Inosine RNA modifications are enriched at the codon wobble position in mouse oocytes and eggs†

Abstract

Mammalian oocytes and eggs are transcriptionally quiescent and depend on post-transcriptional mechanisms for proper maturation. Post-transcriptional mRNA modifications comprise an important regulatory mechanism that can alter protein and miRNA recognition sites, splicing, stability, secondary structure, and protein coding. We discovered that fully grown mouse germinal vesicle oocytes and metaphase II eggs display abundant inosine mRNA modifications compared to growing oocytes from postnatal day 12 oocytes. These inosines were enriched in mRNA protein coding regions (CDS) and specifically located at the third codon base, or wobble position. Inosines, observed at lower frequencies in CDS of somatic tissues, were similarly enriched at the codon wobble position. In oocytes and eggs, inosine modifications lead primarily to synonymous changes in mRNA transcripts. Inosines may ultimately affect maternal mRNA stability by changing codon usage, thereby altering translational efficiency and translationally coupled mRNA degradation. These important observations advance our understanding of post-transcriptional mechanisms contributing to mammalian oocyte maturation.

Loss ofCnot6limpairs inosine RNA modifications in mouse oocytes

“…It has recently been reported that inosine RNA modifications in the coding sequence of mRNA lead to ribosome stalling (Licht et al 2019). In a previous report (Brachova et al 2019), and now here in WT and Btg4 -/oocytes, we identified inosine RNA modifications specifically enriched in at the codon wobble position in oocytes (Brachova et al 2019). Based on our finding and others, we hypothesized that inosine RNA modifications within the coding sequence could alter mRNA stability, potentially through translational mRNA decay.…”

“…Inosine RNA modifications are lost in oocytes from Cnot6l -/mice Using our computational approach (Brachova et al 2019), we identified transcriptome wide inosine RNA modifications (Supplemental Fig. S1) in previously reported RNA-seq datasets (Sha et al 2018) from wild-type (WT), Cnot6l -/-, Btg4 -/oocytes at 0, 8, and 16 h after in vitro maturation.…”

Loss of Cnot6l Impairs Inosine RNA Modifications in Mouse Oocytes

“…Inosine RNA modifications are identified by comparing the transcriptome to the genome to identify A-to-G mismatches, as we and others have shown [ 28 , 30 , 31 , 32 ]. Using our computational approach [ 28 ], we identified transcriptome-wide inosine RNA modifications ( Supplemental Figure S1 ) in previously reported RNA-seq datasets [ 14 ] from wild-type (WT), Cnot6l -/- , and Btg4 -/- oocytes and eggs at 0, 8, and 16 h after in vitro maturation. These three time points correspond to the GV, MI, and MII oocyte and egg stages, respectively.…”

“…Our previous study linked inosine RNA modifications to the process of translation through RNA modifications of codons [ 28 ]. To test if the accumulated and over-translated maternal transcripts found in oocytes/eggs of Cnot6l -/- mice were enriched in inosine RNA modifications, we cataloged inosine RNA modifications in ribosome-associated mRNA (i.e., mRNA bound by multiple ribosomes) at the GV, MI, and MII stages in WT and Cnot6l -/- oocytes/eggs.…”

“…Inosines are a frequent RNA modification generated by the deamination of adenosine and catalyzed by a family of conserved double stranded RNA (dsRNA) binding proteins, adenosine deaminases acting on RNA (ADARs) [ 25 , 26 , 27 ]. Adar /ADAR is the predominant adenosine deaminase detected in mouse GV oocytes and MII eggs, and Adarb2 /ADAR2 is also present [ 28 ]. Although inosine post-transcriptional RNA modifications can impact mRNA stability through a translation mechanism [ 29 ], the relationship of inosines and RNA stability in mouse oocytes has not yet been demonstrated.…”