Assessing serotonin receptor mRNA editing frequency by a novel ultra high-throughput sequencing method.
RNA editing is a post-transcriptional modification of pre-mRNA that results in increased diversity in transcriptomes and proteomes. It occurs in a wide variety of eukaryotic organisms and in some viruses. One of the most common forms of pre-mRNA editing is A-to-I editing, in which adenosine is deaminated to inosine, which is read as guanosine during translation. This phenomenon has been observed in numerous transcripts, including the mammalian 5-HT(2C) receptor, which can be edited at five distinct sites. Methods used to date to quantify 5-HT(2C) receptor editing are labor-intensive, expensive and provide limited information regarding the relative abundance of 5-HT(2C) receptor editing variants. Here, we present a novel, ultra high-throughput method to quantify 5-HT(2C) receptor editing, compare it to a more conventional method, and use it to assess the effect of a range of genetic and pharmacologic manipulations on 5-HT(2C) editing. We conclude that this new method is powerful and economical, and we provide evidence that alterations in 5-HT(2C) editing appear to be a result of regional changes in brain activity, rather than a mechanism to normalize 5-HT(2C) signaling.
Abbas
Atheir I
AI
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
Urban
Daniel J
DJ
Jensen
Niels H
NH
Farrell
Martilias S
MS
Kroeze
Wesley K
WK
Mieczkowski
Piotr
P
Wang
Zefeng
Z
Roth
Bryan L
BL
D000818:Animals; D018076:DNA, Complementary / Q000737:chemistry; D057166:High-Throughput Screening Assays; D051379:Mice; D008810:Mice, Inbred C57BL; D017393:RNA Editing; D012322:RNA Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D044405:Receptor, Serotonin, 5-HT2C / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
D018076:DNA, Complementary; D012322:RNA Precursors; D012333:RNA, Messenger; D044405:Receptor, Serotonin, 5-HT2C