2 DETECTION OF ANTISENSE TO Igf2r (AIR) RNA IN CATTLE

Abstract

The insulin-like growth factor type 2 receptor (Igf2r) regulates fetal growth by removing Igf2 from circulation, thus preventing overgrowth. In mice, expression of the Igf2r gene is imprinted only after implantation and is associated with expression of the antisense non-coding (nc)RNA, Air. In contrast, the human IGF2R gene is not imprinted and AIR ncRNA does not exist. Because it is known that the Igf2r gene is imprinted in cattle, the objectives of this study were to determine if Air ncRNA exists in cattle and, if so, whether bovine Air (bAir) is expressed during both pre- and post-implantation development. For objective 1, primer sets were designed for bAir based on bovine genomic sequence. The primer set, bAir3, was used to amplify a region of bAir corresponding to an antisense segment within intron 1 of Igf2r. Primer set bAir4 amplified a segment of bAir ncRNA corresponding to an antisense region upstream of the 52-untranslated region of Igf2r. Pools of whole-cell RNA were extracted from bovine fetal liver and subjected to DNase treatment, reverse transcription (RT), and PCR. Control RT reactions included RT without superscript and RT without superscript or DNase. Controls confirmed that amplification products resulted from RNA present in the sample and not from genomic DNA contamination. Amplicons were obtained for both the bAir3 and the bAir4 primer sets and were sequence verified, demonstrating that bAir ncRNA does exist in cattle. For objective 2, conceptuses (n = 4; mean ± SEM length: 2.8 ± 0.3 mm) derived from transfer of frozen-thawed in vivo-produced blastocysts were recovered from cows on Day 15 of gestation and snap-frozen for RNA extraction. Samples of liver from in vivo-produced bovine fetuses recovered at Day 70 of gestation (n = 7) were snap-frozen for RNA extraction. Semi-quantitative RT-PCR assays were performed to assess levels of mRNA for Igf2r and H2a, as well as ncRNA for bAir. All conceptus and fetal liver cDNA samples were run in duplicate within the same assay. Relative RNA expression was calculated as the ratio of band intensities of the RNA of interest to that of H2a. Data for relative RNA expression were analyzed by Student's t-test. H2a and Igf2r mRNAs were expressed in all Day 70 fetal liver and Day 15 conceptus samples. Relative levels of Igf2r did not differ (P = 0.19) with stage of development (0.15 ± 0.09 v. 0.36 ± 0.12 for Day 70 v. Day 15). bAir ncRNA was expressed in 7 of 7 samples of Day 70 fetal liver, whereas only 1 of 4 conceptuses expressed a faint bAir ncRNA signal based on either the bAir3 or bAir4 primer sets (χ² = 7.23, P < 0.01). Relative levels of bAir ncRNA were greater (P < 0.001) in Day 70 fetal liver compared to those in Day 15 conceptuses for amplicons bAir3 (0.376 ± 0.039 v. 0.028 ± 0.051) and bAir4 (0.101 ± 0.008 v. 0.003 ± 0.010). In conclusion, the antisense ncRNA, Air, does exist in cattle and its relative expression is greatest following implantation. These observations are consistent with murine data and suggest that bAir may be involved in regulating imprinted expression of Igf2r in cattle.