadenylation. An in silico analysis by Gohin et al () revealed that the MAPS motif appears in transcripts containing a CPE, and that consensus MAPS sites are enriched in deadenylated mRNAs in bovine oocytes. Furthermore, their analysis of the molecular anatomy of 3′UTRs of ATF1 and ATF2 showed that only the second transcript bears a consensus MAPS (Gohin et al, ), which supports the notion that a cis ‐based mechanism delayed GFP translation from ATF2LA− .…”

“…An in silico analysis by Gohin et al () revealed that the MAPS motif appears in transcripts containing a CPE, and that consensus MAPS sites are enriched in deadenylated mRNAs in bovine oocytes. Furthermore, their analysis of the molecular anatomy of 3′UTRs of ATF1 and ATF2 showed that only the second transcript bears a consensus MAPS (Gohin et al, ), which supports the notion that a cis ‐based mechanism delayed GFP translation from ATF2LA− . Therefore, the MAPS may constitute a regulatory mechanism of ATF2 transcripts with a long 3′UTR in early bovine development, delaying its translation until an opportune stage.…”

“…Therefore, the MAPS may constitute a regulatory mechanism of ATF2 transcripts with a long 3′UTR in early bovine development, delaying its translation until an opportune stage. Indeed, Gohin et al, () established that a candidate gene ontology term associated with MAPS is RNA binding (GO:0003723; http://amigo.geneontology.org), which is associated with the exosome protein EXOSC7. The exosome is a structure mainly known for its transcript‐degradation function, although it is also involved in transcript retention (reviewed in Fasken & Corbett, ).…”