Abstract:进入21世纪以来，非编码RNA和非编码基因的研究越来越引起人们的关注。2010年12月7日的《科学》杂志(Science)在评选本世纪前10年的十大科学突破时，首先提到的就是基因组中的暗物质(the genome's "dark matter")。近10年非编码RNA领域发展如此迅速，首先源于20世纪90年代开展的基因组研究和随后的比较基因组研究。海量的基因组序列数据证实：DNA上编码蛋白质的区域(也就是通常说的基因)只占人类和其他高等动、植物基因组的极小部分，在人类不会超过整个基因组的3%，其余部分都不编码蛋白质或多肽。而且，随着生物从简单到复杂、从低级到高级，非编码序列在基因组中所占的比例也单调地增加，说明非编码序列是有功能的；近年来大量转录组的新实验结果表明，基因组中的非编码序列是可以表达的，其表达产物就是非编码RNA，转录组的研究说明基因组中的非编码序列是有信息发放的；此后，越来越多的事实证明非编码RNA具有重要的生物功能，microRNA(miRNA)的研究就是最突出的例子。上述的进展明确地告诉我们数量巨大的非编码RNA是有待挖掘的生物宝库。当今，非编码序列、非编码基因和非编码RNA的研究已成为生物学领域的研究热点，重新唤起了科学家们对“RNA世界”的重视及对“生命起源于RNA分子”这一命题的兴趣。目前，这一领域值得关注的问题很多，比如：
1 长非编码RNA系统发现和功能研究
尽管长度小于50个碱基的非编码RNA(如microRNA和piRNA等)的研究已取得突破性进展，但长度大于100个碱基的非编码RNA，还有数以万计功能尚未发现。长非编码RNA不仅数量巨大，更为重要的是它们能折叠为特定的空间结构，因此它们与其他生物分子相互作用的方式是不同于microRNA的。虽然到目前为止，才知道了几百个长非编码RNA的功能，但已涉及到：转录调控、转录后调控、翻译调控、表观遗传调控、基因印迹以及端粒系统等。近年来还发现了一些新类型的长非编码RNA，如ceRNA和环RNA等。所以，进一步开展非编码序列、非编码基因和非编码RNA的研究，系统发现新的长非编码RNA，研究它们的空间结构与功能，可能为我们带来更多的创新机会。
2 非编码RNA是生物网络的元件
近年来的大量新研究成果表明非编码RNA是许多生命过程中富有活力的参与者。一些科学家认为成千上万非编码RNA分子组成了巨大的分子网络调节着细胞中的生命活动，它们与蛋白质网络相对应，同时这两类网络必然有紧密的相互作用，从而构成更复杂的网络。所以未来的网络至少是双色的才更符合生命活动的实际。
3 非编码RNA调节的多样性
非编码RNA在发挥生物学功能时的作用方式是多种多样的，以miRNA为例：它的负调控作用不仅存在于靶mRNA的3'UTR区，也可发生在5'UTR区；miRNA不仅具有负调控作用，在特定条件下也可以激活基因的表达；最近的研究表明，miRNA不仅可通过调节mRNA的表达发挥生物作用，还可以通过调节蛋白来发挥生物功能。
miRNA的作用方式尚且如此复杂，其他非编码RNA行使功能的途径可能更为多样。
4 非编码RNA与疾病紧密相关
非编码RNA，特别是miRNA，与疾病相关的研究已数不胜数。长非编码RNA也和人类疾病紧密相关，仅以肿瘤为例：PCGEM1与前列腺癌有关；His-1是白血病致病因素，而且参与了癌变代谢通路和细胞周期调控； MALAT-1的转录本是一条8000多个碱基长度的非编码RNA，该基因与非小细胞肺癌有关。上述一些成果正逐渐走向临床。非编码RNA研究是一个紧密结合实际，又可迅速用于实际的领域，应特别关注从基础到应用的转化。 为了让广大读者了解非编码RNA领域的进展，本刊特意组织了这个专辑。邀请国内RNA研究领域的5位知名专家对该领域当前的进展与挑战作了综述。屈良鹄教授介绍了microRNA和细胞信号通路间的相互作用及其对两者功能发挥的关键作用，并探讨了其生物学意义；邵宁生教授的综述介绍了在不同细胞或同一细胞的不同状态下microRNA发挥生物学功能的时序特异性和组织特异性，说明microRNA在机体内工作的复杂性；张辰宇教授结合自己多年的研究实践叙述了人体体液中microRNA的起源与功能，对分泌RNA和循环RNA概念作了论述，并探讨了它们在临床医学领域的潜在诊断功能；施蕴渝教授从结构生物学出发综述了RNA分子伴侣Hfq是如何促进sRNA和mRNA相互配对的；鲁志教授介绍了长非编码RNA研究中的各种生物信息学的工具与方法。希望大家对这些文章感兴趣，也希望它们能给大家带来知识与帮助。

Abstract:MicroRNAs(miRNAs), widely existed in various organisms, play important roles in the regulation of gene expression at the post-transcriptional level. Cell signaling pathways transduce extracellular stimuli into cells and provoke a series of physiological and pathological effects, resulting in cell fate determination. This review summarizes the reciprocal interactions of microRNAs and cell signaling pathways, revealing a miRNA-integrated signaling pathways and network in cells.

Abstract:Recently, numerous studies have documented the importance of microRNAs (miRNAs) as an essential cornerstone of the genetic system. Once thought as unstable RNA molecules, miRNAs are now known to circulate in the bloodstream and other body fluids in a stable, cell-free form. Importantly, extracellular miRNAs are aberrantly present in plasma, serum and other body fluids during the pathogenesis of many diseases and, thus, are promising noninvasive or minimally invasive biomarkers to assess the pathological status of the body. However, the origin and biological function of extracellular miRNAs remains incompletely understood. In this review, we summarize the recent literature on the biogenesis and working models of extracellular miRNAs, and we highlight the impact of extending these ongoing extracellular miRNA studies to clinical applications.

Abstract:MicroRNA(miRNA) are endogenous ～22 nt non- coding small RNAs broadly expressed in plants and mammalians. MiRNAs are involved in the negative regulation of gene expression through the targeting of mRNAs during the process and activity of life such as cell proliferation and differentiation, apoptosis as well as the development of diseases. Recently, numerous studies have demonstrated that the expression of miRNA is of time and tissue specificity. It has been predicted that each miRNA may target more than one or hundreds of mRNAs. In addition, the functional targets of miRNAs may be different in different cells or the different states of the same cell. All these results suggested that miRNA and its functional targets may be dynamically variable with the time and space. MiRNAs may play complex roles in human body. In this paper the dynamic change of miRNA and its targets has been reviewed and it may provide new idea for the study of miRNAs.

Abstract:Hfq is a bacterial post-transcriptional regulator. It facilitates base-pairing between sRNA and target mRNA. The mRNA rpoS, which encodes the master regulator σ^S of general stress response, requires Hfq-facilitated base pairing with DsrA small RNA for efficient translation at low temperatures. Two mutually non-exclusive mechanisms have been proposed to explain the process of how Hfq facilitates base pairing of sRNA DsrA to mRNA rpoS: Hfq may form ternary complex with two RNAs via co-binding to bring the RNA strands into close proximity for optimal annealing; Hfq may bind one or both RNAs, and change its (or their) secondary (or tertiary) structure to facilitate the RNA pairing. Recently, several complex crystal structures of AU₆A-Hfq-ATP, A₇-Hfq, and AU₆A-Hfq-A₇ were acquired, and interesting structural features were extracted from them to deepen our understanding in the RNA binding properties of Hfq and its RNA complexes. Furthermore, the formation of ternary complex sRNA-Hfq-mRNA is proved to be necessary for translation activation of rpoS mRNA in vivo. This mini-review summarizes some recent structural biology advances in the research of DsrA-regulated translation of rpoS and the biological implications of the transient ternary complex are discussed.

Abstract:Noncoding RNA (ncRNA) refers to any RNA that is functional without being translated into proteins. Recently, people have paid more and more attention on various types of novel ncRNAs. Some small ncRNAs have already been identified and well studied, such as microRNAs, snoRNAs, tRNA, etc. However, the fields of long noncoding RNA (lncRNA) have not been well explored. This paper reviewed the research progress of ncRNA, especially the lncRNA, including the historical overview, the basic characteristics and the relationship with diseases. We also compared the prediction methods of ncRNA, and introduced an integrative strategy to combine the high throughput sequencing data in the prediction of ncRNA.

Abstract:A certain concentration of formaldehyde can cause protein misfolding, cell death and biological dysfunction. Though it has been reported that formaldehyde has cytotoxicity, how formaldehyde affects cell cycle of neural cells and the molecular mechanism still needs to be clarified. In this study, neuroblastoma cell line SH-SY5Y was utilized to incubate with formaldehyde and the effect of formaldehyde on cell cycle was in formaldehyde concentration-dependent manner. No significant changes in cell cycle could be detected when [FA]≤0.1 mmol/L (cells were incubated for 48 h), while the percentages of cells in S phase and G2/M phase were markedly increased with the elevation of formaldehyde concentration (0.1 mmol/L <[FA]≤0.2 mmol/L). In the medium with 0.3 mmol/L formaldehyde, 46.28% of cells were in S phase while only 16.05% of them were in G2/M phase, that is, cell proliferation was obviously inhibited under the conditions. When cells were synchronized at G2/M phase, formaldehyde (0.1～0.3 mmol/L) could markedly increase the number of cells in S phase, though, to some extent, the number of cells in G2/M phase decreased. When cells were synchronized at S phase, 0.1 mmol/L formaldehyde could decrease the number of cells in G2/M phase, while 0.3 mmol/L formaldehyde could markedly decrease the number of cells in G2/M phase and significantly increase that in S phase. In the presence of formaldehyde, primary neurons of SD rat exhibited similar changes in cell cycle as that in SH-SY5Y cells. Furthermore, early and late apoptosis was markedly observed when 0.1 mmol/L≤[FA]≤0.2 mmol/L, while DNA were obviously damaged and most cells were apoptosis and some of them underwent necrosis when [FA]≥0.3 mmol/L. In sum, formaldehyde at a low concentration (0.1 mmol/L≤[FA]≤0.2 mmol/L) mainly suppresses DNA synthesis in S phase via hypermethylation of global DNA, while formaldehyde at a higher concentration ([FA]≥ 0.3 mmol/L) causes DNA damage, both of them lead to the aberrant effects on cell cycle.

Abstract:Rat oxidored nitro domain containing protein 1 (rNOR1) was identified and characterized. Isolated rNOR1 cDNA consisted of 1 418 base pairs that encoded a 379-amino acid protein, and the amino acid sequence was 89% and 93% identical to that of human NOR1 (hNOR1) and mouse NOR1 (mNOR1), respectively. The rat NOR1 protein contained a putative conserved domain belong to OSCP1 superfamily. The message for rNOR1 is highly detected in rat testis. Furthermore, human NOR1 homologue is enriched in testis. By immunostaining human NOR1 protein was detected in human testicular tumors of different subtypes on tissue microarray. NOR1 was strongly immunostained in non-cancerous testicular tissues and embryonal carcinomas, while seminomatous tumour cells and differentiated nonseminomatous derivatives (teratoma, yolk sac tumor) stained less intensely. These data suggested that rNOR1 might serve as a testis-selective gene, and that altered NOR1 expression in testicular cancer may help us to elucidate the functions of NOR1 protein in germ line cells carcinogenesis.

Abstract:In natural vision, due to object, body, head, and eye movements, the contrast and orientation information received by our eyes change simultaneously and rapidly, yet our visual system can efficiently and accurately distinguish the contrasts and orientations under the dynamical conditions. The visual processes responsible for discriminating orientations and contrasts occurred at this very short time scale, however, are poorly understood. In the current experiments, we investigated the ability of human subjects to discriminate orientations or contrasts using the briefly presented stimuli in which orientations and contrasts were changing simultaneously in comparison with the stimuli in which orientation changes were discriminated when contrast remained constant or contrast changes were discriminated when orientation was constant. We show that the discrimination threshold for orientation or contrast when both contrast and orientation were varying was significantly lower than that for orientation when contrast was fixed or than that for contrast when orientation was fixed. Moreover, the decreased magnitude of discrimination thresholds to orientation or contrast was negatively correlated to the discrimination threshold to orientation or contrast when only orientation or only contrast was varied. These results indicate that the simultaneous changes in orientation and contrast enhanced the discriminability of subjects to contrast and orientation. The subjects who had lower discriminability to orientation or contrast when only orientation or contrast was changing had a relatively larger enhancement in their ability to discriminate contrasts and orientations when both orientation and contrast were changing simultaneously. We suggest that human visual system is more efficient at discriminating orientation and contrast when both parameters are changing which often occurs in natural environments. Our study sheds light on the underlying visual processes.

Abstract:Opioid receptor (OPR) agonists which interact with specific subtypes of opioid receptors, are attractive pharmaceutical chemicals, and are extensively used in the treatment of severe pain associated with traumatic injuries, cancer or heart attacks. There are three typical subtypes of OPRs, i.e., δ, κ, and μ, which all have their respective agonists. Among them, δ-OPR (DOR) agonists are especially promising pharmaceutical chemicals for their additional anti-depressant and anti-anxiety as well as organ-protective abilities. To investigate the mechanism of δ-OPRs agonists and the receptor, in the present work 102 derivatives of N-substituted spiropiperdines were studied through three-dimensional quantitative structure-activity relationships (3D-QSAR) analysis. In conclusion, PLS analysis (Q²=0.501, R²_ncv=0.787, R²_pre=0.780) of the optimal CoMSIA model (yielded by hydrophobic and hydrogen-bond donor fields) manifesting good inter predictive capacity, excellent inter-consistency, and outstanding predictive ability, implies the rationality of the model. At the same time, the 3D-QSAR contour map analysis results indicate that the hydrophobic group substitution of R₁ is beneficial to the activity of delta opioid agonists, and the hydrophilic or hydrogen-donor substitution of R₂ is also favorable. These conclusions are helpful for understanding the mechanism of DOR agonists, as well as the guiding of design and improvement of new δ-OPR agonists in the future.

Abstract:Protein post-translational modifications play very important roles in the regulation of gene functions and successful site-directed mutagenesis is vital to determine a protein's modification sites and hence, to define its structure and function. Yet, researches are always confronted with the problem on how to generate multiple-site mutations of a target gene rapidly. In this study, a novel strategy to generate multiple-site mutations was developed based on accurate single site mutagenesis with SRrp53 as an example. Firstly, the entire SRrp53 coding sequence was amplified from the human breast cDNA library and then cloned into the expression vector pcDNA3-FLAG. Five sets of primers for K196R, K171R, K163R, K146R and K309R mutations were synthesized and were then phosphorylated at their 5' terminus. In the next step, the first run of inverse PCR was performed with one specific set of phosphorylated primers. Further, the PCR products were subjected to DpnⅠ treatment, agarose gel purification, dam methyltransferase treatment, self-ligation and purification with PCR extraction kit. Afterwards, the second run of inverse PCR was performed and the PCR products were treated as above protocol. The second processed PCR products were then used as the template for the third run of inverse PCR. The exact run times of inverse PCR are according to the number of mutation sites that you wanted. The PCR products of the last run were treated with DpnⅠ, purified with agarose gel extract kit, self-ligated and were transformed into DH5α. Ten colonies were randomly picked up for plasmids extraction and DNA sequencing. The expression of both the SRrp53 wild type and five-site mutants was analyzed by transfection of these plasmids into 293T cells. The DNA sequencing results showed that 9 of 10 extracted plasmids gained the correct mutations. And after these 9 correct plasmids were transfected into 293T cells, all of the mutants were expressed with the right molecular mass. In a word, our novel strategy can be applied to generate multiple-site mutants of a target gene efficiently and conveniently. The method has laid a good foundation for further exploration of this protein's function.