Regulated Intron Retention and Nuclear Pre-mRNA Decay Contribute toPABPN1Autoregulation

The poly(A)-binding protein nuclear 1 is encoded by thePABPN1gene, whose mutations result in oculopharyngeal muscular dystrophy, a late-onset disorder for which the molecular basis remains unknown. Despite recent studies investigating the functional roles of PABPN1, little is known about its regulation. Here, we show that PABPN1 negatively controls its own expression to maintain homeostatic levels in human cells. Transcription from thePABPN1gene results in the accumulation of two major isoforms: an unspliced nuclear transcript that retains the 3′-terminal intron and a fully spliced cytoplasmic mRNA. Increased dosage of PABPN1 protein causes a significant decrease in the spliced/unspliced ratio, reducing the levels of endogenous PABPN1 protein. We also show that PABPN1 autoregulation requires inefficient splicing of its 3′-terminal intron. Our data suggest that autoregulation occurs via the binding of PABPN1 to an adenosine (A)-rich region in its 3′ untranslated region, which promotes retention of the 3′-terminal intron and clearance of intron-retained pre-mRNAs by the nuclear exosome. Our findings unveil a mechanism of regulated intron retention coupled to nuclear pre-mRNA decay that functions in the homeostatic control of PABPN1 expression.

IGF-I and serine protease inhibitor 2·1 nuclear transcript abundance in rat liver during protein restriction

Restriction of dietary protein consumption of young male rats results in decreased growth velocity and a reduction in the abundance of hepatic IGF-I mRNA. It is not known whether the reduction in IGF-I mRNA abundance in the liver of protein-restricted rats results from a decrease in IGF-I gene transcription. In the present study, three experiments were performed with 4-week-old male rats to examine the effect of protein restriction on IGF-I gene transcription in liver. In these experiments, we monitored IGF-I nuclear transcripts (pre-mRNA) within total cellular RNA using a ribonuclease protection assay. In the first experiment, a consistent decrease in IGF-I mRNA from animals fed isocaloric diets containing 20% (control), 12%, 8% and 4% protein (dietary effect, P<0·001) was not paralleled by a decrease (P>0·50) in IGF-I pre-mRNA. Two additional experiments examining the effect of 4% vs 20% protein diets yielded comparable results. Pooled results from these two studies (n=12/treatment) demonstrated that a 64% reduction (P<0·0001) in IGF-I mRNA abundance was not accompanied by a decrease in IGF-I pre-mRNA (1·17 vs 1·31 ±0·21 image density units for 4% and 20% protein treatments). Unlike IGF-I, the abundance of carbamyl phosphate synthetase-I (CPS-I) pre-mRNA and mRNA was comparably reduced (∼70%, P<0·001), indicating that the decrease in mRNA of this urea cycle enzyme during protein restriction occurs predominantly by a transcriptional mechanism. A common feature of all experiments was a pronounced variability in the expression of hepatic IGF-I pre-mRNA among animals, which was not diet specific. To test whether the variability in IGF-I gene transcription was correlated with variability in the transcription of another gene that is regulated by GH, we quantified the abundance of nuclear transcripts for the serine protease inhibitor 2·1 (SPI 2·1 gene. A positive association (r=0·81, P<0·0001) between SPI 2·1 and IGF-I nuclear transcripts was demonstrated. The correlation between IGF-I and SPI 21 transcripts was specific, because the quantity of IGF-I and CPS-I nuclear transcripts was not correlated in this study. Although transcription of the IGF-I and SPI 2·1 genes was similar, the abundance of SPI 21 mRNA was not altered by protein deprivation. In summary, these studies indicated that protein restriction does not substantially alter the mean quantity of IGF-I nuclear transcripts, suggesting that the decrease in IGF mRNA occurs predominantly by a post-transcriptional mechanism. In addition, nuclear transcript abundance of the IGF-I and SPI 21 genes varies in a co-ordinate manner, supporting the hypothesis that transcription of these genes responds rapidly to a common variable factor such as plasma GH. Journal of Endocrinology (1995) 145, 397–407

Three-dimensional localization of poly(A) RNA and splicing components in the nucleus

The physical distribution of active genes has long been a subject of interest and speculation, however technical limitations have necessitated that it be addressed only by indirect approaches with sometimes contradictory results. However, developments in fluorescence in situ hybridization methodologies allow the position of specific genes and RNAs to be visualized directly within intact cells, thus providing a more direct means to study such questions. To address whether compartmentalization occurs during the production and processing of pre-mRNA in mammalian somatic cells we have recently investigated the distribution of nuclear polyadenylated transcripts which represent approximately 90% of all pre-mRNA. We found that poly(A) RNA forms discrete nuclear “transcript domains” which are specifically positioned with respect to the underlying genome and contain snRNP antigens of the pre-mRNA splicing class. Several lines of evidence indicate a close spatial and temporal linkage between transcription and processing of pol II RNAs (reviewed in 5), therefore, it is possible that poly(A) RNA transcript domains reflect a clustering of active genes at these sites.

Multiple inducers of the Drosophila heat shock locus 93D (hsr omega): inducer-specific patterns of the three transcripts.

The Drosophila hsr omega locus produces one of the largest and most active heat shock puffs, yet it does not encode a heat shock protein. Instead, this locus produces a distinctive set of three transcripts, all from the same start site. The largest transcript, omega 1, is limited to the nucleus and appears to have a role there. A second nuclear transcript, omega 2, is produced by alternative termination and contains the sequence found in the 5' 20-25% of omega 1 (depending on the Drosophila species). The cytoplasmic transcript, omega 3, is produced by removal of a 700-bp intron from omega 2. All three hsr omega RNAs are produced constitutively and production is enhanced by heat shock. In addition to being a member of the set of heat shock puffs, the hsr omega puff is induced by agents that do not affect other heat shock loci, suggesting that hsr omega is more sensitive to environmental changes than other loci. We report here that agents that induce puffing of hsr omega loci in polytene nuclei also lead to an increase in hsr omega transcripts in diploid cells. We also show that the relative levels of omega 1 and omega 3 can be modulated independently by several agents. All drugs that inhibit translation, either initiation or elongation, stabilize the omega 3 transcript, which normally turns over within minutes in control cells. Drugs (such as benzamide and colchicine) that induce puffing of hsr omega, but not other heat shock loci, lead to large increases in omega 1. Although the constitutive level of omega 1 is relatively stable, the drug-induced excess is lost rapidly when the drug is withdrawn. The relative levels of hsr omega transcripts may reflect different states in cellular metabolism.