A comprehensive transcript map of the mouse Gnas imprinted complex

Abstract

The recent publication of the FANTOM mouse transcriptome has provided a unique opportunity to study the diversity of transcripts arising from a single gene locus. We have focused on the Gnas complex, as imprinting loci themselves provide unique insights into transcriptional regulation. Thirteen full-length cDNAs from the FANTOM2 set were mapped to the Gnas locus. These represented one previously described transcript and 12 putative new transcripts. Of these, eight were found to be differentially expressed from either the maternal or paternal allele. Two clones extended Nespas in the 3' direction, providing evidence of antisense transcription spanning a 30-kb genomic region from a single allele. The transcripts were summarized into six transcriptional units, Nespas, Nesp, Gnasxl, F7, exon 1A, and Gnas. The resolution of the Gnas transcript map by the FANTOM2 clones revealed a pattern of alternate splicing. In addition to the transcripts described previously as splicing onto exon 2 of Gnas, each new sense transcript had an alternate short 3'UTR independent of Gnas. Both spliced and unspliced variants of the new imprinted sense transcripts were found. Whereas the functional significance of these alternate transcripts is not known, the availability of the FANTOM clones has provided remarkable insights into the repertoire of transcripts in the Gnas complex locus.

Figure 1

Relative position of FANTOM2 cDNAs. Previously identifiedexons (solidboxes) and ESTs (open boxes) are shown above the line and FANTOM2 clones below. Asterisks indicate regions of homology to human A20 and A21 exons. Figure not to scale.

Figure 2

Imprinting status of FANTOM clones F1–F10. Each PCR product is sized with a -kb ladder (Invitrogen). MatDp(dist2) and PatDp(dist2) are abbreviatedto MatDp and PatDp, respectively. + and - refers to samples amplifiedin the presence (+) or absence (-) of reverse transcriptase. Oligo dT-primed cDNA was amplified with: (A) Hprt primers as an amplification control; (B) Nesp primers to confirm the MatDp(dist2) cDNA; and (C) Gnasxl primers to confirm the PatDp(dist2) cDNA. Strand-specific RT-PCR of FANTOM clones 1–10: (D) paternal expression of F1; (E) paternal expression of F2; (F) maternal expression of F3; (G) maternal expression of F4; (H) paternal expression of F5; (I) maternal expression of F7; (J) paternal expression of F8 and F9; and(K) bi-allelic expression of F10. The PCR products from F3 (F), F4 (G), F7 (I), and F8 (J) were blottedand probedas describedin Methods. (L) F1 is part of Nespas. PCR from exon IV of Nespas to F1 reveals a paternal-specific product. The products in A–C, G, H, and L were amplifiedfor 25 cycles. The products in D–F, I and J were amplifiedfor 30 cycles; the product in K was amplifiedfor 20 cycles. (L) 1-kb Ladder (Invitrogen).

Figure 3

Transcript map of the Gnas complex imprintedlocus. Six transcriptional units were definedfrom this study; these are Nespas, Nesp, Gnasxl, F7, exon 1A, and Gnas. Each transcriptional unit is associatedwith a differentially methylatedpromoter, andconsists of several splice variants. F7 is the exception, with unexpectedmaternal expression indicating a link to the Nesp promoter. The arrows show direction of transcription. Figure is not drawn to scale.