Maternally expressed gene 3 (MEG3) noncoding ribonucleic acid: isoform structure, expression, and functions

Abstract

Maternally expressed gene 3 (MEG3) is an imprinted gene highly expressed in the human pituitary. However, MEG3 expression is lost in human gonadotroph-derived pituitary adenomas and most human tumor cell lines. Expression of MEG3 in tumor cells results in growth suppression, p53 protein increase, and activation of p53 downstream targets. The MEG3 gene encodes a noncoding RNA of approximately 1700 nucleotides. There are 12 different MEG3 gene transcripts, generated by alternative splicing. They contain the common exons 1-3 and exons 8-10, but each uses one or more exons 4-7 in a different combination in the middle. MEG3 isoform expression patterns are tissue and cell type specific. Functionally, each isoform stimulates p53-mediated transactivation and suppresses tumor cell growth. We analyzed the secondary RNA folding structure of each MEG3 isoform, using the computer program mfold. All MEG3 RNA isoforms contain three distinct secondary folding motifs M1, M2, and M3. Deletion analysis showed that motifs M2 and M3 are important for p53 activation. Furthermore, a hybrid MEG3 RNA, containing a piece of artificially synthesized sequence different from the wild type but folding into a similar secondary structure, retained the functions of both p53 activation and growth suppression. These results support the hypothesis that a proper folding structure of the MEG3 RNA molecule is critical for its biological functions. This study establishes for the first time the structure-function relationship of a large noncoding RNA and provides a first look into the molecular mechanisms of the biological functions of a large noncoding RNA.

Figure 1

The schematic representation of molecular structures for the human MEG3 gene and its mature isoform transcripts. Each box represents an exon. Exon 3a is an alternative exon that contains 40 extra nucleotides at the 3′-end of exon 3. The regions corresponding to folding motifs M1, M2, M3, and RM2B1 are indicated.

Figure 2

A, Stimulation of p53-mediated transactivation by MEG3 isoforms in reporter assays in HCT116 cells. For each activator (each MEG3 isoform or blank pCI-neo vector as a control), 10 ng of plasmid DNA were used. *, P < 0.01 compared with the control. B, A dose-dependent stimulation of p53-mediated transactivation by MEG3 (left panel) or MEG3a (right panel). For p53-Luc reporter, 50 ng were used in all experiments. The luciferase activity observed from the cotransfection of pCI-neo was designated as 1. Values are mean ± sd from six experiments for each construct. *, P < 0.01 compared with the control. C, Suppression of DNA synthesis by MEG3 isoforms. The expression vector for each MEG3 isoform or blank vector as a control was transfected into HCT116 cells, and BrdU incorporation assays were performed. The relative labeling index from the blank vector control was designated as 100. Values are mean ± sd from four experiments for each construct. *, P < 0.01 compared with the control. D, MEG3 isoform RNA expression in transfected cells measured by Northern blot. For control, a MEG3 expression vector without a promoter (DP) is used. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

Figure 3

Predicted RNA secondary folding structure of MEG3, MEG3a, MEG3b, and MEG3c, determined by mfold. The structures for additional MEG3 RNA isoforms can be found in the Supplemental Folding Structures. The three conserved motifs found in all isoforms, M1, M2, and M3, are indicated.

Figure 4

A, Stimulation of p53-mediated transactivation by MEG3 deletion mutants dM1, dM2, and dM3, in the reporter assays in HCT116 cells. For each activator (each MEG3 deletion mutant, wild-type MEG3, or blank pCI-neo vector as a control), 10 ng of plasmid DNA were used. For p53-Luc reporter, 50 ng were used. The luciferase activity observed from the cotransfection of pCI-neo was designated as 1. Values are mean ± sd from six experiments for each construct. *, P < 0.01 compared with the wild type. B, Suppression of DNA synthesis by MEG3 deletion mutants. The expression vector for MEG3, each deletion mutant or blank vector as a control was transfected into HCT116 cells, and BrdU incorporation assays were performed. Values are mean ± sd from four experiments for each construct. C, Expression of MEG3 and deletion mutants in transfected cells measured by Northern blot. For control, a MEG3 expression vector without a promoter (DP) is used. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

A, Comparison of the wild-type primary sequence in MEG3 cDNA (top) from position 542 to 698, composed of the major folding branch in M2, and the artificially synthesized primary cDNA sequence (bottom), which replaces the original sequence in MEG3-RM2B1. B, The folding structure of M2 in wild-type MEG3 RNA (left) and that of the hybrid MEG3-RM2B1 (right). C, Stimulation of p53-mediated transactivation by MEG3-RM2B1. For each activator, 250 ng of plasmid DNA were used. The luciferase activity observed from the cotransfection of pCI-neo was designated as 1. Values are mean ± sd from six experiments for each construct. *, P < 0.01 compared with the control. D, Suppression of DNA synthesis by MEG3-RM2B1. The expression vector for MEG3, MEG3-dM2, MEG3-RM2B1, or blank vector as a control was transfected into HCT116 cells, and BrdU incorporation assays were performed. Values are mean ± sd from four experiments for each construct. *, P < 0.01 compared with the control. E, Expression of MEG3, dM2, and RM2B1 in transfected cells measured by Northern blot. For control, a MEG3 expression vector without a promoter (DP) is used. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.