环状RNA在抗病毒固有免疫和甲型流感病毒感染中的作用

环状RNA(circular RNA,circRNA)是一种新型的非编码RNA,具有特殊的环状结构,由mRNA前体通过反向剪接产生。随着高通量测序技术的发展,人们发现环状RNA几乎存在于所有的生物体内,目前的研究表明,环状RNA具有多种生物学功能,在癌症、心脑血管等多种疾病中都发挥着重要作用。更重要的是,环状RNA也被证实参与病毒感染,并在抗病毒固有免疫中具有重要作用。本文对环状RNA的生物合成、分类和生物学功能以及其在抗病毒免疫和甲型流感病毒感染中的研究进展进行综述,旨在提供基于环状RNA的抗病毒新策略。     

tRNA衍生片段和tRNA半分子的生物学功能及其在疾病发生中的作用

tRNA衍生片段(tRNA-derived RNA fragment,t RF)和tRNA半分子(tRNA halves,ti RNA)由成熟tRNA或其前体tRNA在不同位点特异性剪切产生,它们是一类广泛存在于原核生物和真核生物转录组中的非编码小RNA分子.t RF主要有tRF-5、tRF-3和tRF-1等3亚类,分别来自成熟tRNA的D环至反密码环茎区间切割至5′端、T环开始至3′端和前体tRNA的3′端尾部,其长度为14~30个核苷酸(nucleotide,nt).ti RNA主要有5′ti RNA和3′ti RNA等2亚类,是在成熟tRNA反密码子环处切割分别产生,其长度为29~50 nt.t RF和ti RNA具有多种生物学功能,既可以在应激反应中作为信号分子,又可以作为基因表达的调节者.它们与人类多种疾病(如肿瘤、神经退行性疾病、代谢性疾病和传染病等)的发生密切相关,有希望成为疾病诊断的新型标志物.本文就t RF和ti RNA的分类、生物学功能以及与人类疾病的关系作一综述.     

环状RNA及其在结直肠癌中的相关研究

环状RNA是长链非编码RNA的一种特殊类型,是首尾共价闭合环化而形成的环状RNA分子,在生理和疾病过程中具有特殊的生物学功能。环状RNA与肿瘤的发生发展关系密切,其高度稳定性及特异性使其有希望成为恶性肿瘤潜在的生物标志物和新的治疗靶点。目前仅有少量关于环状RNA在结直肠癌中的生物学功能及其相关调控机制的研究报道。本文拟从环状RNA的形成、生物学特征、生物学功能以及其结直肠癌中的相关研究等方面进行简要综述。     

原核生物16S rRNA基因多重拷贝及其序列异化

核糖体小亚基RNA(16S rRNA)分子存在于所有细胞生物中,在细胞中执行恒定的功能,其分子序列既有高度保守性片段,又有相对可变性部分,因而成为研究原核生物系统发育的理想分子,其基因已成为原核生物系统分类学研究的核心标识基因。但是,近年来的大量研究表明,原核生物基因组内16S rRNA基因是多拷贝的。16SrRNA基因拷贝数在种属水平上基本是稳定的,但在更高分类阶元上则是不确定的。在拷贝数多的菌株中各拷贝间还存在差异,这种差异有时会大于不同菌株间甚至不同种间的差异。原核生物基因组内16S rRNA基因拷贝数和异化与其利用环境资源的生态策略和对环境的适应性有关。原核生物16S rRNA基因拷贝数及其异化研究对深入理解原核生物的环境生态功能具有重要意义。     

植物中的一种内源小RNA——ta-siRNA

随着对miRNA和siRNA研究的逐步深入,越来越多的新型小RNA被科学家们所认识和了解。内源ta-siRNA是新近在植物中发现的依赖于miRNA、长度为21个碱基的小RNA,对植物的生长发育过程具有重要的表达调控功能。ta-siRNA的产生需要miRNA的剪切引发,之后通过siRNA途径形成,但产生的ta-siRNA生物学功能上不同于其他siRNA,其作用机制类似于miRNA。文章通过综述ta-siRNA的一些研究进展,对ta-siRNA的形成机制和功能进行简单介绍。     

新型隐球酵母非编码小RNAs的研究进展

新型隐球酵母是一种担子菌病原真菌,主要感染免疫功能缺陷的人群,例如HIV-1感染病人,最终会引起致命隐球菌性脑膜炎。非编码小RNAs一般指长度为20-30nt的小RNAs,具有调节功能。新型隐球酵母能够产生大量的小RNAs,但是其生成（biogenesis）过程以及生物学功能尚未完全阐述。本文就新型隐球酵母中小RNAs的特征和产生、以及在新型隐球酵母中的生物学作用和机制进行综述。     

脱氧核酶的研究进展

脱氧核酶是的年来利用体外分子进化技术合成的一种具有催化功能的单链DNA片段,它能催化RNA特定部位的切割反应,从mRNA水平对基因灭活,从而调控蛋白的表达,可能成为对抗RNA病毒感染、肿瘤等疾病的新型工具。     

A/U富集片段RNA结合蛋白的固相纯化与鉴定

RNA与细胞靶蛋白结合是RNA发挥其生物学功能的重要基础,因此,分离和鉴定RNA结合蛋白是研究RNA功能的必要步骤．目前,RNA结合蛋白的分离和鉴定方法较多,但各有优缺点．本实验利用可溶性碳二亚胺(EDC)介导的缩合反应,将A/U富集片段RNA共价偶联到固相介质amine M-270上,再用固定化RNA经亲和层析从细胞抽提物中分离纯化RNA结合蛋白,并以SDS-PAGE联合质谱分析和Western blotting等方法鉴定RNA特异性结合蛋白．最后通过荧光原位杂交和共聚焦显微镜证明这些RNA特异性结合蛋白确与RNA在细胞内结合．实践证明这一方法简单、高效、易于掌握．     

非编码小RNA的多样性

RNA是一类广泛存在的极其重要的生物大分子,它不仅种类繁多,而且不同种类的RNA在结构方面有着显著的差异。RNA种类和结构的多样性决定了RNA具有很多重要的生物学功能。随着对非编码RNA(non-coding RNA,ncRNA)研究的不断深入,ncRNAs同样呈现出前所未有的复杂性和多样性。主要介绍了tRNA、snRNA、scRNA、rRNA、siRNA、miRNA、pi RNA和nat-si RNA等两大类持家ncRNA和调控ncRNA的结构和功能,为便于了解生物体内小的非编码RNA的多样性,进一步挖掘和利用ncRNAs提供一定的参考,促使人们对RNA的认识和地位作出新的思考。     

运用紫外交联免疫沉淀技术构建RNA结合蛋白的靶基因文库

紫外交联免疫沉淀(crosslinking-immunoprecipitation,CLIP)是一种研究RNA结合蛋白的技术,它通过特异性抗体富集靶RNA片段,然后逆转录构建cDNA文库,最后进行高通量测序。它可以在基因组水平上全景式研究靶RNA的特点,为揭示该蛋白生物学功能与分子功能提供新的依据。本研究针对一个已知的RNA结合蛋白MOV10(moloney leukemia virus 10),通过CLIP技术构建cDNA文库,然后小规模克隆后测序,得到小部分靶RNA信息,并利用RNA免疫沉淀技术(RNA-immunoprecipitation,RIP)进行验证。测序提示MOV10结合mRNA,而其中有部分是其已知靶标。说明该文库可以用于后续深度测序,为研究该蛋白在精子发生领域内的功能和机制奠定基础。     

A short fragment of 23S rRNA containing the binding sites for two ribosomal proteins, L24 and L4, is a key element for rRNA folding during early assembly

Previously we described an in vitro selection variant abbreviated SERF (in vitro selection from random rRNA fragments) that identifies protein binding sites within large RNAs. With this method, a small rRNA fragment derived from the 23S rRNA was isolated that binds simultaneously and independently the ribosomal proteins L4 and L24 from Escherichia coli. Until now the rRNA structure within the ternary complex L24-rRNA-L4 could not be studied due to the lack of an appropriate experimental strategy. Here we tackle the issue by separating the various complexes via native gel-electrophoresis and analyzing the rRNA structure by in-gel iodine cleavage of phosphorothioated RNA. The results demonstrate that during the transition from either the L4 or L24 binary complex to the ternary complex the structure of the rRNA fragment changes significantly. The identified protein binding sites are in excellent agreement with the recently reported crystal structure of the 50S subunit. Because both proteins play a prominent role in early assembly of the large subunit, the results suggest that the identified rRNA fragment is a key element for the folding of the 23S RNA during early assembly. The introduced in-gel cleavage method should be useful when an RNA structure within mixed populations of different but related complexes should be studied.     

Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes

Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into the origin and functions of methylation guide small nucleolar RNAs and illuminate the still elusive role of rRNA ribose methylations.     

tRNA cleavage is a conserved response to oxidative stress in eukaryotes

Recent results have identified a diversity of small RNAs in a wide range of organisms. In this work, we demonstrate that Saccharomyces cerevisiae contains a small RNA population consisting primarily of tRNA halves and rRNA fragments. Both 5′ and 3′ fragments of tRNAs are detectable by Northern blot analysis, suggesting a process of endonucleolytic cleavage. tRNA and rRNA fragment production in yeast is most pronounced during oxidative stress conditions, especially during entry into stationary phase. Similar tRNA fragments are also observed in human cell lines and in plants during oxidative stress. These results demonstrate that tRNA cleavage is a conserved aspect of the response to oxidative stress.     

The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy

The Ribosomal Database Project-II (RDP-II) pro-vides data, tools and services related to ribosomal RNA sequences to the research community. Through its website (http://rdp.cme.msu.edu), RDP-II offers aligned and annotated rRNA sequence data, analysis services, and phylogenetic inferences (trees) derived from these data. RDP-II release 8.1 contains 16 277 prokaryotic, 5201 eukaryotic, and 1503 mitochondrial small subunit rRNA sequences in aligned and annotated format. The current public beta release of 9.0 debuts a new regularly updated alignment of over 50 000 annotated (eu)bacterial sequences. New analysis services include a sequence search and selection tool (Hierarchy Browser) and a phylogenetic tree building and visualization tool (Phylip Interface). A new interactive tutorial guides users through the basics of rRNA sequence analysis. Other services include probe checking, phylogenetic placement of user sequences, screening of users' sequences for chimeric rRNA sequences, automated alignment, production of similarity matrices, and services to plan and analyze terminal restriction fragment polymorphism (T-RFLP) experiments. The RDP-II email address for questions or comments is rdpstaff@msu.edu.     

Cloning and biochemical characterization of Bacillus subtilis YxiN, a DEAD protein specifically activated by 23S rRNA: delineation of a novel sub-family of bacterial DEAD proteins.

DEAD, DEAH and DExH proteins are involved in almost every facet of RNA biochemistry. Members of these protein families exhibit an RNA-dependent ATPase activity and some possess an ATP-dependent RNA helicase activity. Although genetic studies have identified specific functions for certain DEx(D)/(H)proteins from which an RNA substrate can be reasonably inferred, only DbpA from Escherichia coli has been shown to exhibit significant RNA specificity in vitro. Here we describe the characterization of YxiN from Bacillus subtilis, the second DEx(D)/(H)protein to show significant RNA specificity as an isolated, homogenous protein. The ATPase activity of YxiN, like that of DbpA, is stimulated by a 154 nt fragment of 23S rRNA. YxiN has a 2 nM apparent binding constant for this fragment, yet its ATPase activity shows 1800-fold RNA specificity. Along with the conserved motifs shared among all DEAD proteins, YxiN and DbpA have a conserved C-terminal extension. This extension is highly conserved in several additional DEAD proteins. We propose that the C-terminus identifies a protein sub-family whose members bind 23S rRNA and that proteins of this family are likely to function in rRNA maturation/ribosome biogenesis or an unappreciated aspect of translation.     

Specific cleavage of ribosomal RNA caused by alpha sarcin.

Alpha sarcin causes the specific cleavage of RNA from 80S ribosomes and 60S subunits of yeast, but not from the 40S subunits to produce a small RNA fragment. The fragment was also produced on treatment of the 60S subunits of wheat germ ribosomes. The fragment has a molecular weight of 100,000 and is a cleavage product of the large RNA species in the 60S subunits. The fragment is not derived from the 5'end of the yeast 25S RNA nor does it bind to 5.8S RNA and we propose that the fragment represents the 3' terminal 320 nucleotides of 25S rRNA. The ability to produce fragment could not be separated from the ability of alpha sarcin to inhibit protein synthesis. Alpha sarcin also causes the specific cleavage of the 23S RNA of the E. coli subunit to produce a smaller fragment of RNA than that produced from eukaryote ribosomes.     

N-terminal domain, residues 1-91, of ribosomal protein TL5 from Thermus thermophilus binds specifically and strongly to the region of 5S rRNA containing loop E.

In this work we show for the first time that the overproduced N-terminal fragment (residues 1-91) of ribosomal protein TL5 binds specifically to 5S rRNA and that the region of this fragment containing residues 80-91 is a necessity for its RNA-binding activity. The fragment of Escherichia coli 5S rRNA protected by TL5 against RNase A hydrolysis was isolated and sequenced. This 39 nucleotides fragment contains loop E and helices IV and V of 5S rRNA. The isolated RNA fragment forms stable complexes with TL5 and its N-terminal domain. Crystals of TL5 in complex with the RNA fragment diffracting to 2.75 A resolution were obtained.