Evidence for evolutionarily conserved secondary structure in the H19 tumor suppressor RNA

Abstract

The molecular basis for function of the mammalian H19 as a tumor suppressor is poorly understood. Large, conserved open reading frames (ORFs) are absent from both the human and mouse cDNAs, suggesting that it may act as an RNA. Contradicting earlier reports, however, recent studies have shown that the H19 transcript exists in polysomal form and is likely translated. To distinguish between possible functional roles for the gene product, we have characterized the sequence requirements for H19-mediated in vitro suppression of tumor cell clonogenicity and analyzed the sequence of the gene cloned from a range of mammals. A cDNA version of the human gene, lacking the unusually short introns characteristic of imprinted genes, is as effective as a genomic copy in blocking anchorage-independent growth by G401 cells. The first 710 nucleotides of the gene can be deleted with no effect on in vitro activity. Further truncations from either the 5'- or 3'-end, however, cause a loss of suppression of clonogenicity. Using conserved sequences within the H19 gene as PCR primers, genomic DNA fragments were amplified from a range of mammalian species that span the functional domain defined by deletion analysis. Sequences from cat, lynx, elephant, gopher and orangutan complement the previous database of sequences from human, mouse, rat and rabbit. Hypothetical translation of the resulting sequences shows an absence of conserved ORFs of any size. Free energy and covariational analysis of the RNA sequences was used to identify potential helical pairings within the H19 transcript. A set of 16 helices are supported by covariation (i.e. conservation of base pairing potential in the absence of primary sequence conservation). The predicted RNA pairings consist largely of local hairpins but also include several long range interactions that bridge the 5'- and 3'-ends of the functional domain. Given the evolutionary conservation of structure at the RNA level and the absence of conservation at the protein level, we presume that the functional product of the H19 gene is a structured RNA.

Figure 1

Mapping regions of the H19 RNA required for tumor suppressor activity. G401 cells were transfected with the indicated pMEP4-H19 constructs. Proliferation rates were quantified following plating at low density and monitoring growth over the period of 1 week. Effects of H19 on clonogenicity were determined after allowing transfected cells to grow for 3 weeks on soft agar as described in Materials and Methods. Shown are transfection results for vector control, H19 genomic DNA, H19 cDNA and deletion constructs obtained by restriction digestion using KpnI (K), XhoI (X) or EagI (E).

Figure 2

Sequence conservation in the H19 RNA. (A) Regions marking the primer sites used to generate the PCR products are shown along the H19 gene. The non-conserved 5′-extension in the database H19 sequences is indicated by shading. (B) A representative portion of the ClustalW alignment of H19 cDNAs shows regions of very high and low sequence conservation at the DNA level. Complete conservation is marked by an asterisk.

Figure 3

Average pairwise identity at the nucleotide level. The average pairwise identity of nucleotides in the alignments of erythropoeitin mRNA (A), 16S rRNA (B) and H19 mRNA (C) are calculated as described in Results and plotted as a function position along the sequence.

Figure 4

Average pairwise identity of ORFs. The average pairwise identity of all predicted ORFs in erythropoeitin (A), 16S rRNA (B) and H19 (C) are plotted as a function of position within the gene.

Figure 5

Secondary structure of the H19 RNA. (A) A sampling of helical pairings in the H19 alignment identified by X2s. Analogous base pairs are aligned horizontally. Pairs displaying covariation are colored. (B) A schematic diagram of the proposed secondary structure. The location of restriction sites used to generate truncated forms of H19 are marked with arrowheads. The length of unstructured linkers is not to scale.