Regulation of glucose transporter messenger RNA in insulin-deficient states

Recent studies have indicated that a family of structurally related proteins with distinct but overlapping tissue distributions are responsible for facilitative glucose transport in mammalian tissues. Insulin primarily stimulates glucose transport by inducing the redistribution of a unique glucose transporter protein from an intracellular pool to the plasma membrane. This 509-amino-acid integral membrane protein, termed GLUT-4, is the main insulin-responsive glucose transporter in adipose and muscle tissues. We have observed a dramatic decrease (tenfold) in the steady-state levels of GLUT-4 messenger RNA in adipose tissue from fasted rats or rats made insulin deficient with streptozotocin. Insulin treatment of the streptozotocin-diabetic rats or refeeding the fasted animals causes a rapid recovery of the GLUT-4 mRNA to levels significantly above those observed in untreated control animals. By contrast, the levels of the erythrocyte/HepG2/rat brain-type glucose transporter mRNA remain essentially unchanged under these conditions. These data suggest that the in vivo expression of GLUT-4 mRNA in rat adipose tissue is regulated by insulin.     

An intron with a constitutive transport element is retained in a Tap messenger RNA

Alternative splicing is a key factor contributing to genetic diversity and evolution. Intron retention, one form of alternative splicing, is common in plants but rare in higher eukaryotes, because messenger RNAs with retained introns are subject to cellular restriction at the level of cytoplasmic export and expression. Often, retention of internal introns restricts the export of these mRNAs and makes them the targets for degradation by the cellular nonsense-mediated decay machinery if they contain premature stop codons. In fact, many of the database entries for complementary DNAs with retained introns represent them as artefacts that would not affect the proteome. Retroviruses are important model systems in studies of regulation of RNAs with retained introns, because their genomic and mRNAs contain one or more unspliced introns. For example, Mason-Pfizer monkey virus overcomes cellular restrictions by using a cis-acting RNA element known as the constitutive transport element (CTE). The CTE interacts directly with the Tap protein (also known as nuclear RNA export factor 1, encoded by NXF1), which is thought to be a principal export receptor for cellular mRNA, leading to the hypothesis that cellular mRNAs with retained introns use cellular CTE equivalents to overcome restrictions to their expression. Here we show that the Tap gene contains a functional CTE in its alternatively spliced intron 10. Tap mRNA containing this intron is exported to the cytoplasm and is present in polyribosomes. A small Tap protein is encoded by this mRNA and can be detected in human and monkey cells. Our results indicate that Tap regulates expression of its own intron-containing RNA through a CTE-mediated mechanism. Thus, CTEs are likely to be important elements that facilitate efficient expression of mammalian mRNAs with retained introns.     

A tripeptide 'anticodon' deciphers stop codons in messenger RNA

The two translational release factors of prokaryotes, RF1 and RF2, catalyse the termination of polypeptide synthesis at UAG/UAA and UGA/UAA stop codons, respectively. However, how these polypeptide release factors read both non-identical and identical stop codons is puzzling. Here we describe the basis of this recognition. Swaps of each of the conserved domains between RF1 and RF2 in an RF1-RF2 hybrid led to the identification of a domain that could switch recognition specificity. A genetic selection among clones encoding random variants of this domain showed that the tripeptides Pro-Ala-Thr and Ser-Pro-Phe determine release-factor specificity in vivo in RF1 and RF2, respectively. An in vitro release study of tripeptide variants indicated that the first and third amino acids independently discriminate the second and third purine bases, respectively. Analysis with stop codons containing base analogues indicated that the C2 amino group of purine may be the primary target of discrimination of G from A. These findings show that the discriminator tripeptide of bacterial release factors is functionally equivalent to that of the anticodon of transfer RNA, irrespective of the difference between protein and RNA.