|The 21st Annual Meeting of the RNA Society|
|International Conference Center, Kyoto, Japan|
|Jun 30, 2016|
|Epitranscriptome of human placental tissue|
Kosuke Taniguchi1, Tomoko Kawai1, Kohji Okamura2, Hiroko Ogata1, Michiyo Ohashi1, Kazuhiko Nakabayashi1, and Kenichiro Hata1|
1Dept. of Maternal-Fetal Biol., NCCHD, Japan, 2Dept. of Sys. BioMed., NCCHD., Japan
N6-Methyladenosine (m6A) is known as one of major mRNA modifications.
It was first discovered in 1970s and its function has been explosively investigated since the advent of next-generation sequencers (NGSs) (Dominissini D et al. Nature 2012, Meyer KD et al. Cell 2012).
Several reports showed that m6A accumulates around 3' UTRs, especially in the vicinity of stop codons of various transcripts and that the adenosine within GGAC motif could be a major m6A target.
It is suggested that m6A has a relation to post-transcriptional functions, especially for mRNA stability.
As an RNA modifying enzyme, Fat Mass and Obesity Associated (FTO) is known to demethylate m6A.
FTO was initially identified to be associated with obesity since SNPs in its 1st intron were relevant to BMI.
In perinatal field, placental FTO expression has been shown to correlate with newborn's birth weight.
We also validated that the level of FTO expressions at placental villi positively correlated with correspondent newborn's birth weights using 47 human postpartum placentas (r = 0.3293, p = 0.0238).
Therefore, we hypothesized that the status of m6A modifications of placental transcripts may involve in fetal growth through placental functions.
To address this question, we performed Methylated RNA immunoprecipitation followed by sequencing (MeRIP-seq) using human placental tissues.
Methods: We recruited term, low-risk pregnant subjects who planned to have caesarean section.
Just after delivery, we collected villi from postpartum placenta and homogenized them in TRIzol® within 15 minutes.
After isolation of total RNA, mRNA was purified using Dynabeads® Oligo (dT) twice.
Fragmented mRNA was immunoprecipitated (IP) with anti-m6A antibody (SySy polyclonal).
Then precipitated RNA was employed to prepare NGS libraries and sequenced with HiSeq 2000 (100 bp, paired end).
We processed data via our m6A peak-detection pipeline.
Results: We confirmed that most of the m6A peak regions resided around 3' UTR, especially in the vicinity of stop codons and possessed the motif 'GGAC' in their regions as previously reported.
In addition, we found placental transcripts whose m6A modifications were regulated in relation to fetal growth.|