Vector systems of RNA interference

RNA interference is a mechanism of posttranslational (at the level of mRNA) gene silencing. Sequence-specific mRNA degradation is realized with the help of small interfering RNAs produced by processing of a precursor using Dicer, an enzyme from the RNAse III family. This mechanism is now widely used in vitro on cultures of mammalian cells in order to elucidate functions of individual genes by gene specific knockdown. Analogs of small interference RNAs are intensely expressed during embryogenesis. The mechanism of RNA interference plays an especially important role in embryogenesis of invertebrates. Identification of the functions of small noncoding RNAs is essential for understanding the genetic mechanisms underlying individual developmental stages. In order to integrate small interference RNAs in mammalian cells, various systems have been developed that allow both transient (for 48 h) and stable expression in vitro. These systems are considered in the present review.     

Localization of mitochondrial ribosomal RNA on the chromatoid bodies of marine planarian polyclad embryos

Electron-dense cytoplasmic structures, referred to as chromatoid bodies, are observed in the somatic stem cells, called neoblasts, and germline cells in adult planarians. Although it has been revealed that the chromatoid bodies morphologically resemble germline granules in Drosophila and Xenopus embryos, what essential role it plays in the planarian has remained unclear. In the present study, to examine whether chromatoid bodies in planarian embryos are responsible for germline formation, the presence and behavior of chromatoid bodies during embryogenesis were examined. Mitochondrial large ribosomal RNA and mitochondrial small ribosomal RNA were used as candidate markers for components of the chromatoid body. Starting from the fertilized egg, extramitochondrial signals of both RNA (mtrRNA) were observed. At the ultrastructural level, mtrRNA were localized on the surface of the chromatoid bodies. At subsequent stages, the signals of mtrRNA were observed in certain restricted blastomeres that contribute to the formation of larval structures. The signals gradually decreased from the gastrula stage. These results suggest that the chromatoid bodies associated with mtrRNA in embryogenesis are not germline granules. The chromatoid bodies of blastomeres may be concerned with the toti- or pluripotency and cell differentiation as proposed in adult planarian neoblasts.     

The vertebrate E1/U17 small nucleolar ribonucleoprotein particle

Each of the many different box H/ACA ribonucleoprotein particles (RNPs) present in eukaryotes and archaea consists of four common core proteins and one specific H/ACA small RNA, which bears the sequence elements H (ANANNA) and ACA. Most of the H/ACA RNPs are small nucleolar RNPs (snoRNPs), which are localized in nucleoli, and are one of the two major classes of snoRNPs. Most H/ACA RNPs direct pseudouridine synthesis in pre-rRNA and other RNAs. One H/ACA small nucleolar RNA (snoRNA), vertebrate E1/U17 (snR30 in yeast), is required for pre-rRNA cleavage processing that generates mature 18S rRNA. E1 snoRNA is encoded in introns of protein-coding genes, and the evidence suggests that human E1 RNA undergoes uridine insertional RNA editing. The vertebrate E1 RNA consensus secondary structure shows several features that are absent in other box H/ACA snoRNAs. The available UV-induced RNA-protein crosslinking results suggest that the E1 snoRNP is asymmetrical in vertebrate cells, in contrast to other H/ACA snoRNPs. The vertebrate E1 snoRNP in cells is surprisingly complex: (i) E1 RNA contacts directly and specifically several proteins which do not appear to be any of the H/ACA RNP four core proteins; and (ii) multiple E1 RNA sites are needed for E1 snoRNP formation, E1 RNA stability, and E1 RNA-protein direct interactions.     

真菌中RNA干扰的生物学功能

RNA干扰(RNA interference,RNAi)是一种保守的真核生物基因调控机制,它利用小的非编码RNA介导转录/转录后的基因沉默。虽然部分真菌丢失了RNAi系统,但随着对真菌RNAi机制研究的增加,越来越多的证据表明,真菌的RNAi系统不但参与维持基因组完整性,其在调节真菌生长发育、介导异染色质组装、促进着丝粒进化、调节真菌耐药性与毒力等方面也具有重要作用。本文主要对真菌中RNAi的生物学功能进行综述,以期为进一步深入研究真菌RNA干扰机制提供一定的理论与研究基础。     

Proteins that interact with bacterial small RNA regulators

Small regulatory RNAs have been identified in a wide range of organisms, where they modify mRNA stability, translation or protein function. Small RNA regulators (sRNAs) either pair with mRNA targets or modify protein activities. Here we discuss current knowledge of the various proteins that interact with RNA regulators and review the physiologic implications of sRNA-protein complexes in DNA, RNA and protein metabolism, as well as in RNA and protein quality control in prokaryotes. Proteins that interact with the sRNAs can possess catalytic activity, induce conformational changes of the sRNA, or be sequestered by the sRNA to prevent the action of the protein.     

Phase separation of chromatin and small RNA pathways in plants

Gene expression can be modulated by epigenetic mechanisms, including chromatin modifications and small regulatory RNAs. These pathways are unevenly distributed within a cell and usually take place in specific intracellular regions. Unfortunately, the fundamental driving force and biological relevance of such spatial differentiation is largely unknown. Liquid–liquid phase separation (LLPS) is a natural propensity of demixing liquid phases and has been recently suggested to mediate the formation of biomolecular condensates that are relevant to diverse cellular processes. LLPS provides a mechanistic explanation for the self-assembly of subcellular structures by which the efficiency and specificity of certain cellular reactions are achieved. In plants, LLPS has been observed for several key factors in the chromatin and small RNA pathways. For example, the formation of facultative and obligate heterochromatin involves the LLPS of multiple relevant factors. In addition, phase separation is observed in a set of proteins acting in microRNA biogenesis and the small interfering RNA pathway. In this Focused Review, we highlight and discuss the recent findings regarding phase separation in the epigenetic mechanisms of plants.     

The ribonuclease polynucleotide phosphorylase can interact with small regulatory RNAs in both protective and degradative modes

In all bacterial species examined thus far, small regulatory RNAs (sRNAs) contribute to intricate patterns of dynamic genetic regulation. Many of the actions of these nucleic acids are mediated by well-characterized chaperones such as the Hfq protein, but genetic screens have also recently identified the 3′-to-5′ exoribonuclease polynucleotide phosphorylase (PNPase) as an unexpected stabilizer and facilitator of sRNAs in vivo. To understand how a ribonuclease might mediate these effects, we tested the interactions of PNPase with sRNAs and found that the enzyme can readily degrade these nucleic acids in vitro but, nonetheless, copurifies from cell extracts with the same sRNAs without discernible degradation or modification to their 3′ ends, suggesting that the associated RNA is protected against the destructive activity of the ribonuclease. In vitro, PNPase, Hfq, and sRNA can form a ternary complex in which the ribonuclease plays a nondestructive, structural role. Such ternary complexes might be formed transiently in vivo, but could help to stabilize particular sRNAs and remodel their population on Hfq. Taken together, our results indicate that PNPase can be programmed to act on RNA in either destructive or stabilizing modes in vivo and may form complex, protective ribonucleoprotein assemblies that shape the landscape of sRNAs available for action.     

Novel function of C5 protein as a metabolic stabilizer of M1 RNA

Escherichia coli RNase P is a ribonucleoprotein composed of a large RNA subunit (M1 RNA) and a small protein subunit (C5 protein). We examined if C5 protein plays a role in maintaining metabolic stability of M1 RNA. The sequestration of C5 protein available for M1 RNA binding reduced M1 RNA stability in vivo, and its reduced stability was recovered via overexpression of C5 protein. In addition, M1 RNA was rapidly degraded in a temperature-sensitive C5 protein mutant strain at non-permissive temperatures. Collectively, our results demonstrate that the C5 protein metabolically stabilizes M1 RNA in the cell.     

Air proteins control differential TRAMP substrate specificity for nuclear RNA surveillance

RNA surveillance systems function at critical steps during the formation and function of RNA molecules in all organisms. The RNA exosome plays a central role in RNA surveillance by processing and degrading RNA molecules in the nucleus and cytoplasm of eukaryotic cells. The exosome functions as a complex of proteins composed of a nine-member core and two ribonucleases. The identity of the molecular determinants of exosome RNA substrate specificity remains an important unsolved aspect of RNA surveillance. In the nucleus of Saccharomyces cerevisiae, TRAMP complexes recognize and polyadenylate RNAs, which enhances RNA degradation by the exosome and may contribute to its specificity. TRAMPs contain either of two putative RNA-binding factors called Air proteins. Previous studies suggested that these proteins function interchangeably in targeting the poly(A)-polymerase activity of TRAMPs to RNAs. Experiments reported here show that the Air proteins govern separable functions. Phenotypic analysis and RNA deep-sequencing results from air mutants reveal specific requirements for each Air protein in the regulation of the levels of noncoding and coding RNAs. Loss of these regulatory functions results in specific metabolic and plasmid inheritance defects. These findings reveal differential functions for Air proteins in RNA metabolism and indicate that they control the substrate specificity of the RNA exosome.     

Multiple Modes of Protein–Protein Interactions Promote RNP Granule Assembly

Eukaryotic cells are known to contain a wide variety of RNA–protein assemblies, collectively referred to as RNP granules. RNP granules form from a combination of RNA–RNA, protein–RNA, and protein–protein interactions. In addition, RNP granules are enriched in proteins with intrinsically disordered regions (IDRs), which are frequently appended to a well-folded domain of the same protein. This structural organization of RNP granule components allows for a diverse set of protein–protein interactions including traditional structured interactions between well-folded domains, interactions of short linear motifs in IDRs with the surface of well-folded domains, interactions of short motifs within IDRs that weakly interact with related motifs, and weak interactions involving at most transient ordering of IDRs and folded domains with other components. In addition, both well-folded domains and IDRs in granule components frequently interact with RNA and thereby can contribute to RNP granule assembly. We discuss the contribution of these interactions to liquid–liquid phase separation and the possible role of phase separation in the assembly of RNP granules. We expect that these principles also apply to other non-membrane bound organelles and large assemblies in the cell.     

抑制基因组转座子活性的小RNAs在生育调节中的作用

小RNAs可以在转录和转录后水平沉默转座子, 最近发现的几类小RNAs(piRNAs、endo-siRNAs)均具有抑制转座子活性的作用, 其中, 果蝇piRNAs、小鼠piRNAs、小鼠endo-siRNAs及其相关蛋白主要在生殖系表达, 说明小RNAs作用途径在生殖系转座子沉默中扮演着重要角色。最新的研究揭示了小RNAs、转座子沉默、生殖生育调节之间的联系, 然而其具体作用机制尚未得到阐明。文章综述了小RNAs对基因组转座子活性的控制及其在生育调节中的作用。     

Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8

Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.     

细菌非编码小RNA分子RyhB的结构与功能

RyhB是一种大小为90个核苷酸的细菌非编码小RNA分子(small noncoding RNA, sRNA).当铁缺乏时,RyhB通过下调一系列与铁的储存和利用相关蛋白的表达水平以维持体内的铁平衡,而其本身的表达则受到负调控因子Fur(ferric uptake regulator)的调节.在体内,RyhB与Hfq蛋白和核糖核酸酶E (ribonuclease E, RNase E)形成核蛋白复合物sRNP来发挥活性.sRNP通过RyhB与靶基因的互补配对序列作用于靶基因的核糖体结合位点,阻断靶mRNA的翻译,并迅速引起靶mRNA的降解.此外,RyhB还可以通过影响致病菌的生物膜形成、趋化性、耐酸性等方面的能力对细菌的致病力进行调节.本文综述了RyhB的结构、功能及作用机制方面的研究进展,并对其存在的生理意义进行了探讨.     

RNA 解旋酶调控rRNA内稳态: 水稻耐热 新机制、分子育种新资源

高温热害是影响水稻(Oryza sativa)产量形成的重要限制因子。DEAD-box RNA解旋酶在核糖体RNA前体加工及植物抗逆中扮演着重要角色。最近, 中国科学家在DEAD-box RNA解旋酶调控水稻耐热性分子机理研究方面取得了突破性进展。     

RNA‐directed DNA methylation efficiency depends on trigger and target sequence identity

RNA‐directed DNA methylation (RdDM) in plants has been extensively studied, but the RNA molecules guiding the RdDM machinery to their targets are still to be characterized. It is unclear whether these molecules require full complementarity with their target. In this study, we have generated Nicotiana tabacum (Nt) plants carrying an infectious tomato apical stunt viroid (TASVd) transgene (Nt‐TASVd) and a non‐infectious potato spindle tuber viroid (PSTVd) transgene (Nt‐SB2). The two viroid sequences exhibit 81% sequence identity. Nt‐TASVd and Nt‐SB2 plants were genetically crossed. In the progeny plants (Nt‐SB2/TASVd), deep sequencing of small RNAs (sRNAs) showed that TASVd infection was associated with the accumulation of abundant small interfering RNAs (siRNAs) that mapped along the entire TASVd but only partially matched the SB2 transgene. TASVd siRNAs efficiently targeted SB2 RNA for degradation, but no transitivity was detectable. Bisulfite sequencing in the Nt‐SB2/TASVd plants revealed that the TASVd transgene was targeted for dense cis‐RdDM along its entire sequence. In the same plants, the SB2 transgene was targeted for trans‐RdDM. The SB2 methylation pattern, however, was weak and heterogeneous, pointing to a positive correlation between trigger–target sequence identity and RdDM efficiency. Importantly, trans‐RdDM on SB2 was also detected at sites where no homologous siRNAs were detected. Our data indicate that RdDM efficiency depends on the trigger–target sequence identity, and is not restricted to siRNA occupancy. These findings support recent data suggesting that RNAs with sizes longer than 24 nt (>24‐nt RNAs) trigger RdDM.     

表观遗传学修饰在血液肿瘤中的研究进展

血液肿瘤作为一类常见恶性肿瘤疾病主要包括各类白血病、多发性骨髓瘤以及恶性淋巴瘤.随着现代社会高速发展,人群发病率呈逐年升高趋势,且发病年龄逐渐低龄化,发病原因与环境因素以及遗传因素密不可分.近年来研究发现,表观遗传学修饰在血液肿瘤发生发展的过程中扮演了重要角色,一些相关修饰基因作为血液肿瘤的治疗靶点在临床应用上取得了重要进展.针对近年来表观遗传学修饰在血液肿瘤中的研究新进展,本文将系统综述DNA甲基化、组蛋白修饰、非编码RNA及RNA修饰等在血液肿瘤发病机制方面的研究进展.