Identification and characterization of small RNAs involved in RNA silencing

Double-stranded RNA (dsRNA) is a potent trigger of sequence-specific gene silencing mechanisms known as RNA silencing or RNA interference. The recognition of the target sequences is mediated by ribonucleoprotein complexes that contain 21- to 28-nucleotide (nt) guide RNAs derived from processing of the trigger dsRNA. Here, we review the experimental and bioinformatic approaches that were used to identify and characterize these small RNAs isolated from cells and tissues. The identification and characterization of small RNAs and their expression patterns is important for elucidating gene regulatory networks.     

Identification of a novel cis-acting RNA element involved in nuclear export of hY RNAs

Ro RNPs are small cytoplasmic RNA-protein complexes of unknown function that have been found in all metazoan cells studied so far. In human cells, Ro RNPs consist of one of four small RNA molecules, termed hY RNAs and at least two well-characterized proteins, Ro60 and La. In previous Xenopus laevis oocyte microinjection studies, we showed that an intact Ro60 binding site (Stem-loop 1) is a prerequisite for efficient nuclear export of hY1 RNA, whereas an intact La-binding site promotes nuclear retention (Simons et al. RNA, 1996, 2:264-273). Here we present evidence that the distal half (Stem 2) of the conserved base-paired stem structure found in all hY RNAs also plays a critical role in the export process. A minimal RNA molecule containing this region, L1S2 RNA, competes effectively for the export of full-length hY1 RNAs and is itself exported very rapidly in a Ro60-independent and RanGTP-dependent manner. Mutational analyses of this RNA shows that a 5'/3' terminal double-stranded stem structure (>10 bp) of no specific nucleotide sequence constitutes a novel nuclear export element (NEE). Cross-competition studies indicate that this type of NEE may also be involved in export of other classes of RNAs. Like full-length hY1 RNA, L1S2 RNA also competes for export of ET-202 RNA, an RNA that was selected for its efficient nuclear export in the presence of the nuclear transport inhibitor, VSV Matrix protein (Grimm et al. Proc Natl Acad Sci USA, 1997, 94:10122-10127). However, export of L1S2 RNA is strongly inhibited by VSV-M protein, showing that these RNAs use partially overlapping, but not identical export pathways. We propose that export of Y RNAs is mediated by two contiguous cis-acting elements in the 5'/3' double-stranded stem region that is conserved between different Y RNAs.     

Esre: a novel essential non-coding RNA in Escherichia coli

YigP gene (GeneID: 948915) locates between ubiquinone biosynthetic genes ubiE and ubiB in Escherichia coli. GeneBank annotates yigP as a putative protein-coding gene. In this study, we found a new essential sRNA gene, esre, locates within the region of yigP. The E. coli strain with inactive esre must rely on a complementary plasmid to survive. Moreover, RACE experiments showed esre encodes an RNA molecule of 252 nt. Further experiments revealed esre gene is immune to frame shift mutations and the function of esre depends mostly on the RNA secondary structure, which are typical traits of sRNA. Since it is difficult to predict the target of an essential sRNA, more research is needed to reveal the function and mechanism of esre.     

Identification and characterization of SNX15, a novel sorting nexin involved in protein trafficking

Sorting nexins are a family of phox homology domain containing proteins that are homologous to yeast proteins involved in protein trafficking. We have identified a novel 342-amino acid residue sorting nexin, SNX15, and a 252-amino acid splice variant, SNX15A. Unlike many sorting nexins, a SNX15 ortholog has not been identified in yeast or Caenorhabditis elegans. By Northern blot analysis, SNX15 mRNA is widely expressed. Although predicted to be a soluble protein, both endogenous and overexpressed SNX15 are found on membranes and in the cytosol. The phox homology domain of SNX15 is required for its membrane association and for association with the platelet-derived growth factor receptor. We did not detect association of SNX15 with receptors for epidermal growth factor or insulin. However, overexpression of SNX15 led to a decrease in the processing of insulin and hepatocyte growth factor receptors to their mature subunits. Immunofluorescence studies showed that SNX15 overexpression resulted in mislocalization of furin, the endoprotease responsible for cleavage of insulin and hepatocyte growth factor receptors. Based on our data and the existing findings with yeast orthologs of other sorting nexins, we propose that overexpression of SNX15 disrupts the normal trafficking of proteins from the plasma membrane to recycling endosomes or the trans-Golgi network.     

地中海富盐菌中非编码RNA的鉴定与分析

【目的】通过转录组高通量测序技术(即RNA-seq),结合生物信息学分析和分子生物学方法,在组学水平鉴定极端嗜盐菌中可能的非编码RNA(nc RNA)。【方法】将培养至对数中期的地中海富盐菌在不同盐浓度下处理30分钟,提取RNA,进行链特异的转录组测序和5′端区分的转录组测序,通过生物信息学分析在全基因组范围内鉴定nc RNA,预测其转录边界;然后通过Northern blot和环化RNA反转录聚合酶链式反应(CR-RT-PCR)对部分预测的nc RNA进行实验验证。【结果】比较两种RNA-seq技术在不同培养条件下的RNA测序结果和对转录单元的精细分析,共鉴定到105个高可信度的nc RNA,并发现4个在不同盐度下表达差异较大的nc RNA,通过Northern blot和CR-RT-PCR验证了inc RNA1436和inc RNA1903的表达情况、转录本、转录起始位点及终止位点等。【结论】首次在组学水平鉴定了地中海富盐菌中的nc RNA,不同盐浓度刺激下部分nc RNA的转录差异暗示其有可能参与地中海富盐菌对盐胁迫的适应,高可信度nc RNA的组学发现为今后全面开展嗜盐古菌nc RNA的功能机制研究提供了基础数据及重要的切入点。     

Identification and functional characterization of pfm, a novel gene involved in swimming motility of Pseudomonas aeruginosa

Pseudomonas aeruginosa, an important opportunistic pathogen, has a single polar flagellum which is an important virulence and colonization factor by providing swimming motility. This paper describes the functional characterization of a novel gene pfm (PA2950) of P. aeruginosa. The pfm encodes a protein that is similar to a number of short-chain alcohol dehydrogenases of other bacterial species. Mutation of this gene results in a defect in swimming motility which can be completed back to that of wild type by a plasmid containing the pfm. Interestingly, the pfm mutant possesses an intact flagellum which does not rotate, thus giving rise to a non-motile phenotype. The pfm gene is encoded on an operon together with two upstream genes which code for electron transfer flavoprotein (ETF). Yeast two-hybrid tests indicated that the PFM interacts with the ETF, suggesting that the putative dehydrogenase (PFM) is involved in energy metabolism that is critical for the rotation of flagellum in P. aeruginosa.     

Identification of Cox20p, a novel protein involved in the maturation and assembly of cytochrome oxidase subunit 2

We have identified Cox20p, a 23.8-kDa protein of the mitochondrial inner membrane that is involved in the biogenesis of the yeast cytochrome oxidase complex. Cytochrome oxidase subunit 2 (Cox2p) accumulates as a precursor in cox20 mutants, suggesting a defect in biogenesis of this mitochondrially encoded protein. The inability of cox20 mutants to process the subunit 2 precursor (pCox2p) is not due to impaired export of the protein across the inner membrane or to an inactive Imp1p/Imp2p peptidase. Rather, Cox20p specifically binds the newly synthesized pCox2p, a step required to present the exported pCox2p as a substrate to the Imp1p peptidase. All of the endogenous pCox2p accumulated in an Deltaimp1 mutant, and a small fraction of Cox2p in wild type yeast, is detected in a complex with Cox20p. Following maturation Cox2p remained associated with Cox20p, prior to assembling into the cytochrome oxidase complex. We propose that Cox20p acts as a membrane-bound chaperone necessary for cleavage of pCox2p and for interaction of the mature protein with other subunits of cytochrome oxidase in a later step of the assembly process.