Antisense regulation by transposon‐derived RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus

We report the first example of antisense RNA regulation in a hyperthermophilic archaeon. In Sulfolobus solfataricus, the transposon‐derived paralogous RNAs, RNA‐257_1–4, show extended complementarity to the 3′ UTR of the 1183 mRNA, encoding a putative phosphate transporter. Phosphate limitation results in decreased RNA‐257₁ and increased 1183 mRNA levels. Correspondingly, the 1183 mRNA is faster degraded in vitro upon duplex formation with RNA‐257₁. Insertion of the 1183 3′ UTR downstream of the lacS gene results in strongly reduced lacS mRNA levels in transformed cells, indicating that antisense regulation can function in trans.     

Inactivation of the type I interferon pathway reveals long double‐stranded RNA‐mediated RNA interference in mammalian cells

RNA interference (RNAi) elicited by long double‐stranded (ds) or base‐paired viral RNA constitutes the major mechanism of antiviral defence in plants and invertebrates. In contrast, it is controversial whether it acts in chordates. Rather, in vertebrates, viral RNAs induce a distinct defence system known as the interferon (IFN) response. Here, we tested the possibility that the IFN response masks or inhibits antiviral RNAi in mammalian cells. Consistent with that notion, we find that sequence‐specific gene silencing can be triggered by long dsRNAs in differentiated mouse cells rendered deficient in components of the IFN pathway. This unveiled response is dependent on the canonical RNAi machinery and is lost upon treatment of IFN‐responsive cells with type I IFN. Notably, transfection with long dsRNA specifically vaccinates IFN‐deficient cells against infection with viruses bearing a homologous sequence. Thus, our data reveal that RNAi constitutes an ancient antiviral strategy conserved from plants to mammals that precedes but has not been superseded by vertebrate evolution of the IFN system.     

Broadening the mission of an RNA enzyme

The “RNA World” hypothesis suggests that life developed from RNA enzymes termed ribozymes, which carry out reactions without assistance from proteins. Ribonuclease (RNase) P is one ribozyme that appears to have adapted these origins to modern cellular life by adding protein to the RNA core in order to broaden the potential functions. This RNA‐protein complex plays diverse roles in processing RNA, but its best‐understood reaction is pre‐tRNA maturation, resulting in mature 5' ends of tRNAs. The core catalytic activity resides in the RNA subunit of almost all RNase P enzymes but broader substrate tolerance is required for recognizing not only the diverse sequences of tRNAs, but also additional cellular RNA substrates. This broader substrate tolerance is provided by the addition of protein to the RNA core and allows RNase P to selectively recognize different RNAs, and possibly ribonucleoprotein (RNP) substrates. Thus, increased protein content correlated with evolution from bacteria to eukaryotes has further enhanced substrate potential enabling the enzyme to function in a complex cellular environment. J. Cell. Biochem. 108: 1244–1251, 2009. © 2009 Wiley‐Liss, Inc.     

天然反义转录物及其调控基因的表达机制

天然反义转录(NATs)是一组编码蛋白质或非编码蛋白质的RNAs, 与其他(有义)转录物具有互补序列, 可以调节有义链的表达。这种调节可以发生在转录水平或转录后水平, 调节方式有转录干扰、RNA封闭、双链依赖机制或染色质重建(修饰)等。正义链和反义链分别加工成小RNAs调节基因表达, 也是NATs调节基因表达的重要方式, 如piRNAs的“乒乓机制”。实验或计算机研究已经证明了NATs在生物中广泛存在, 是一种重要的基因表达调节方式。文章论述了NATs的重要作用和机理, 重点论述了NATs的调节机制和相关的小RNAs。     

双链RNA在基因沉默中的作用

介绍依赖于同源识别的基因沉默 .依赖于同源识别的基因沉默 ,是指向生物体内导入外源核酸时引起相应序列的内源基因的表达被特异性抑制的一种基因调控现象 .基因沉默分为转录基因沉默和转录后基因沉默 ,二者都通过双链RNA介导 .它们是真核生物中普遍存在的抵抗病毒入侵、抑制转座子活动、调控基因表达的监控机制 .这些机制具有巨大的应用前景 .     

Evidence for a major role of antisense RNAs in cyanobacterial gene regulation

Information on the numbers and functions of naturally occurring antisense RNAs (asRNAs) in eubacteria has thus far remained incomplete. Here, we screened the model cyanobacterium Synechocystis sp. PCC 6803 for asRNAs using four different methods. In the final data set, the number of known noncoding RNAs rose from 6 earlier identified to 60 and of asRNAs from 1 to 73 (28 were verified using at least three methods). Among these, there are many asRNAs to housekeeping, regulatory or metabolic genes, as well as to genes encoding electron transport proteins. Transferring cultures to high light, carbon‐limited conditions or darkness influenced the expression levels of several asRNAs, suggesting their functional relevance. Examples include the asRNA to rpl1, which accumulates in a light‐dependent manner and may be required for processing the L11 r‐operon and the SyR7 noncoding RNA, which is antisense to the murF 5′ UTR, possibly modulating murein biosynthesis. Extrapolated to the whole genome, ～10% of all genes in Synechocystis are influenced by asRNAs. Thus, chromosomally encoded asRNAs may have an important function in eubacterial regulatory networks.     

Recognition of double-stranded RNA by proteins and small molecules

Molecular recognition of double-stranded RNA (dsRNA) is a key event for numerous biological pathways including the trafficking, editing, and maturation of cellular RNA, the interferon antiviral response, and RNA interference. Over the past several years, our laboratory has studied proteins and small molecules that bind dsRNA with the goal of understanding and controlling the binding selectivity. In this review, we discuss members of the dsRBM class of proteins that bind dsRNA. The dsRBM is an approximately 70 amino acid sequence motif found in a variety of dsRNA-binding proteins. Recent results have led to a new appreciation of the ability of these proteins to bind selectivity to certain sites on dsRNA. This property is discussed in light of the RNA selectivity observed in the function of two proteins that contain dsRBMs, the RNA-dependent protein kinase (PKR) and an adenosine deaminase that acts on dsRNA (ADAR2). In addition, we introduce peptide-acridine conjugates (PACs), small molecules designed to control dsRBM-RNA interactions. These intercalating molecules bear variable peptide appendages at opposite edges of an acridine heterocycle. This design imparts the potential to exploit differences in groove characteristics and/or base-pair dynamics at binding sites to achieve selective binding.     

An embryonic story: Analysis of the gene regulative network controlling Xist expression in mouse embryonic stem cells

In mice, dosage compensation of X‐linked gene expression is achieved through the inactivation of one of the two X‐chromosomes in XX female cells. The complex epigenetic process leading to X‐inactivation is largely controlled by Xist and Tsix, two non‐coding genes of opposing function. Xist RNA triggers X‐inactivation by coating the inactive X, while Tsix is critical for the designation of the active X‐chromosome through cis‐repression of Xist RNA accumulation. Recently, a plethora of trans‐acting factors and cis‐regulating elements have been suggested to act as key regulators of either Xist, Tsix or both; these include ubiquitous factors such as Yy1 and Ctcf, developmental proteins such as Nanog, Oct4 and Sox2, and X‐linked regulators such as Rnf12. In this paper we summarise recent advances in our knowledge of the regulation of Xist and Tsix in embryonic stem (ES) and differentiating ES cells.     

PU.1 antisense lncRNA against its mRNA translation promotes adipogenesis in porcine preadipocytes

Antisense long non‐coding RNAs (AS lncRNAs) play important roles in refined regulation of animal gene expression. However, their functions and molecular mechanisms for domestic animal adipogenesis are largely unknown. Here, we found a novel AS lncRNA transcribed from the porcine PU.1 gene (also known as SPI1) by strand‐specific RT‐PCR. Results showed that PU.1 AS lncRNA was expressed and generally lower than the level of PU.1 mRNA in porcine subcutaneous adipose, heart, liver, spleen, lympha, skeletal muscle and kidney tissues. We further found that the levels of PU.1 mRNA and PU.1 protein were significantly lower in subcutaneous and intermuscular adipose than in mesenteric and greater omentum adipose, whereas the levels of PU.1 AS lncRNA showed no difference in porcine adipose tissues from four different parts of the body. During porcine adipogenesis, levels of PU.1 mRNA increased at day 2 and then gradually decreased. Meanwhile, PU.1 AS lncRNA exhibited an expression trend similar to PU.1 mRNA but sharply decreased after day 2. Interestingly, PU.1 protein level rose during differentiation. In addition, at day 6 after differentiation, knockdown of endogenous PU.1 promoted adipogenesis, whereas knockdown of endogenous PU.1 AS lncRNA had the opposite effect. Moreover, peroxisome proliferator‐activated receptor gamma (PPARG) and fatty acid synthase (FASN) were significantly upregulated in the PU.1 shRNA treatment group (P < 0.05), whereas they were downregulated in the PU.1 AS shRNA treatment group (P < 0.05). Adipose triglyceride lipase [ATGL; also known as patatin‐like phospholipase domain containing 2 (PNPLA2)] and hormone‐sensitive lipase [HSL; also known as lipase, hormone‐sensitive (LIPE)] contrasted with PPARG and FASN. Finally, the PU.1 mRNA/PU.1 AS lncRNA duplex was detected by an endogenous ribonuclease protection assay combined with RT‐PCR. Based on the above results, we suggest that PU.1 AS lncRNA (vs. its mRNA translation) promotes adipogenesis through the formation of a sense–antisense RNA duplex with PU.1 mRNA.     

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG‐I‐like receptor (RLR) family, which signal to induce production of type I interferons (IFN). These key antiviral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon‐stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG‐I, MDA5 and, the least‐studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus‐derived double‐stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals, and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We show that LGP2 associates with Dicer and inhibits cleavage of dsRNA into siRNAs both in vitro and in cells. Further, we show that in differentiated cells lacking components of the IFN response, ectopic expression of LGP2 interferes with RNAi‐dependent suppression of gene expression. Conversely, genetic loss of LGP2 uncovers dsRNA‐mediated RNAi albeit less strongly than complete loss of the IFN system. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs. LGP2‐mediated antagonism of dsRNA‐mediated RNAi may help ensure that viral dsRNA substrates are preserved in order to serve as targets of antiviral ISG proteins.