The methylation of internal adenosine residues in mRNA only occurs within GAC or AAC sequences. Although both of these sequence motifs are utilized, a general preference has been noted for the extended sequence RGACU. Not all RGACU sequences in an mRNA are methylated and the mechanisms that govern the selection of methylation sites in mRNA remain unclear. To address this problem we have examined the methylation of transcripts containing sequences of a natural mRNA, namely, bovine prolactin mRNA. In this mRNA, a specific AGACU sequence in the 3' untranslated region is the predominant site of methylation both in vivo and in vitro. The degree to which N6-adenosine methyltransferase recognizes the sequence context of the consensus methylation site was explored by mutational analysis of the nucleotides adjacent to the core sequence as well as the extended regions in which the core element was found. Our results indicate that efficient methylation depends on the extended five nucleotide consensus sequence but is strongly influenced by the context in which the consensus sequence occurs within the overall mRNA molecule. Furthermore, consensus methylation sites present in an RNA duplex are not recognized by the methyltransferase.
Two isoforms of the human growth hormone receptor (hGHR), which differ in the presence (hGHRwt) or absence (hGHRd3) of exon 3, are expressed in the placenta. Specifically, three expression patterns are observed: only hGHRwt, only hGHRd3, or an approximately 1:1 combination of both isoforms. We investigated several potential regulatory mechanisms which might account for the expression of the hGHR isoforms. The frequency of hGHRd3 expression did not change when placentas from differing stages of gestation were examined, suggesting splicing was not developmentally regulated. However, when hGHR isoform expression patterns were examined in each component of a given placenta, it was evident that alternative splicing of exon 3 is individualspecific. Surprisingly, the individual-specific regulation of hGHR isoforms appears to be the result of a polymorphism in the hGHR gene. We analyzed hGHRwt and hGHRd3 expression in Hutterite pedigrees, and our results are consistent with a simple Mendelian inheritance of two differing alleles in which exon 3 is spliced in an "all-or-none" fashion. We conclude the alternative splicing of exon 3 in hGHR transcripts is the result of an unusual polymorphism which significantly alters splicing of the hGHR transcript and that the relatively high frequency ("10O%) of homozygous hGHRd3 expression suggests the possibility it may play a role in polygenic determined events.In humans, chorionic somatomammotropin (hCS) and growth hormone (hGH) are encoded by a family of genes clustered on chromosome 17 (1). At least two forms of each hormone are encoded by separate genes for both hCS (hCS-A and hCS-B) and hGH (hGH-N and hGH-V). hGH-N, which is expressed only in the anterior lobe of the pituitary, is best known for its effects on the growth of skeletal and soft tissues and for its metabolic actions. The remaining proteins are expressed exclusively in the placenta. Their precise function remains obscure, and the picture is further complicated by the presence of alternatively spliced mRNAs that can give rise to additional isoforms.The synthesis and secretion of hCS, hGH-V, and their multiple isoforms in the placenta raises an important question as to whether a single receptor mediates the activity of these proteins or if different receptors exist. A receptor for hGH (hGHR) has been cloned (2) and shown to belong to the cytokine receptor family (3). A second, alternatively
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