In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.

Mammalian bombesin-like peptides are widely distributed in the central nervous system as well as in the gastrointestinal tract, where they modulate smooth-muscle contraction, exocrine and endocrine processes, metabolism and behaviour. They bind to G-protein-coupled receptors on the cell surface to elicit their effects. Bombesin-like peptide receptors cloned so far include, gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor (NMB-R), and bombesin receptor subtype-3 (BRS-3). However, despite the molecular characterization of BRS-3, determination of its function has been difficult as a result of its low affinity for bombesin and its lack of an identified natural ligand. We have generated BRS-3-deficient mice in an attempt to determine the in vivo function of the receptor. Mice lacking functional BRS-3 developed a mild obesity, associated with hypertension and impairment of glucose metabolism. They also exhibited reduced metabolic rate, increased feeding efficiency and subsequent hyperphagia. Our data suggest that BRS-3 is required for the regulation of endocrine processes and metabolism responsible for energy balance and adiposity. BRS-3-deficient mice provide a useful new model for the investigation of human obesity and associated diseases.

RNA interference (RNAi) is the process of long, double-stranded (ds), RNA-dependent posttranscriptional gene silencing (PTGS). In lower eukaryotes, dsRNA introduced into the cytoplasm is cleaved by the RNaseIII-like enzyme, Dicer, to 21-23 nt RNA (short interfering [si] RNA), which may serve as guide for target mRNA degradation. In mammals, long-dsRNA-dependent PTGS is applicable only to a limited number of cell types, whereas siRNA synthesized in vitro is capable of effectively inducing gene silencing in a wide variety of cells. Although biochemical and genetic analyses in lower eukaryotes showed that Dicer and some PIWI family member proteins are essential for long-dsRNA-dependent PTGS, little is known about the molecular mechanisms underlying siRNA-based PTGS. Here, we show that Dicer and eIF2C translation initiation factors belonging to the PIWI family (eIF2C1-4) play an essential role in mammalian siRNA-mediated PTGS, most probably through synergistic interactions. Immunoprecipitation experiments suggest that, in human and mouse cells, complex formation occurs between Dicer and eIF2C1 or 2 and that the PIWI domain of eIF2C is essential for the formation of this complex.

Amphibian bombesin and its related peptides consist a family of neuropeptides in many vertebrate species. Bombesin and two major bombesin-like peptide in mammals, gastrinreleasing peptide (GRP) and neuromedin B (NMB), have been shown to elicit various physiological effects. These include inhibition of feeding, smooth muscle contraction, exocrine and endocrine secretions, thermoregulation, blood pressure and sucrose regulations and cell growth. Receptors for GRP and NMB (GRP-R and NMB-R), as well as third subtype of bombesin-like peptide receptor (BRS-3) have been cloned. These receptors are G-protein-coupled receptors and are expressed in various brain regions and in the digestive tract. In this paper, we will summarize studies on these peptides and their receptors, with special reference to research using gene-knockout mice. These studies clearly demonstrated the role of three receptors in vivo and in vitro. We will also discuss the phylogeny of these receptors. KEY WORDS: gastrin-releasing peptide, neuromedin B, bombesin-like peptide receptor subtype-3 (BRS-3)Int.

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Neuromedin B (NMB) is one of the bombesin (BN)-related peptides in mammals. It was originally purified from pig spinal cords, and it has been shown to be present in central nervous system as well as in gastrointestinal tract. BN and its related peptides have various physiological effects. These include regulation of exocrine and endocrine secretions, smooth muscle contraction, feeding, blood pressure, blood glucose, body temperature and cell growth. NMB exerts its effect by binding to the cell surface receptor. A high affinity receptor, NMB receptor (NMB-R) has been identified. This is a G-protein coupled receptor with seven membrane-spanning regions. Upon agonist binding, several intracellular signaling cascades including phospholipase activation, calcium mobilization and protein kinase C (PKC) activation lead to expression of several genes, DNA synthesis or cellular effects such as secretion. Existence of NMB-R has been demonstrated in several brain regions, notably in olfactory and thalamic regions, and in gastrointestinal tracts. Recent analysis using NMB-R-deficient mice, generated by gene-targeting technique, enables to distinguish functional properties of NMB-R from GRP-R. In this review, molecular characterization, anatomical distribution and pharmacological properties of NMB and NMB-R will be presented. Moreover, physiological roles of NMB and its receptor demonstrated by the analysis of NMB-R-deficient mice will be reported. Comparison with GRP/GRP-R system will provide important information about BN-like peptide systems in mammals.

The neuromedin B-preferring receptor (NMB-R) is one of the members of the bombesin (BN)-like peptide receptor subfamily in mammals. Previously, we have generated and characterized mice with targeted disruption of the two other BN-like peptide receptors, bombesin receptor subtype-3 (BRS-3) and gastrin-releasing peptide-preferring receptor (GRP-R). Here we describe the generation and analysis of NMB-R-deficient mice to investigate how NMB-R differs from BRS-3 and GRP-R. Compensation for NMB-R deficiency by overexpression of GRP-R and/or BRS-3 was not detected. Although the hypothermic effect of NMB was reduced by 50% in NMB-R-deficient mice, the effect of GRP infusion was comparable to the wild-type mice. In contrast, fundic smooth muscle contraction on stimulation with NMB or GRP was normal in NMB-R-deficient mice. Administration of GRP but not NMB suppressed glucose intake in both normal and NMB-R-deficient mice. These results suggest that the NMB-R has an essential role in thermoregulation, but not for smooth muscle contraction of the fundus or for the suppression of feeding behavior. In addition, the behavioral phenotypes of GRP-R-deficient mice were not observed in NMB-R-deficient mice. These data show that the functions of NMB-R and GRP-R are distinct, with only partial overlap.