Sequence Characterization of Bovine Antisense to Insulin-Like Growth Factor Type 2 Receptor Non-Coding RNA (AIRN)

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. Bovine AIRN (AIRN) is expressed in post-implantation fetal tissues coinciding with imprinted expression of IGF2R. Although expression patterns of bovine AIRN have been reported based on PCR analysis, characteristics of this transcript are unknown. Therefore, the objective of this work was to sequence characterize the AIRN ncRNA transcript. PCR primer sets (n = 19) were designed based on genomic sequence to “walk” down the predicted AIRN ncRNA sequence. Total RNA extracted from gestational Day 150 bovine fetal liver was used as source material for analysis. Extracted RNA was DNase-treated prior to cDNA synthesis, PCR amplified, and sequenced. A putative bAIRN promoter was located 623 base-pairs upstream of differentially methylated region 2 (DMR2) within intron 2 of IGF2R. A polyadenylation signal was found 117 kb downstream of the promoter. Primer sets designed upstream of the promoter as well as downstream of the polyadenylation signal yielded no PCR amplicons, suggesting that the length of AIRN is approximately 117 kb. In conclusion, bovine AIRN appears to be an antisense transcript of approximately 117 kb in length with a promoter region located 623 bp upstream of DMR2 within intron 2 of IGF2R.


Introduction
Transfer of in vitro produced (IVP) or somatic cell nuclear transfer (SCNT) manipulated bovine embryos results in a proportion of conceptuses, fetuses, and offspring that exhibit developmental abnormalities collectively referred to as Abnormal Offspring Syndrome (AOS) [1].Live offspring are generated from 45% of transferred IVP embryos with 5% to 20% of those exhibiting abnormalities depending on the culture system used [1].Some of the abnormalities are hypothesized to be derived from the inadequacy of the in vitro culture environment and disruption of epigenetic patterns regulating parent-specific expression of imprinted genes [2].
The insulin-like growth factor type 2 receptor (IGF2R) is an imprinted gene whose aberrant expression has been directly related to the overgrowth phenotype following the transfer of IVP embryos in sheep and implicated in overgrowth of bovine IVP fetuses [3][4][5][6].However, little is known about how imprinted expression of

cDNA synthesis
Prior to cDNA synthesis, 2 µg of wcRNA from a previously extracted fetal liver sample was DNase treated by incubation of the wcRNA with 1.5 µl of DNase and 2 µl of DNase buffer at 37 °C for 20 minutes.Following the manufacturer's instructions, 2 µg of DNase-treated wcRNA was incubated with 1 µg of random primers (Promega; Madison, WI), 1 µl of 10 mM dNTP mix (PCR Nucleotide Mix, Roche; Mannheim, Germany) and distilled water 40 mg) was removed from -80 °C storage, weighed, placed in a mortar, covered with liquid nitrogen, and subsequently crushed to a fine powder.The powder was homogenized (Brinkmann Homogenizer PT 10/35; Westbury, NY) and dissociated in lysis solution with mercapto-ethanol.RNA was extracted using a total RNA extraction kit (Sigma, GenElute Total Mammalian RNA Extraction Kit) following the manufacturer's protocols.The quality and integrity of the wcRNA was assessed by nanodrop using ratio of absorbances at 260 nm and 280 nm, which yielded an A260/A280 ratio of 2.11 for gestational Day 150 bovine fetal liver.genomic DNA sequence of chromosome 9 UMD 3.1 Primary Assembly AC_000166.1 (97,540,000 bp to 97,710,000 bp).All AIRN primer sets (Table 1) were designed using the primer design program, Primer3Plus (Untegrasser and Nijveen 2007, Wageninger University).All amplification products were sequence verified.

Reverse transcription-polymerase chain reaction (RT-PCR)
PCR reactions consisted of a 20 µl reaction volume that contained 100 ng of cDNA, 10 mM dNTP mix, Taq DNA polymerase (1.25 U per 20 µl reaction), sense and anti-sense primers (20 ng of each) and PCR water.PCR reactions were performed in a PTC-100 thermocycler (MJ Research, Inc., Watertown, MA).Annealing temperatures varied (Table 1) and were specific for each primer set evaluated in the primer walk.

Results and Discussion
In cattle, AIRN ncRNA is transcribed from the paternal allele in an antisense direction from a transcriptional start site located in exon 2 of IGF2R continuing into intron 1 of the neighboring proteincoding gene, MAS1 (Figure 1A).Within intron 2 lies a differentially methylated region, DMR2 (Figure 1B), that is 2620 bps in length and is unmethylated on the paternal allele and methylated on the at 65 °C for 5 minutes.After placement on ice for 1 minute, samples were incubated with 4 µl of 5X First Strand Buffer, 1 µl of 0.1 M DTT, and 1 µl of reverse transcriptase (200 U/µl); (Superscript III, Invitrogen; Carlsbad, CA) at 25 °C for 5 minutes.This was followed by incubation at 50 °C for 60 minutes and inactivation by heating to 70 °C for 15 minutes.The synthesized cDNA was subjected to purification using the QIAquick Purification Kit (Qiagen; Qiagen Sciences, MD) according to the manufacturer's instructions.

Bovine AIRN primers
Bovine AIRN primers were designed using the known bovine  maternal one [5].Moving in an antisense direction, a putative AIRN transcriptional start site was identified 623 bp upstream of the DMR.The promoter region was identified by analyzing the genomic DNA sequence (AC_000166, 97664989 to 97664875) upstream of the DMR for essential promoter elements.A TATA-box was identified 20 bp upstream of the putative transcriptional start site [11,12] (Figure 2).An Activator Protein-1 (AP1) binding site, also known as a 12-O-Tetradecanoylphorbol-13-acetate (TPA) response element, was located upstream of the TATA-box [13].In addition, downstream of the TATA-box we found an initiation response element (INR) and a down-stream promoter element.
Interestingly, as has been observed in the murine Airn sequence, there are 4 initiation sites that are clustered together in a 200 bp region in the bovine AIRN promoter region [11].Also intriguing, is the observation that spread out over a 500 bp region are various promoter elements.Within this region are additional TATA boxes, CAAT boxes, AP1 binding sites, INR elements, and DPEs.These observations are consistent with those made of the murine promoter for Airn [11].
Primers for AIRN (Sets 1 and 2) were designed to amplify regions upstream of the putative promoter and downstream of IGF2R exon 3 (Figure 3A).These upstream primers were subjected to varying PCR conditions, including varied concentrations of cDNA template (50 ng, 100 ng, 150 ng), varied annealing temperatures (50 to 65 °C), and altered annealing and extension times (10 to 60s).All attempts to generate a PCR amplicon within this region failed.This suggests that the true promoter region for bovine AIRN is located approximately 623 bp upstream of DMR2 within intron 2 of IGF2R.
A series of primers for AIRN (n = 19) were designed to PCR amplify regions throughout the predicted length of bAIRN (Figure 3).The PCR amplicons generated covered a total of 27,202 bps of the predicted AIRN transcript.Representative gel images of amplicons produced from PCR primer sets are illustrated in Figure 4.
A putative poly-A signal (AATAAA) was identified 270 bps downstream of AIRN primer set 17 (Figure 5).Two additional AIRN primer sets (18 and 19) were designed to amplify products downstream of the poly-A signal (Figure 3C).Amplicons were not obtained from PCR amplification using these primer sets.Therefore, the putative length of bovine AIRN is 117 kb.Amplification of PCR products from primers spread over a 120 kb region from a putative promoter in intron 2 of IGF2R to a putative poly-A signal in intron 1 of MAS1 suggest that AIRN is intronless.However, because only 23% of the putative AIRN noncoding RNA was sequenced in this effort, the possibility remains that the bovine AIRN transcript may be shorter than 117 kb or may contain introns.Additional poly-A signals (n = 3) can be found throughout the region between the second exon of MAS1 and the first exon of IGF2R.These additional poly-A signals may represent additional transcript termination sites.
The murine Airn ncRNA sequence has been reported and was found to be 118 kb in length [11,14].Although the length of bovine AIRN ncRNA appears to be similar to that of the mouse, there is a lack of sequence homology between the two species and some of the core promoter elements also differ.Eukaryotic genomes exhibit a vast range of ncRNAs, however, closer examination of these lncRNAs demonstrated that they may not share close sequence homology between mammalian species [15].Differences in sequence homology may arise from the presence of transposable elements that are common to mature lncRNAs but rarely occur in transcripts from proteincoding genes [15][16][17].Therefore, it may not be surprising that bovine AIRN and murine Airn do not share extensive sequence homology throughout their given lengths.

Conclusion
Bovine AIRN has been characterized as a long non-coding RNA that is estimated to be approximately 117 kb in length.The transcriptional start site is located outside of the imprint control region located within intron 2 of IGF2R.Transcription appears to be continuous from the putative promoter to a poly-A site within intron 1 of MAS1.The AIRN transcript is repeat rich and collinear with the genomic sequence.Based on the known function of murine Airn [18,19], it is presumed that bovine AIRN functions to regulate imprinted expression of IGF2R [20,21].Further studies will be needed to confirm the functional role of bovine AIRN in regulating imprinted expression of IGF2R or other protein-coding genes.

Reverse 5 ' 1 Reverse 5 'Figure 1 :
Figure 1: Illustration of the bovine gene region on Chromosome 9 specific to IGF2R and AIRN. A. Region illustrating the orientation of the sense genes MAS1 and IGF2R to the antisense gene for the non-coding RNA, AIRN.Numbers indicate exons 1 and 2 or exons 1, 2, and 3 of the MAS1 and IGF2R gene sequences, respectively; B. Illustration of the overlap between IGF2R and AIRN.The numbered blocks represent exons 1, 2, and 3 of the IGF2R gene sequence.The open rectangles indicate the location of Differentially Methylated Regions 1 and 2 (DMR1 and DMR2) with the IGF2R gene sequence.

Figure 2 :
Figure 2: Putative promoter for AIRN.Sequence showing transcription initiation site and consensus binding sites of core promoter elements.Nucleotide numbers refer to the location on chromosome 9 Primary Assembly UMD 3.1 (ACC_000166).

Figure 3 :
Figure 3: (A) Illustration of the overlap between IGF2R and AIRN at exons 2 and 3 of IGF2R.Vertical Bars depict exons 2 and 3 of the IGF2R gene sequence connected by arrows indicating direction of transcription.Horizontal Bars 1 to 8 indicate the location of PCR primer products for AIRN through the first 10 kb of transcription; (B) Illustration of the overlap between IGF2R and AIRN between exons 1 and 2 of IGF2R.Vertical bars depict Exons 1 and 2 of the IGF2R gene sequence connected by arrows indicating the direction of transcription.Horizontal Bars 8 to 11 indicate the location of PCR primer products for AIRN from 10 kb to 29 kb of the putative AIRN transcript; (C) Illustration of the overlap between MAS1 and AIRN and depiction of PCR amplicons for AIRN for the terminal portion of transcription.Vertical Bars depict the exons 1 and 2 or exon 1 of the MAS1 and IGF2R gene sequences, respectively.Horizontal Bars 12 to 19 indicate the location of PCR primer products for AIRN through the last 23 kb of AIRN transcription.For all panels; (A-C) open horizontal bars indicate that amplicons were not generated, closed horizontal bars indicate that PCR amplicons for AIRN were produced.

Figure 6 :
Figure 6: Supplemental Material: Individual gel images of PCR amplicons for bovine AIRN non-coding RNA.Set numbers correspond to PCR primer sets listed in table 1 (W, Water Blank; M, Ladder Marker).

Table 1 :
Primers used for RT-PCR to detect the non-coding RNA, AIRN.
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