基因表达调控多面手——microRNA和siRNA的作用机制

miRNA(microRNA)是一类广泛存在于高等真核细胞中的长度约为21个碱基的小分子非编码RNA,参与调控三分之一以上基因的表达,并与多种人类疾病存在重要关联。而siRNA(small interfering RNA)是RNA干扰(RNA interference,RNAi)中的效应分子,其结构和作用机制与miRNA存在许多类似之处。由于miRNA和siRNA具有重要的生物学功能。因此,对它们作用机制的理解具有非常重要的理论意义和应用指导价值。该综述将对它们作用机制的研究进展做一总结和回顾。     

Processing by endoplasmic reticulum mannosidases partitions a secretion-impaired glycoprotein into distinct disposal pathways

In the early secretory pathway, a distinct set of processing enzymes and family of lectins facilitate the folding and quality control of newly synthesized glycoproteins. In this regard, we recently identified a mechanism in which processing by endoplasmic reticulum mannosidase I, which attenuates the removal of glucose from asparagine-linked oligosaccharides, sorts terminally misfolded alpha(1)-antitrypsin for proteasome-mediated degradation in response to its abrogated physical dissociation from calnexin (Liu, Y., Choudhury, P., Cabral, C., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861-5867). In the present study, we examined the quality control of genetic variant PI Z, which undergoes inappropriate polymerization following biosynthesis. Here we show that in stably transfected hepatoma cells the additional processing of asparagine-linked oligosaccharides by endoplasmic reticulum mannosidase II partitions variant PI Z away from the conventional disposal mechanism in response to an arrested posttranslational interaction with calnexin. Intracellular disposal is accomplished by a nonproteasomal system that functions independently of cytosolic components but is sensitive to tyrosine phosphatase inhibition. The functional role of ER mannosidase II in glycoprotein quality control is discussed.     

Nitric oxide activates diverse signaling pathways to regulate gene expression

Nitric oxide signaling is crucial for effecting long lasting changes in cells, including gene expression, cell cycle arrest, apoptosis, and differentiation. We have determined the temporal order of gene activation induced by NO in mammalian cells and have examined the signaling pathways that mediate the action of NO. Using microarrays to study the kinetics of gene activation by NO, we have determined that NO induces three distinct waves of gene activity. The first wave is induced within 30 min of exposure to NO and represents the primary gene targets of NO. It is followed by subsequent waves of gene activity that may reflect further cascades of NO-induced gene expression. We verified our results using quantitative real time PCR and further validated our conclusions about the effects of NO by using cytokines to induce endogenous NO production. We next applied pharmacological and genetic approaches to determine the signaling pathways that are used by NO to regulate gene expression. We used inhibitors of particular signaling pathways, as well as cells from animals with a deleted p53 gene, to define groups of genes that require phosphatidylinositol 3-kinase, protein kinase C, NF-kappaB, p53, or combinations thereof for activation by NO. Our results demonstrate that NO utilizes several independent signaling pathways to induce gene expression.     

Distinct calcium signaling pathways regulate calmodulin gene expression in tobacco

Cold shock and wind stimuli initiate Ca(2+) transients in transgenic tobacco (Nicotiana plumbaginifolia) seedlings (named MAQ 2.4) containing cytoplasmic aequorin. To investigate whether these stimuli initiate Ca(2+) pathways that are spatially distinct, stress-induced nuclear and cytoplasmic Ca(2+) transients and the expression of a stress-induced calmodulin gene were compared. Tobacco seedlings were transformed with a construct that encodes a fusion protein between nucleoplasmin (a major oocyte nuclear protein) and aequorin. Immunocytochemical evidence indicated targeting of the fusion protein to the nucleus in these plants, which were named MAQ 7.11. Comparison between MAQ 7.11 and MAQ 2.4 seedlings confirmed that wind stimuli and cold shock invoke separate Ca(2+) signaling pathways. Partial cDNAs encoding two tobacco calmodulin genes, NpCaM-1 and NpCaM-2, were identified and shown to have distinct nucleotide sequences that encode identical polypeptides. Expression of NpCaM-1, but not NpCaM-2, responded to wind and cold shock stimulation. Comparison of the Ca(2+) dynamics with NpCaM-1 expression after stimulation suggested that wind-induced NpCaM-1 expression is regulated by a Ca(2+) signaling pathway operational predominantly in the nucleus. In contrast, expression of NpCaM-1 in response to cold shock is regulated by a pathway operational predominantly in the cytoplasm.     

Multiple phospholipid N-methyltransferases with distinct substrate specificities are encoded in Bradyrhizobium japonicum

Phosphatidylcholine (PC) is the major phospholipid in eukaryotic membranes. In contrast, it is found in only a few prokaryotes including members of the family Rhizobiaceae. In these bacteria, PC is required for pathogenic and symbiotic plant-microbe interactions, as shown for Agrobacterium tumefaciens and Bradyrhizobium japonicum. At least two different phospholipid N-methyltransferases (PmtA and PmtX) have been postulated to convert phosphatidylethanolamine (PE) to PC in B. japonicum by three consecutive methylation reactions. However, apart from the known PmtA enzyme, we identified and characterized three additional pmt genes (pmtX1, pmtX3, and pmtX4), which can be functionally expressed in Escherichia coli, showing different substrate specificities. B. japonicum expressed only two of these pmt genes (pmtA and pmtX1) under all conditions tested. PmtA predominantly converts PE to monomethyl PE, whereas PmtX1 carries out both subsequent methylation steps. B. japonicum is the first bacterium known to use two functionally different Pmts. It also expresses a PC synthase, which produces PC via condensation of CDP-diacylglycerol and choline. Our study shows that PC biosynthesis in bacteria can be much more complex than previously anticipated.     

Mitochondria/nuclear signaling of alternative oxidase gene expression occurs through distinct pathways involving organic acids and reactive oxygen species

Cultured cells of tobacco (Nicotiana tabacum L. cv Petit Havana) were used to investigate signals regulating the expression of the model nuclear gene encoding the alternative oxidase (AOX) (AOX1), the terminal oxidase of the mitochondrial alternative respiratory pathway. Several conditions shown to induce AOX1 mRNA accumulation also result in an increase in cellular citrate concentrations, suggesting that citrate and/or other tricarboxylic acid (TCA) cycle intermediates may be important signal metabolites. In addition, mitochondrial reactive oxygen species (ROS) production has recently been shown to be a factor mediating mitochondria-to-nucleus signaling for the expression of AOX1. We found that the exogenously supplied TCA cycle organic acids citrate, malate and 2-oxoglutarate caused rapid and dramatic increases in the steady-state level of AOX1 mRNA at low, near physiological concentrations (0.1 mM). Furthermore, an increase in AOX1 induced by the addition of organic acids occurs independently of mitochondrial ROS formation. Our results demonstrate that two separate pathways for mitochondria-to-nucleus signaling of AOX1 may exist, one involving ROS and the other organic acids.