Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs

In response to infection, invertebrates process replicating viral RNA genomes into siRNAs of discrete sizes to guide virus clearance by RNA interference. Here, we show that viral siRNAs sequenced from fruit fly, mosquito, and nematode cells were all overlapping in sequence, suggesting a possibility of using siRNAs for viral genome assembly and virus discovery. To test this idea, we examined contigs assembled from published small RNA libraries and discovered five previously undescribed viruses from cultured Drosophila cells and adult mosquitoes, including three with a positive-strand RNA genome and two with a dsRNA genome. Notably, four of the identified viruses exhibited only low sequence similarities to known viruses, such that none could be assigned into an existing virus genus. We also report detection of virus-derived PIWI-interacting RNAs (piRNAs) in Drosophila melanogaster that have not been previously described in any other host species and demonstrate viral genome assembly from viral piRNAs in the absence of viral siRNAs. Thus, this study provides a powerful culture-independent approach for virus discovery in invertebrates by assembling viral genomes directly from host immune response products without prior virus enrichment or amplification. We propose that invertebrate viruses discovered by this approach may include previously undescribed human and vertebrate viral pathogens that are transmitted by arthropod vectors.     

RNA, but not protein partners, is directly responsible for translational silencing by a bacterial Hfq-binding small RNA

SgrS is an Hfq-binding small RNA that is induced under glucose phosphate stress in Escherichia coli. It forms a specific ribo nucleo protein complex with Hfq and RNase E resulting in translational repression and rapid degradation of ptsG mRNA, encoding the glucose transporter. Here, we report translational silencing of ptsG mRNA in a defined in vitro system. We demonstrate that SgrS and Hfq are the minimum components for translational silencing to faithfully reproduce the reaction in cells. We show that ptsG-SgrS base pairing is sufficient to cause translational repression when the ptsG mRNA is forced to base pair with SgrS without the help of Hfq. The extent of translational repression correlates with the extent of duplex formation. We conclude that base pairing itself but not Hfq is directly responsible for translational silencing and the major role of Hfq in gene silencing is to stimulate the base pairing between SgrS and ptsG mRNA. This simple mechanism is in striking contrast to miRNA action in eukaryote in which the RNA is believed to act only as a guide of protein partners.     

Characterization of corticotropin-releasing hormone receptors on human pituitary corticotroph adenomas and their correlation with endogenous glucocorticoids

Specific receptors for CRH were identified in five freshly excised pituitary adenomas causing Cushing's disease. Their kinetic properties and mean affinity constant [1.45 +/- 0.38 (+/- SE) nmol/L] were comparable to the characteristics of rat and monkey anterior pituitary CRH receptors. No correlation was found between the immediate preoperative plasma and urinary cortisol levels and the number of pituitary adenoma CRH receptors, which ranged from 6-96 fmol/mg protein, unlike in rats, in which corticosterone modulates the number of anterior pituitary CRH receptors. The lack of correlation between the concentration of CRH receptors and plasma cortisol levels may reflect the inability of glucocorticoids to down-regulate CRH receptors in these tumors. Thus, corticotroph adenomas are resistant not only to the feedback actions of glucocorticoids on proopiomelanocortin synthesis and secretion but also to their actions on CRH receptors.     

RNA interference-mediated silencing of Sod2 in Drosophila leads to early adult-onset mortality and elevated endogenous oxidative stress

Oxidative stress has been widely implicated as an important factor in the aging process. Because mitochondrial respiration is the principal source of reactive oxygen within cells, the mitochondrially localized superoxide dismutase (SOD) 2 is thought to play an important front-line defensive role against aging-related oxidative stress. Although genetic studies with mutants deficient in SOD1, the predominantly cytosolic isoform of SOD, have been instrumental in elucidating the role of reactive oxygen metabolism in aging in Drosophila, the lack of available mutations in the Sod2 gene has hampered an equivalent analysis of the participation of this important antioxidant enzyme in the Drosophila aging model. Here we report that ablation of mitochondrial SOD2 through expression of a GAL4-regulated, inverted-repeat Sod2 RNA-interference transgene in an otherwise normal animal causes increased endogenous oxidative stress, resulting in loss of essential enzymatic components of the mitochondrial respiratory chain and the tricarboxylic acid cycle, enhances sensitivity to applied oxidative stress, and causes early-onset mortality in young adults. In sharp contrast, ablation of SOD2 has no overt effect on the development of larvae and pupae, which may reflect a fundamental transition in oxygen utilization andor reactive oxygen metabolism that occurs during metamorphosis from larval to adult life.     

Reversal of endogenous dopamine receptor silencing in pituitary cells augments receptor-mediated apoptosis

Dopamine (DA)-agonist targeting of the DA D(2) receptor (D2R) in prolactinomas is the first-line treatment choice for suppression of prolactin and induction of tumor shrinkage. Resistance to DA agonists seems to be related to receptor number. Using the MMQ and GH3 pituitary cell lines, that either do or do not express D2R, respectively, we explored the epigenetic profile associated with the presence or absence of D2R in these cells lines. These studies led us to explore pharmacological strategies designed to restore receptor expression and thereby potentially augment DA agonist-mediated apoptosis. We show in GH3 cells that the D2R harbors increased CpG island-associated methylation and enrichment for histone H3K27me3. Conversely, MMQ cells and normal pituitaries show enrichment for H3K9Ac and barely detectable H3K27me3. Coculture of GH3 cells with the demethylating agent zebularine and the histone deacetylase inhibitor trichostatin A was responsible for a decrease in CpG island methylation and enrichment for the histone H3K9Ac mark. In addition, challenge of GH3 cells with zebularine alone or coculture with both agents led to expression of endogenous D2R in these cells. Induced expression D2R in GH3 cells was associated with a significant increase in apoptosis indices to challenge with either DA or bromocriptine. Specificity of a receptor-mediated response was established in coincubations with specific D2R antagonist and siRNA approaches in GH3 cell and D2R expressing MMQ cell lines. These studies point to the potential efficacy of combined treatment with epigenetic drugs and DA agonists for the medical management of different pituitary tumor subtypes, resistant to conventional therapies.     

Characterization and partial nucleotide sequence of endogenous type C retrovirus segments in human chromosomal DNA.

Twenty-six different murine leukemia virus (MuLV)-related clones have been isolated from a human DNA library and characterized by restriction enzyme mapping and reciprocal nucleic acid hybridization reactions. The sequence of approximately 2,600 nucleotides, spanning more than 4.0 kilobases, of one of the MuLV-related cloned human DNAs was also determined. The deduced amino acid sequence permitted the alignment of this prototype cloned human DNA segment with the p12 gag, p30 gag, p10 gag, and pol regions of Moloney MuLV. A majority of the endogenous type C retrovirus-related segments present in human DNA are approximately 6.0 kilobases in size and appear to contain a deletion of env sequences.     

RNAi抑制存活素基因表达对前列腺癌细胞侵袭的影响

目的 探讨存活素(survivin)基因对前列腺癌细胞侵袭的影响和可能机制. 方法 采用survivin基因小干扰RNA(small interfering RNA,siRNA)转染处理人前列腺癌细胞系PC-3后,分别采用实时-定量RF PCR和Western blot检测survivin基因和基质金属蛋白酶家族-2、-9(matrixmetalloproteinases-2、-9)mRNA和蛋白水平,分别采用软琼脂集落培养试验和Boyden小室模型试验检测癌细胞的锚着不依赖性增殖和侵袭能力;其次,将转染48 h的细胞接种裸鼠,观察对癌细胞体内侵袭的影响. 结果 软琼脂集落形成试验显示,3.125、6.250 nmol/L和12.500 nmol/L siRNA组集落形成数分别为17.8±1.6、13.6±1.5、8.8±1.4,而对照组为22.65±1.8(P<0.05);Boyden小室模型试验显示,3.125、6.250 nmol/L和12.500/ nmol/L siRNA组穿过滤膜的细胞数分别为33.6±2.1、19.5±1.9、8.1±1.8,而对照组为49.4±2.3(P<0.05).体内试验显示,对照组裸鼠组织癌细胞侵袭横纹肌和血管,而转染组未见这些现象.同时发现,转染组细胞MMP-2、-9基因mRNA和蛋白水平明显下调,呈浓度和时间依赖性(P<0.01). 结论 采用survivin siRNA转染可抑制前列腺癌细胞侵袭转移,其机制可能与下调MMP-2和MMP-9表达有关.     

Determinants of specific RNA interference-mediated silencing of human beta-globin alleles differing by a single nucleotide polymorphism

A single nucleotide polymorphism (SNP) in the sickle beta-globin gene (beta(S)) leads to sickle cell anemia. Sickling increases sharply with deoxy sickle Hb concentration and decreases with increasing fetal gamma-globin concentration. Measures that decrease sickle Hb concentration should have an antisickling effect. RNA interference (RNAi) uses small interfering (si)RNAs for sequence-specific gene silencing. A beta(S) siRNA with position 10 of the guide strand designed to align with the targeted beta(S) SNP specifically silences beta(S) gene expression without affecting the expression of the gamma-globin or normal beta-globin (beta(A)) genes. Silencing is increased by altering the 5' end of the siRNA antisense (guide) strand to enhance its binding to the RNA-induced silencing complex (RISC). Specific beta(S) silencing was demonstrated by using a luciferase reporter and full-length beta(S) cDNA transfected into HeLa cells and mouse erythroleukemia cells, where it was expressed in the context of the endogenous beta-globin gene promoter and the locus control region enhancers. When this strategy was used to target beta(E), silencing was not limited to the mutant gene but also targeted the normal beta(A) gene. siRNAs, mismatched with their target at position 10, guided mRNA cleavage in all cases except when two bulky purines were aligned. The specific silencing of the beta(S)-globin gene, as compared with beta(E), as well as studies of silencing SNP mutants in other diseases, indicates that siRNAs developed to target a disease-causing SNP will be specific if the mutant residue is a pyrimidine and the normal residue is a purine.     

长链非编码RNA的竞争性内源RNA调控模式在动脉粥样硬化中的研究进展

动脉粥样硬化是一种缓慢进行性的血管炎症性病变.越来越多的研究表明长链非编码RNA(lncRNA)作为竞争性内源RNA(ceRNA)与微小RNA(miRNA)结合,通过ceRNA网络调控靶基因信使RNA(mRNA)分子的表达水平和功能,在动脉粥样硬化的病理生理过程中发挥重要作用.lncRNA、miRNA和mRNA之间的互...