Regulation of insulin preRNA splicing by glucose

Abstract

Glucose tightly regulates the synthesis and secretion of insulin by ␤ cells in the pancreatic islets of Langerhans. To investigate whether glucose regulates insulin synthesis at the level of insulin RNA splicing, we developed a method to detect and quantify a small amount of RNA by using the branched DNA (bDNA) signal-amplification technique. This assay is both sensitive and highly specific: mouse insulin II mRNA can be detected from a single ␤ cell (␤TC3 cells or mouse islets), whereas 1 million non-insulinproducing ␣ cells (␣TC1.6 cells) give no signal. By using intron and exon sequences, oligonucleotide probes were designed to distinguish the various unspliced and partially spliced insulin preRNAs from mature insulin mRNA. Insulin RNA splicing rates were estimated from the rate of disappearance of insulin preRNA signal from ␤ cells treated with actinomycin D to block transcription. We found that the two introns in mouse insulin II are not spliced with the same efficiency. Intron 2 is spliced out more efficiently than intron 1. As a result, some mRNA retaining intron 1 enters the cytoplasm, making up Ϸ2-10% of insulin mRNA in the cell. This partially spliced cytoplasmic mRNA is quite stable, with a half-life similar to the completely spliced form. When islets grown in high glucose are shifted to low glucose medium, the level of insulin preRNA and the rate of splicing fall significantly. We conclude that glucose stimulates insulin gene transcription and insulin preRNA splicing. Previous estimates of insulin transcription rates based on insulin preRNA levels that did not consider the rate of splicing may have underestimated the effect of glucose on insulin gene transcription.Insulin, the key signal for energy storage, is synthesized and secreted by the ␤ cells of the pancreatic islets of Langerhans in mammals. Insulin synthesis and secretion is tightly regulated by glucose, which serves as a signal of the energy state of the organism. Synthesis of mature insulin is a multistep process and glucose regulates synthesis at several of these steps, including transcription (1), mRNA stabilization (2), translation (3, 4), and processing proinsulin to insulin (5).The level of insulin mRNA available in the cytoplasm for translation to preproinsulin depends on the relative rates of insulin mRNA production and degradation. Glucose regulates both insulin gene transcription and insulin mRNA degradation (1, 2). Transcription rate alone, however, does not determine the production rate of cytoplasmic insulin mRNA. Prior to export from the nucleus, preRNAs are spliced and a 5Ј m 7 GpppN cap and 3Ј adenosines are added. These processes are not independent: splicing accelerates polyadenylylation, and both are important for nuclear export (6-8).Insulin preRNA in most species contains two introns. The sequences of the introns show little evidence of evolutionary conservation, but the positions of the introns are highly conserved (9, 10). The length of intron 2 varies widely among species, but intron 1 is generally small (118 ...

Glucose Regulation of Insulin Gene Transcription and Pre-mRNA Processing in Human Islets

“…3A-C) as well as mature mRNA (Fig. 3D and I) did not show significant changes in abundance after 60 min of exposure to actinomycin D. These data suggest that mature and intron 1-containing mRNAs are relatively long-lived species, with half-lives well in excess of 60 min, and are consistent with observations made in mouse islets (20,23). By con- …”

Variants in the Human Insulin Gene That Affect Pre-mRNA Splicing

“…Uridine substitutions in PPT position Ϫ6 have been shown to increase exon skipping and retention of weakly spliced introns (16), most likely by diminishing interaction between the PPT and the 65-kDa subunit of the U2 small nuclear RNA auxiliary factor or competing polypyrimidine binding proteins. INS intron 1 is weakly spliced in both the rat (17) and the mouse gene, where transcripts retaining this intron have been reported with higher translational efficiency (18). Absence of introns reduced the cytoplasmic level of rat insulin mRNA and protein expression by approximately sixfold (19), which is comparable with our observation (Fig.…”

Genetic analysis reveals that PAX6 is required for normal transcription of pancreatic hormone genes and islet development.

“…The net result is a marked signal amplification that is proportional to the concentration of the target sequence. Detailed methods for the mouse insulin II mRNA assay have been described (Wang et al 1997).…”