We report the identification of bovine miRNAs by cloning small RNAs from adipose tissue and the mammary gland. Fifty-nine distinct miRNAs were identified, five of them were not homologous to known mammalian miRNAs, and many of them had 3 0 and/or 5 0 end variants. Ribonuclease protection assays indicated that miR-23a and miR-24, whose genes are closely located on the same chromosome, were co-expressed in different tissues. The assays also suggested a role for several miRNAs in the mammary gland and a role for miR-133, a previously known skeletal and cardiac muscle-specific miRNA, in the rumen, an organ unique to the ruminant.

The transcription factor signal transducer and activator of transcription (STAT)-5 mediates GH stimulation of IGF-I gene expression in the liver. Previous studies suggested that STAT5 might exert this effect by binding to an IGF-I intron 2 region and a distal 5'-flanking region each containing two STAT5 binding sites. Here we report the identification of three additional chromosomal regions containing a total of five putative STAT5 binding sites that may mediate GH-induced STAT5 activation of IGF-I gene expression in the mouse liver. By comparing an 170-kb mouse genomic DNA containing the IGF-I gene with the corresponding human sequence, we identified 19 putative STAT5 binding sites that bear the consensus sequence of STAT5 binding site and are conserved across the two species. Chromatin immunoprecipitation assays indicated that five chromosomal regions containing a total of nine of the 19 putative STAT5 binding sites were bound by STAT5 in the mouse liver in response to GH administration and that these bindings preceded or coincided with GH-increased IGF-I gene transcription. Two of the five chromosomal regions correspond to those previously identified in other species, and the three new chromosomal regions that contain a total of five putative STAT5 binding sites are IGF-I intron 3 regions located at least 26 kb from the transcription start site. Gel-shift assays confirmed the binding of the five new putative STAT5 binding sites as well as three of the four previously identified STAT5 binding sites to GH-activated STAT5 from the mouse liver. Cotransfection analyses indicated that, although each of the five chromosomal regions was able to mediate STAT5 activation of reporter gene expression, together they mediated greater STAT5 activation of reporter gene expression in response to GH. Overall, these results suggest that GH-induced STAT5 activation of IGF-I gene expression in the mouse liver might be collectively mediated by at least eight STAT5 binding sites located in distal intronic and 5'-flanking regions of the IGF-I gene.

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