Expression and stability of insulin-like growth factor-I (IGF-I) mRNA splicing variants in the GH3 rat pituitary cell line

We have employed Northern blot analyses and solution hybridization/RNase protection assays to evaluate the presence and stability of IGF-I mRNA splicing variants in the GH3 rat pituitary cell line. All of the IGF-I mRNA size classes and IGF-I mRNAs with alternately-spliced 5'-untranslated regions and E-peptide coding regions seen in adult rat liver also were present in GH3 cells, although the proportions of the 5' splicing variants were significantly altered. In actinomycin D-treated cells, all IGF-I mRNA splicing variants were equally stable; thus, changes in the levels of some splicing variants were not due to differential mRNA stability. Additionally, all IGF-I mRNA size classes seen on Northern blots were equally stable; this data suggests that the large IGF-I mRNA species is not a precursor of the smaller species.     

Donor site competition is involved in the regulation of alternative splicing of the rat beta-tropomyosin pre-mRNA

The rat beta-tropomyosin (beta-TM) gene encodes both skeletal muscle beta-TM mRNA and nonmuscle TM-1 mRNA via alternative RNA splicing. This gene contains eleven exons: exons 1-5, 8, and 9 are common to both mRNAs; exons 6 and 11 are used in fibroblasts as well as in smooth muscle, whereas exons 7 and 10 are used in skeletal muscle. Previously we demonstrated that utilization of the 3' splice site of exon 7 is blocked in nonmuscle cells. In this study, we use both in vitro and in vivo methods to investigate the regulation of the 5' splice site of exon 7 in nonmuscle cells. The 5' splice site of exon 7 is used efficiently in the absence of flanking sequences, but its utilization is suppressed almost completely when the upstream exon 6 and intron 6 are present. The suppression of the 5' splice site of exon 7 does not result from the sequences at the 3' end of intron 6 that block the use of the 3' splice site of exon 7. However, mutating two conserved nucleotides GU at the 5' splice site of exon 6 results in the efficient use of the 5' splice site of exon 7. In addition, a mutation that changes the 5' splice site of exon 7 to the consensus U1 snRNA binding site strongly stimulates the splicing of exon 7 to the downstream common exon 8. Collectively, these studies demonstrate that 5' splice site competition is responsible, in part, for the suppression of exon 7 usage in nonmuscle cells.     

Elements of the rat tropoelastin gene associated with alternative splicing

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA.     

Distribution and regulation of rat insulin-like growth factor I messenger ribonucleic acids encoding alternative carboxyterminal E-peptides: evidence for differential processing and regulation in liver

Alternative splicing of insulin-like growth factor I (IGF-I)/somatomedin C mRNAs generates two IGF-I mRNAs coding for IGF-I peptides with different sequences in the E domain of the IGF-I prohormone. These two mRNAs encode alternative E peptides due to the presence (IGF-Ib) or absence (IGF-Ia) of a 52-base insert in the region coding for the E domain. We have used a solution hybridization/RNase protection assay to determine the tissue distribution and regulation by GH of the expression of these alternative IGF-I mRNAs. IGF-Ib mRNAs are present in low abundance (representing approximately 2.5% of the total IGF-I mRNA) in heart, lung, muscle, testes, stomach, kidney, and brain, but represent approximately 13% of the IGF-I mRNA in liver. GH treatment of hypophysectomized rats increased steady-state IGF-I mRNA levels in liver, kidney, lung, and heart. In kidney, lung, and heart, IGF-Ia and IGF-Ib mRNA levels were coordinately regulated by GH, but, in liver, the fold increase in IGF-Ib mRNA levels was approximately three times greater than the fold increase in IGF-Ia mRNA levels. These data suggest that the processing of IGF-I mRNA in liver is different than in nonhepatic tissues. These results also further elucidate the organization of the rat IGF-I gene as well as the generation of multiple IGF-I mRNAs by alternative splicing.