Identification of Intron and Exon Sequences Involved in Alternative Splicing of Insulin Receptor Pre-mRNA

Abstract

The insulin receptor exists as two isoforms, A and B, that result from alternative splicing of exon 11 in the primary transcript. We have shown previously that the alternative splicing is developmentally and hormonally regulated. Consequently, these studies were instigated to identify sequences within the primary RNA transcript that regulate the alternative splicing. Minigenes containing exons 10, 11, and 12 and the intervening introns were constructed and transfected into HepG2 cells, which contain both isoforms of the insulin receptor. The cells were able to splice the minigene transcript to give both A (؊ exon 11) and B-like (؉ exon 11) RNAs. A series of internal deletions within intron 10 were tested for their ability to give A and B RNAs. Intron 10 contained two sequences that modulated exon 11 inclusion; a 48-nucleotide purine-rich sequence at the 5 end of intron 10 that functions as a splicing enhancer and causes an increase in exon 11 inclusion, and a 43-nucleotide sequence at the 3 end of intron 10 upstream of the branch point sequence that favors skipping of exon 11. Increasing the length of the polypyrimidine tract at the 3 end of intron 10 caused exon 11 to be spliced constitutively, indicating that a weak splice site is required for alternative splicing. Finally, point mutations, insertions, and deletions within exon 11 itself were able to regulate inclusion of the exon both positively and negatively. The human insulin receptor (IR)1 is encoded by a single gene that is located on chromosome 19 and composed of 22 exons. The mature IR exists as two isoforms, designated A and B, which result from alternative splicing of the primary transcript (1-3). The A isoform lacks exon 11, is expressed ubiquitously, and is the only isoform in lymphocytes, brain, and spleen; the B isoform contains exon 11 and is expressed predominantly in liver, muscle, adipocytes, and kidney (4 -6). Exon 11 is composed of 36 nucleotides that encode a 12-amino acid segment (residues 717-728) of the carboxyl terminus of the ␣-subunit of IR. A number of investigators have suggested that the isoform ratio could be altered in non-insulin-dependent diabetes mellitus (7-10), but other studies have produced conflicting results (11-14). We have found that alterations in isoform ratio in skeletal muscle were associated with hyperinsulinemia rather than diabetes (15). Similar results have been found in the rhesus monkey (16). Along these lines, Sell and co-workers (17) have shown that alternative splicing of the IR gene is regulated by insulin in the Fao hepatoma cell line. Furthermore, we have shown that the alternative splicing is hormonally and developmentally regulated in both the HepG2 hepatoma and 3T3-L1 adipocyte cell lines (18). The changes in splicing were accompanied by increases in insulin sensitivity, as measured by a number of parameters (19). These data indicate that regulation of the alternative splicing of the IR is important for insulin sensitivity and responsiveness.Splicing of pre-mRNA depends on the presence of relatively...

Natural variation in ARF18 gene simultaneously affects seed weight and silique length in polyploid rapeseed

“…Comparative analysis showed that the possible branchpoints differed between the ARF18 genes from zy72360 and R1, suggesting that the disruption of the branchpoint in the sixth intron induced the loss of splicing function at the 3′ site in zy72360. In fact, exon skipping caused by an insertion in the sequence upstream of the 3′ splice site of the intron has been reported previously (32,33).…”

Post-transcriptional Regulation of Thyroid Hormone Receptor Expression by cis-Acting Sequences and a Naturally Occurring Antisense RNA

“…These elements appear to act as a decoy by directing the partial assembly of competing but non-functional spliceosomes. However, examples of purine-rich intronic splicing enhancers have also been identified, suggesting that purine-rich sequences within an intron can regulate splicing in a positive as well as negative manner (36,37).…”

Insulin Receptor and its Relationship with Different Forms of Insulin Resistance

“…The splicing site selection model during the insulin receptor transcript maturation was proposed in 1998 [72]. According to this model, the GA-rich sequence located on the 5' end of the intron 10 otherwise known as the intronic splicing enhancer sequence ISE, favours exon 11 inclusion in the mature transcript by direct interaction with 3' splicing site.…”

“…According to this model, the GA-rich sequence located on the 5' end of the intron 10 otherwise known as the intronic splicing enhancer sequence ISE, favours exon 11 inclusion in the mature transcript by direct interaction with 3' splicing site. It is possible that due to its location (more than 2 kb upstream branch point, BP), this region may interact with an adjacent 5' splice site (UAG:GUCAGGAC) considerably different from a consensus sequence (CAG:GUAAGUAU) [72]. Thus, the role of this enhancer may rely on the strengthening the interaction of U1snRNP (U-rich 1 small nuclear ribonucleoprotein particle) -one of the component of the spliceosome, with 5' splicing site [74].…”

“…43 bp upstream the branch point in intron 10, there is an intronic splicing enhancer sequence (ISE) as well as intronic splicing silencing sequence (ISS) located at its 3' end responsible for exon skipping. ISE sequence located at 5' end of intron 10 contains a GA-rich sequence 48 bp long, crucial for exon 11 inclusion in the mature mRNA molecule [72].…”