Identification of G protein‐coupled receptors by RNase H‐mediated hybrid depletion using Xenopus laevis oocytes as expression system

Abstract

A method has been developed for rapidly identifying putative G protein-coupled receptors isolated initially as small cDNA fragments, following reverse transcription and polymerase chain reaction (PCR) amplification of mRNA. The method is based upon the use of synthetic oligonucleotides deduced from the sequence of the amplified receptor fragments, to direct a RNase H-mediated specific degradation of hybrids formed between the oligonucleotides and the corresponding receptor-encoding mRNA. Loss of an agonist-dependent receptor response in the Xenopus laevis oocyte expression system identifies the amplified receptor fragment. Taking in vitro synthesised serotonin HT2-receptor (SR)-encoding mRNA as a model, it was shown that following incubation with RNase H and SR antisense oligonucleotides, injection of this message no longer caused the acquisition of agonist-dependent membrane currents in voltage-clamped oocytes. In contrast, when corresponding sense oligonucleotides were used, the serotonin-evoked membrane responses in oocytes were acquired as normal. The method should allow the identification of receptors which can functionally be expressed and measured in Xenopus oocytes.

Relative contributions of Na+-dependent phosphate co-transporters to phosphate transport in mouse kidney: RNase H-mediated hybrid depletion analysis

“…Oligonucleotides (see Table 1) were synthesized, purified with an Applied Biosystems DNA synthesizer (model 380B) and purified by HPLC. Poly(A) + RNA (5 µg) from mouse kidney cortex was incubated for 5 min at 70 mC with 40 ng of oligonucleotide in 20 µl of 100 mM KCl [15]. The annealing mixture was allowed to cool to room temperature over a period of 10 min, placed on ice, and diluted with 20 µl of a solution containing 70 units of RNase inhibitor (TaKaRa, Kyoto, Japan), 1 unit of RNase H (Gibco-BRL), 5 µg of tRNA, 6 mM MgCl # , 25 mM Tris\HCl (pH 7.5) and 0.75 mM dithiothreitol.…”

“…We have now evaluated the relative contributions of various Na + \P i co-transporters in P i transport in mouse kidney cortex with the use of in itro RNase H-mediated hybrid depletion. In this approach, polyadenylated [poly(A) + ] RNA from mouse kidney cortex was incubated in itro first with synthetic antisense oligonucleotides specific for the transcripts of transporter genes and then with RNase H, which catalyses the degradation of hybrids formed between the oligonucleotides and the corresponding transporters mRNAs [15]. The effect of this specific degradation on P i uptake was then measured in Xenopus oocytes injected with the RNA preparation.…”

Serotonin 2C Receptor (5-HT2CR) mRNA Editing–Induced Down-Regulation of 5-HT2CR Function in Xenopus Oocytes: the Significance of Site C Editing

“…Xenopus oocytes have been used to evaluate the functions of membrane proteins such as receptors and ion channels which can be translated from mRNAs in oocytes (21,22). The present study took advantage of this model to clarify the functional significance of 5-HT2CR mRNA editing in the central nervous system.…”

Expression cloning human and rat renal cortex Na/Pi cotransporters: behind the scenes in the Murer laboratory

“…An elegant strategy to test the specific contribution of NaPi-IIa to overall P i transport in a mix of renal cortical RNA was adopted using RNaseH-mediated hybrid depletion [22]. Kidney RNA samples were hybridized with NaPi-II-specific DNA oligonucleotides and treated with RNase H, an endonuclease that specifically hydrolyses RNA hybridized to DNA.…”

“…An essential question remained unanswered, whether the identified gene/protein would respond to mediators of P i homeostasis such as P i availability and parathyroid hormone (PTH). An elegant strategy to test the specific contribution of NaPi-IIa to overall P i transport in a mix of renal cortical RNA was adopted using RNaseH-mediated hybrid depletion [ 22 ]. Kidney RNA samples were hybridized with NaPi-II-specific DNA oligonucleotides and treated with RNase H, an endonuclease that specifically hydrolyses RNA hybridized to DNA.…”