Quantification of RNA editing of the serotonin 2C receptor (5-HT(₂C)R) ex vivo.
The 5-HT(₂C)R receptor (5-HT(₂C)R) exerts tonic and phasic inhibitory influence over brain circuitry, and dysregulation of this influence contributes to the neurochemical underpinnings in the etiology of a variety of neuropsychiatric disorders including addiction, depression, and schizophrenia. A strategically important regulator of the 5-HT(₂C)R function and protein diversity is mRNA editing, a type of posttranscriptional modification that alters codon identity and thus the translation of distinct, though closely related, isoforms of 5-HT(₂C)R from a single, original transcript. The 5-HT(₂C)R mRNA can be edited at five closely spaced sites, altering the identity of up to three amino acids in the predicted second intracellular loop of the receptor to modulate receptor:G-protein coupling and constitutive activity. Methods to study changes in mRNA editing based upon direct DNA sequencing are both time and labor intensive. To streamline the acquisition of mRNA editing data and improve quantification, we have adapted real-time reverse transcription polymerase chain reaction (qRT-PCR) to detect and quantify mRNA editing in 5-HT(₂C)R transcripts by utilizing TaqMan® probes modified with a minor groove binder (MGB). The method is very sensitive, detecting as little as 10⁻¹⁸ g (1 attogram) of standard cDNA template and can discriminate closely related 5-HT(₂C)R mRNA edited isoforms. This technique expands the breadth of available quantification methods for mRNA editing and is particularly useful for the ex vivo analyses of mRNA editing of the 5-HT(₂C)R by allowing the rapid collection of data on large numbers of tissue samples. In addition, the general technique can be adapted easily to investigate edited mRNA from other genes, thus facilitating the development of a broader knowledge base of the physiological role of mRNA editing.
Center for Addiction Research, University of Texas Medical Branch, Galveston, Texas, USA.
Maria Fe
MF
Lanfranco
Noelle C
NC
Anastasio
Patricia K
PK
Seitz
Kathryn A
KA
Cunningham
D000818:Animals; D001921:Brain / Q000378:metabolism; D006801:Humans; D051379:Mice; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000302:isolation & purification; D017393:RNA Editing; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D044405:Receptor, Serotonin, 5-HT2C / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D020033:Protein Isoforms; D012333:RNA, Messenger; D044405:Receptor, Serotonin, 5-HT2C; D012313:RNA