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.     

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.     

Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels

Mutations in the period (per) gene of Drosophila melanogaster affect both circadian and ultradian rhythms. Levels of per gene product undergo circadian oscillation, and it is now shown that there is an underlying oscillation in the level of per RNA. The observations indicate that the cycling of per-encoded protein could result from per RNA cycling, and that there is a feedback loop through which the activity of per-encoded protein causes cycling of its own RNA.     

Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses

IN mammalian muscle, the subunit composition of the nicotinic acetylcholine receptor (AChR) and the distribution of AChRs along the fibre are developmentally regulated. In fetal muscle, AChRs are distributed over the entire fibre length whereas in adult fibres they are concentrated at the end-plate. We have used in situ hybridization techniques to measure the development of the synaptic localization of the messenger RNAs (mRNAs) encoding the alpha-subunit and the epsilon-subunit of the rat muscle AChR. The alpha-subunit is present in both fetal and adult muscle, whereas the epsilon-subunit appears postnatally and specifies the mature AChR subtype. The synaptic localization of alpha-subunit mRNA in adult fibres may arise from the selective down-regulation of constitutively expressed mRNA from extrasynaptic fibre segments. In contrast, epsilon-subunit mRNA appears locally at the site of neuromuscular contact and its accumulation at the end-plate is not dependent on the continued presence of the nerve terminal very early during synapse formation. This suggests that epsilon-subunit mRNA expression is induced locally via a signal which is restricted to the end-plate region and is dependent on the presence of the nerve only during a short period of early neuromuscular contact. Evidently, several mechanisms operate to confine AChR mRNAs to the adult end-plate region, and the levels of alpha-subunit and epsilon-subunit mRNAs depend on these mechanisms to differing degrees.     

Selective localization of messenger RNA for cytoskeletal protein MAP2 in dendrites

For nerve cells to develop their highly polarized form, appropriate structural molecules must be targeted to either axons or dendrites. This could be achieved by the synthesis of structural proteins in the cell body and their sorting to either axons or dendrites by specific transport mechanisms. For dendrites, an alternative possibility is that proteins could be synthesized locally in the dendritic cytoplasm. This is an attractive idea because it would allow regulation of the production of structural molecules in response to local demand during dendritic development. The feasibility of dendritic protein synthesis is suggested both by the existence of dendritic polyribosomes and by the recent demonstration that newly synthesized RNA is transported into the dendrites of neurons differentiating in culture. However, to date there has been no demonstration of the selective synthesis of an identified dendrite-specific protein in the dendritic cytoplasm. Here, we use in situ hybridization with specific complementary DNA probes to show that messenger RNA for the dendrite-specific microtubule-associated protein MAP2 (refs 3-5) is present in dendrites in the developing brain. By contrast the mRNA for tubulin, a protein present in both axons and dendrites is located exclusively in neuronal cell bodies.     

Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes

The product of the yeast PRP22 gene acts late in the splicing of yeast pre-messenger RNA, mediating the release of the spliced mRNA from the spliceosome. The predicted PRP22 protein sequence shares extensive homology with that of PRP2 and PRP16 proteins, which are also involved in nuclear pre-mRNA splicing. The homologous region contains sequence elements characteristic of several demonstrated or putative ATP-dependent RNA helicases. A putative RNA-binding motif originally identified in bacterial ribosomal protein S1 and Escherichia coli polynucleotide phosphorylase has also been found in PRP22.     

A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis

The telomerase enzyme of Tetrahymena synthesizes repeats of the telomeric DNA sequence TTGGGG de novo in the absence of added template. The essential RNA component of this ribonucleoprotein enzyme has now been cloned and found to contain the sequence CAACCCCAA, which seems to be the template for the synthesis of TTGGGG repeats.     

RNA干涉及其在肿瘤研究中的应用

RNAi是双链RNA介导的转录后基因沉默的过程,是一种高效的高特异性抑制基因表达的新途径。通过双链小干涉RNA(siRNA)与一系列蛋白质结合形成siRNA诱导的沉默复合体(RISC)并活化,然后,RISC对靶基因进行识别、降解。与反义方法相比,siRNA具有更好的抑制效果。RNAi的应用将为癌症的基因治疗提供新的方法。