The product of the H19 gene may function as an RNA.

Abstract

The mouse H19 gene was identified as an abundant hepatic fetal-specific mRNA under the transcriptional control of a trans-acting locus termed raf. The For the majority of cloned eucaryotic genes, their protein products were already known prior to cloning. More recently, differential hybridization screens of cDNA libraries have been used to clone genes with specific tissue and temporal patterns of expression, without regard to their products. The murine H19 gene is such a gene. It was originally identified in a screen of a fetal liver cDNA library to find genes that were coordinately regulated with the a-fetoprotein (AFP) gene (25). The aim of the screen was to find fetal-specific mRNAs under the control of raf, a transacting locus that determines, at least in part, the adult basal level of AFP mRNA (2, 24). The only cDNA that fulfilled these criteria was the H19 cDNA. This gene was expressed at high levels in fetal and neonatal liver, and its 600-fold repression in the adult was under the control of raf. Its coordinate expression with AFP extended to the visceral endoderm of the yolk sac and the fetal gut. However, unlike AFP, H19 mRNA was also found in both fetal skeletal and cardiac muscle at high levels and at reduced levels in all adult muscle, although in these tissues it is not under the influence of raf (2).Sequence analysis of the single-copy H19 gene revealed the presence of multiple small open reading frames (ORFs), none of which spanned more than two of its five exons. The largest of these, called ORF5 (referred to as ORF1 in reference 26), was located entirely within the first exon and could potentially encode a 132-amino-acid protein. This ORF begins 680 bases downstream of the cap site and is preceded by four small ORFs, the largest of which could encode a protein of 27 amino acids. As there was no precedent for such a gene organization, we decided to investigate its possible significance.As a first step, we cloned and sequenced the H19 gene from the human genome, reasoning that a comparison between the murine and human homologs would identify regions that are conserved and therefore important to the function of the gene. The demonstration that the genes shared no ORF led us to investigate the subcellular distribution of H19 mRNA, which indicated that the majority of the mRNA is located in a cytoplasmic particle. * Corresponding author. MATERIALS AND METHODScDNA library construction. A 20-,Ig sample of poly(A)+ RNA from Hep3B human hepatoma cells in 90 ,l of distilled H20 was heat denatured at 65°C for 3 min and then placed at 4°C. To this was added 20 ,ug of oligo(dT) (Boehringer Mannheim Biochemicals), 80 U of RNasin (Promega Biotec), 1.5 mM each dGTP, dATP, dCTP, and dTTP, 50 mM Tris hydrochloride (pH 8.3), 10 mM MgCl2, 70 mM KCI, 10 mM dithiothreitol, and 300 U of reverse transcriptase (Life Sciences, Inc.) to a final reaction volume of 200 ,ul; the mixture was incubated at 42°C for 1 h. This preparation was then mixed with 400 ,u1 of a solution containing 20 mM Tris hydrochloride (pH 7.5), 5...

The expression of the imprinted genes H19 and IGF-2 in choriocarcinoma cell lines. Is H19 a tumor suppressor gene?

“…H19 RNA appears as 2.3 kb band corresponding to the sum of the ®ve exons of the H19 gene (Brannan et al, 1990). In the majority of the blots done by us and others a second band of approximately 4 kb is visible.…”

“…It is bound in 28S cytoplasmic RNPs and not to ribosomes (Brannan et al, 1990). Its function is enigmatic.…”

High density oligonucleotide array analysis of interferon-α2a sensitivity and transcriptional response in melanoma cells

“…Genes involved in the regulation of cell proliferation, such as IFI16, h19 and RCC1, but also hox2 (Acampora et al, 1989;Brannan et al, 1990;Bischoff and Ponstingl 1991;Trapani et al, 1994), were found to be preferentially expressed in sensitive cell lines. Intriguingly, genes encoding SHB and PKC-ζ proteins, known components of defined signal transduction pathways (Barbee et al, 1993;Welsh et al, 1994), appear to be preferentially expressed in IFN-α resistant cells (Figure 4, panels A and B).…”

“…2 of them, IFI16 and RCC1 encode nuclear proteins endowed with mitotic regulation and transcriptional activation capacities, respectively (Bischoff and Ponstingl, 1991;Trapani et al, 1994). A third is the hox2 homeobox gene (Acampora et al, 1989), whereas the fourth, h19 gene, encodes an untranslated RNA, involved in the DNA methylation and genetic imprinting processes (Brannan et al, 1990). Notably, however, RCC1 gene was not expressed in one IFN-α sensitive cell line (ME51).…”

Hypervariable allelic expression patterns of the imprinted IGF2 gene in tumor cells

“…Whereas IGF2 produces a growth factor in both mouse and man (Stewart and Rotwein, 1996), the function of H19 is enigmatic. In the mouse, sequences¯anking H19 control the activity of Igf2 in cis while the structural gene has no documented function in trans (Brannan et al, 1990;Leighton et al, 1995).…”