140 CHARACTERIZATION OF THE BOVINE ANTISENSE TO INSULIN-LIKE GROWTH FACTOR TYPE 2 RECEPTOR (bAIRN) NONCODING RNA

Bovine insulin-like growth factor type 2 receptor (IGF2R) is an imprinted gene whose aberrant expression has been implicated in development of abnormal offspring syndrome. Gene IGF2R is a member of the IGF2R/AIRN imprinted gene cluster located on chromosome 9 in cattle. This imprinting cluster is flanked on the proximal 5′ end by MAS1. In the mouse, the antisense to Igf2r noncoding RNA, Airn, is a 108-kb polyadenylated transcript that regulates imprinted expression of Igf2r and other genes within the Igf2r/Airn cluster in association with implantation. Bovine AIRN (bAIRN) is expressed in post-implantation fetal tissues with expression coincident with imprinted expression of IGF2R. Further characterisation of bAIRN is needed before investigation of its potential role in regulating imprinted expression of IGF2R in cattle can be pursued. Therefore, the objective of this study was to identify the length and key characteristics of the bAIRN ncRNA transcript. The PCR primer sets (n = 26) were designed based on genomic sequences to walk down the predicted bAIRN ncRNA sequence by amplifying key segments located along the transcript. Total RNA, extracted from gestational day 150 bovine fetal liver, was DNase-treated to eliminate contaminating genomic DNA. The DNase-treated RNA was then used to generate cDNA as a template for PCR amplification. A putative promoter region containing the transcriptional start site for bAIRN was identified based on NCBI sequence data. This bAIRN promoter is located 441 bp upstream of differentially methylated region 2 (DMR2) within intron 2 of IGF2R and contains a CAAT-box, GC-box and TATA-box. The PCR primer sets (n = 2) designed to amplify products upstream of the putative promoter failed to produce any PCR amplicons. However, PCR primer sets (n = 6) designed to amplify segments located from 4 to 10 kb downstream of the putative promoter region produced amplicons that were sequence verified to be bAIRN ncRNA. Additional PCR primer sets (n = 3) produced sequence-verified amplicons that were located ∼27, 45 and 52 kb downstream of the putative bAIRN transcription start site. Between 27 and 44 kb downstream of the bAIRN promoter, a series of sequence duplications from MAS1 were identified within the bAIRN ncRNA. The role of these duplicated regions is currently unknown. Three PCR primer sets (n = 3) designed to amplify consecutive regions spanning from 93.4 to 101 kb downstream of the bAIRN promoter failed to produce amplicons, suggesting prior termination of the bAIRN transcript. In addition, a strong putative polyadenylation signal was identified at 65.4 kb downstream from the bAIRN promoter. In summary, the full-length bAIRN ncRNA has been characterised. The bAIRN promoter is located ∼440 bp upstream of DMR2 on the IGF2R antisense strand. Transcription from the bAIRN promoter results in a noncoding RNA with a length of ∼65 kb. The central third of the bAIRN transcript contains a series of sequence duplications from MAS1. The function of these duplications within the bAIRN ncRNA is currently unknown. This research was supported by the North Carolina Agricultural Experiment Station.