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An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region.

ABSTRACT: The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.

SUBMITTER: Thakur N

PROVIDER: S-EPMC515059 | biostudies-other | 2004 Sep

REPOSITORIES: biostudies-other

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Project description:BackgroundGenomic imprinting leads to maternal expression of IGF2R in both mouse and opossum. In mouse, the antisense long noncoding (lnc) RNA Airn, which is paternally expressed from the differentially methylated region (DMR) in the second intron of Igf2r, is required to silence the paternal Igf2r. In opossum, however, intriguingly, the DMR was reported to be in a different downstream intron (intron 11) and there was no antisense lncRNA detected in previous analyses. Therefore, clarifying the imprinting mechanism of marsupial IGF2R is of great relevance for understanding the origin and evolution of genomic imprinting in the IGF2R locus. Thus, the antisense lncRNA associated with the marsupial DMR can be considered as the 'missing link'. In this study, we identified a novel antisense lncRNA, ALID, after detailed analysis of the IGF2R locus in an Australian marsupial, the tammar wallaby, Macropus eugenii, and compared it to that of the grey short-tailed opossum, Monodelphis domestica.ResultsTammar IGF2R showed maternal expression and had a maternally methylated CpG island (CGI) in intron 12 as well as a promoter CGI without differential methylation, but none in the second intron. Re-analysis of the IGF2R of opossum detected the CGI in intron 12, not intron 11, as previously reported, confirming that the DMR in intron 12 is conserved between these marsupials and so is the putative imprinting control region of marsupial IGF2R. ALID is paternally expressed from the middle of the DMR and is approximately 650 bp long with a single exon structure that is extremely short compared to Airn. Hence, the lncRNA transcriptional overlap of the IGF2R promoter, which is essential for the Igf2r silencing in the mouse, is likely absent in tammar. This suggests that fundamental differences in the lncRNA-based silencing mechanisms evolved in eutherian and marsupial IGF2R and may reflect the lack of differential methylation in the promoter CGI of marsupial IGF2R.ConclusionsOur study thus provides the best candidate factor for establishing paternal silencing of marsupial IGF2R without transcriptional overlap, which is distinct from the Igf2r silencing mechanism of Airn, but which may be analogous to the mode of action for the flanking Slc22a2 and Slc22a3 gene silencing in the mouse placenta.

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An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region.

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