West, Cyrille Andrew; (1997) Regulation of insulin-like growth factor-I: Gene expression in bone cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The rat insulin-like growth factor-I (IGF-I) gene is characterised by the synthesis of multiple mRNA transcripts differing in the 5'- and 3'-untranslated regions (UTRs). Transcription is initiated from either of the leader exons, exons 1 or 2, with further heterogeneity in the 5'-UTR conferred by multiple transcription start sites and differential splicing within exon 1. Differences in the nature of transcript expression are likely to reflect cell-and hormone-response-specific regulation. IGF-I is important in skeletal growth and development, therefore, the initial aim of this study was to determine the pattern of transcript expression in osteoblast-enriched primary cultures from rat long bones. Subsequently, a possible role for RNA-protein interactions in the regulation of translation of IGF-I was examined. The presence of all IGF-I transcripts was demonstrated by RT-PCR, while Northern analysis and RNase protection assays revealed that transcript expression in osteoblasts was heterogeneous and differed from that in liver. The relative proportion of transcripts varying in the 5'-UTR was quantitated and revealed a lower expression of exon 2-derived transcripts in bone cells compared to liver and that individual start site usage in exon 1 differed greatly. Transcripts synthesised by osteoblasts and whole bone were predominantly initiated from start site 3. In contrast, the majority of the transcripts in liver were the alternatively spliced mRNAs. Interestingly, the transcripts predominating in bone cells are the less translatable mRNA variants. Such heterogeneity is often indicative of post-transcriptional regulation, which is also likely to involve regulatory proteins. Indeed, crosslinking experiments revealed proteins with apparent molecular masses of 48, 63 and 69 kDa binding with different affinities to the different 5'-UTRs. In summary, since the mRNA variants exhibit different properties, including half-life and translatability, such cell-specific variation in their relative expression and the transcript-specific interactions with cytosolic proteins may reflect differential regulation of IGF-I synthesis in these tissues.

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