Hervé Seitz ¹, Neil Youngson ², Shau-Ping Lin ², Simone Dalbert ³, Martina Paulsen ³, Jean-Pierre Bachellerie ¹, Anne C Ferguson-Smith ² and Jérôme Cavaillé ¹

¹ LBME, Centre National de la Recherche Scientifique (UMR 5099), IFR 109, Université P. Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France.
² Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK.
³ Universität des Saarlandes, FR Genetik, Postfach 151150, D-66041 Saarbrücken, Germany.

Correspondence should be addressed to J Cavaillé.
e-mail:   cavaille@ibcg.biotoul.fr

We mapped four recently reported mouse tissue-specific miRNA genes [1] to an imprinted locus on mouse distal chromosome 12/human 14q32 (Fig. 1).

Figure 1: Several microRNA genes map within an imprinted locus on mouse distal 12 chromosome/human 14q32.

The position of several imprinted genes is indicated by squares (Meg3 and protein-coding genes), vertical bars (small nucleolar RNA genes) or triangles (miRNA genes). Experimentally detected [1] or predicted repeated gene copies of miRNAs (with hairpin structure 70 nt long; data not shown) are indicated by filled (red) or open triangles, respectively. Maternally expressed (red) and paternally expressed (blue) genes indicate the imprinting status. Several methylated regions are represented by circles (filled, hypermethylated; open, hypomethylated). In Mirg, spliced exons detected by genomic/cDNA comparison are represented by red boxes with dotted lines denoting splicing events. The maternally expressed antisense transcript to Rtl1 was initially suggested in sheep [14] (called anti-PEG11). The schematic is not drawn to scale.

Two of these, mir-136 and mir-127, are located near two CpG islands in a mouse retrotransposon-like gene, Rtl1, which is expressed exclusively from the paternal allele (Fig. 2a,b).

Figure 2: miR-127 and miR-136, two miRNAs expressed from the maternal chromosome, are antisense to Rtl1, a retrotransposon-like gene expressed only from the paternally inherited chromosome.

(a) Reciprocal imprinting of Rtl1, mir-127 and mir-136. Total or poly(A)+ RNAs were isolated from normal embryos and embryos with maternal or paternal uniparental disomy for chromosome 12 (mUPD12 and pUPD12, respectively). Paternal allelic expression of Rtl1 imprinting is evident on northern blots of poly(A)⁺ RNAs using normal and UPD12 embryos (top). Maternal-specific expression of the miRNAs is indicated by northern blots for miR-127 (middle) or by primer extension for miR-136 (bottom). P, ³²P-labeled primer. Probes for Gapd and let-7 were used as internal gel loading controls. Size is indicated in nucleotides. For unknown reasons, miR-136 could be detected only by primer extension.

(b) Strand-specificity of maternally and paternally transcribed transcripts at Rtl1. Total RNA from mUPD12 or pUPD12 embryos was subjected to strand-specific RT–PCR with appropriate primers (depicted on top). The antisense RNA was detected only in mUPD12 RNA, whereas the sense RNA was detected only in pUPD12 RNA (bottom left). miR-127 and miR-136 are transcribed as a large precursor (bottom right). In each case, the primers used are indicated under the corresponding lanes (01–04; sequences available on request). RT, reverse transcriptase.

(c) Mirg is maternally expressed in mouse embryos. Total RNA from normal, mUPD12 or pUPD12 embryos was reverse transcribed with primer Mirg8R. Subsequent PCR with primers Mirg7F and Mirg8R (primer sequences available on request) showed that Mirg was detected only in normal and mUPD12 RNA. Mirg7F and Mirg8R are located in the last two exons of the transcript (Fig. 1). As an additional control, RT–PCR (30 cycles) was done on the biallelically expressed gene Actb (encoding b-actin). RT, reverse transcriptase.

(d) Schematic representation of miR-127 and miR-136 complementary sites in the Rtl1 open reading frame (nucleotide numbering begins at the start codon).

These miRNA genes are transcribed in an antisense orientation to Rtl1 and are expressed exclusively from the maternal chromosome (Fig. 2a,b). The mechanism by which miR-127 and miR-136 are generated has yet to be determined, but miR-127 does not seem to be processed from long double-stranded RNAs formed by Rtl1 and anti-Rtl1 RNAs, as we did not detect any RNA species 21–23 nt long corresponding to anti-miR-127 (data not shown). Moreover, RT–PCR analysis indicates that these miRNAs are generated from a larger host transcript (Fig. 2b). The other two miRNA genes, mir-154 and mir-134, are located in a cluster of conserved repeated sequences [2] (A-repeats; Fig. 1). The gene mir-154 (A13) and several copies of related sequences are intron-encoded in Mirg (microRNA-containing gene), a novel maternally expressed gene that lacks an open
reading frame (Figs. 1 and 2c). As we could not detect them by northern-blot analysis, their imprinted status and mode of biosynthesis from Mirg introns have yet to be resolved.

Although some Rtl1 RNA is clearly detectable (Fig. 2a), the perfect complementarity between miR-136, miR-127 and the Rtl1 open reading frame (Fig. 2d) suggests they might function as small interfering RNAs [3, 4] to silence Rtl1, presumably in a tissue-specific manner. For instance, such a role could be achieved in the embryo brain or in the placenta, in which mir-127 and Rtl1 are co-expressed and reciprocally imprinted (data not shown). Moreover, an involvement of miRNAs in Rtl1 imprinting cannot be formally ruled out. Although RNA interference is believed to be a trans-acting mechanism, preferential nuclear degradation of the Rtl1 transcript from the maternal allele only, mediated by the sense/antisense gene organization of the Rtl1 miRNA genes, could account for Rtl1 imprinting. Alternatively or additionally, miRNAs might mediate chromatin epigenetic changes specifically at the maternal Rtl1 allele, consistent with the involvement of the RNAi machinery in heterochromatin formation [5-7]. RNA-directed allele-specific methylation of the two CpG islands in Rtl1 is unlikely, as they are hypermethylated on both alleles and this occurs even in pUPD12 embryos in which miRNAs are not expressed (N.Y., S.-P.L. and A.C.F.-S., manuscript in preparation). Efforts are underway to decipher whether these miRNAs function in the regulation of Rtl1 gene expression during mouse development or in adults.

The reciprocal imprinting of protein-coding genes and non-coding RNAs in imprinted clusters is a recurrent theme and suggests a regulatory role for non-coding RNAs in autosomal euchromatic epigenetic control [8]. Except for a direct role of the Air antisense RNA in the silencing of imprinted genes on mouse chromosome 17 (ref. 9), the involvement of RNA per se has not yet been shown. In contrast to plant miRNAs [3, 10], none of the metazoan miRNAs reported so far has perfect complementarity to any mRNA. Thus, the imprinted miR-127 and miR-136 are the first miRNAs fully complementary to an endogenous cellular mRNA in animals, suggesting that the RNAi machinery could regulate this gene. RNA interference is considered a defense
mechanism against mobile parasitic elements, including retrotransposons and retroviral-like sequences [11]; several imprinted genes share some features of these types of sequences [12, 13], notably the mouse gene Rtl1 identified as imprinted in this study. Thus, these findings identify a potential link between miRNAs, retrotransposon silencing and genomic imprinting.

Animal care.

All animal research was conducted in accordance with UK Government Home Office Licensing procedures.

GenBank accession numbers.

Mirg cDNA sequence, AJ517767; Rtl1, BK001261.

REFERENCES

1. Lagos-Quintana, M. et al. Curr. Biol. 12, 735-739 (2002).

2. Cavaille, J., Seitz, H., Paulsen, M., Ferguson-Smith, A.C. & Bachellerie, J.P. Hum. Mol. Genet. 11, 1527-1538 (2002).

3. Llave, C., Xie, Z., Kasschau, K.D. & Carrington, J.C. Science 297, 2053-2056 (2002).

4. Hutvagner, G. & Zamore, P.D. Science 297, 2056-2060 (2002).

5. Reinhart, B.J. & Bartel, D.P. Science 297, 1831 (2002).

6. Volpe, T.A. et al. Science 297, 1833-1837 (2002).

7. Hall, I.M. et al. Science 297, 2232-2237 (2002).

8. Rougeulle, C. & Heard, E. Trends Genet. 18, 434-437 (2002).

9. Sleutels, F., Zwart, R. & Barlow, D.P. Nature 415, 810-813 (2002).

10. Rhoades, M.W. et al. Cell 110, 513-520 (2002).

11. Plasterk, R.H. Science 296, 1263-1265 (2002).

12. Neumann, B., Kubicka, P. & Barlow, D.P. Nat. Genet. 9, 12-13 (1995).

13. Yoder, J.A., Walsh, C.P. & Bestor, T.H. Trends Genet. 13, 335-340 (1997).

14. Charlier, C. et al. Genome Res. 11, 850-862 (2001). 

This work was supported by grants from the Toulouse Génopole, the Programme Interdisciplinaire du Centre National de la Recherche Scientifique Dynamique et Réactivité des Assemblages Biologiques and La Ligue contre le Cancer/Comité de Haute-Garonne (to J.C.) and by laboratory funds from the Centre National de la Recherche Scientifique and Université Paul-Sabatier, Toulouse (to J.-P.B.). Work in M.P.'s laboratory was supported by the Deutsche Forschungs Gemeinschaft. N.Y. is supported by a BBSRC studentship and S.-P. L. by a scholarship from the Taiwanese government. H.S. is supported by a PhD fellowship Allocation de Moniteur
Normalien (Ministère de l'Education Nationale, de la Recherche et de la Technologie).

Competing interests statement:

The authors declare that they have no competing financial interests.

Additional References for RNA in gene imprinting:

1. Dallosso AR, Hancock AL, Brown KW, Williams AC, Jackson S, and Malik K, "Genomic imprinting at the WT1 gene involves a novel coding transcript (AWT1) that shows deregulation in Wilms' tumours".

2. Sleutels F, Zwart R, and Barlow DP, "The non-coding Air RNA is required for silencing autosomal imprinted genes".

3. Nikaido I, Saito C, Wakamoto A, Tomaru Y, Arakawa T, Hayashizaki Y, and Okazaki Y, "EICO (Expression-based Imprint Candidate Organizer): finding disease-related imprinted genes".

4. Han M-H, Goud S, Song L, and Fedoroff N, "The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation".

5. Lai EC, Wiel C, and Rubin GM, "Complementary miRNA pairs suggest a regulatory role for miRNA:miRNA duplexes".

6. Sen G, Wehrman TS, Myers JW, and Blau HM, "Restriction enzyme-generated siRNA (REGS) vectors and libraries".

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