DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

Design of LNA-modified siRNAs against the highly structured 5' UTR of coxsackievirus B3

This study describes a strategy to develop LNA-modified small interfering RNA (siRNAs) against the highly structured 5' UTR of coxsackievirus B3 (CVB-3), which is an attractive target site due to its high degree of conservation. Accessible sites were identified based on structural models and RNase H assays with DNA oligonucleotides. Subsequently, LNA gapmers, siRNAs, siLNAs and small internally segmented interfering RNA (sisiLNAs) were designed against sites, which were found to be accessible in the in vitro assays, and tested in reporter assays and experiments with the infectious virus. The best siLNA improved viability of infected cells by 92% and exerted good antiviral activity in plaque reduction assays.     

植物小RNA的研究进展

在植物中发现大量内源性的小RNA,它们与真核生物中的内源性的微RNA和外源性的干扰小RNA有类似的性质和功能。本对植物中小RNA分子的分布、作用机制、功能以及信号传导等方面作一概述。     

Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA

Photoperiod- and thermo-sensitive genic male sterility (PGMS and TGMS) are the core components for hybrid breeding in crops. Hybrid rice based on the two-line system using PGMS and TGMS lines has been successfully developed and applied widely in agriculture. However, the molecular mechanism underlying the control of PGMS and TGMS remains obscure. In this study, we mapped and cloned a major locus, p/tms12-1 (photo- or thermo-sensitive genic male sterility locus on chromosome 12), which confers PGMS in the japonica rice line Nongken 58S (NK58S) and TGMS in the indica rice line Peiai 64S (PA64S, derived from NK58S). A 2.4-kb DNA fragment containing the wild-type allele P/TMS12-1 was able to restore the pollen fertility of NK58S and PA64S plants in genetic complementation. P/TMS12-1 encodes a unique noncoding RNA, which produces a 21-nucleotide small RNA that we named osa-smR5864w. A substitution of C-to-G in p/tms12-1, the only polymorphism relative to P/TMS12-1, is present in the mutant small RNA, namely osa-smR5864m. Furthermore, overexpression of a 375-bp sequence of P/TMS12-1 in transgenic NK58S and PA64S plants also produced osa-smR5864w and restored pollen fertility. The small RNA was expressed preferentially in young panicles, but its expression was not markedly affected by different day lengths or temperatures. Our results reveal that the point mutation in p/tms12-1, which probably leads to a loss-of-function for osa-smR5864m, constitutes a common cause for PGMS and TGMS in the japonica and indica lines, respectively. Our findings thus suggest that this noncoding small RNA gene is an important regulator of male development controlled by cross-talk between the genetic networks and environmental conditions.     

The RNA phosphatase PIR-1 regulates endogenous small RNA pathways in C. elegans

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The Cdc42p GTPase is targeted to the site of cell division in the fission yeast Schizosaccharomyces pombe

The Rho-family GTPase Cdc42p regulates many aspects of cell polarity and growth in eukaryotic cells, including the organization of the actin cytoskeleton. To further examine Cdc42p function in the fission yeast Schizosaccharomyces pombe, a functional green fluorescent protein (GFP)-Cdc42p fusion protein was generated. GFP-Cdc42p was observed at the medial region of the cell at the cell-division site early in cytokinesis and remained there through cell separation, and was also localized to the periphery of the cell and to internal membranes. Unexpectedly, treatment with the actin-depolymerizing drug latrunculin-A disrupted the medial region targeting pattern, and cells deficient in the actin-binding proteins tropomyosin and profilin also did not exhibit medial GFP-Cdc42p staining. In addition, medial GFP-Cdc42p localization was eliminated in a number of cytokinesis mutants, including strains defective in assembling the medial actinomyosin ring, medial ring contraction, and septum assembly. GFP-Cdc42p targeting was less affected in mutants that formed misplaced or multiple septa. These results suggest that the localization of Cdc42p at the cell-division site was dependent upon the actin cytoskeleton and that Cdc42p may function in the interdependent processes of cytokinesis and septation.     

RNA干涉现象的发现与研究进展——2006年诺贝尔生理学或医学奖简介

RNA干涉现象以一种非常明确的方式抑制了基因表达,对于基因表达的调控、病毒感染的防护、控制跳跃基因具有重要的意义.它已被作为一种强大的“基因沉默”技术被用于全球的实验室,而且,会推动新的医疗技术的出现.     

Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway

In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character—as is found in small interfering RNAs (siRNAs)—directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide.     

The fission yeast rpa17+ gene encodes a functional homolog of AC19, a subunit of RNA polymerases I and III of Saccharomyces cerevisiae

Eukaryotic RNA polymerases I and III consist of multiple subunits. Each of these enzymes includes two distinct and evolutionarily conserved subunits called α-related subunits which are shared only by polymerases I and III. The α-related subunits show limited homology with the α-subunit of prokaryotic RNA polymerase. To gain further insight into the structure and function of α-related subunits, we cloned and characterized a gene from Schizosaccharomyces pombe that encodes a protein of 17 kDa which can functionally replace AC19 – an α-related subunit of RNA polymerases I and III of Saccharomyces cerevisiae– and was thus named rpa17 ⁺. RPA17 has 125 amino acids and shows 63% identity to AC19 over a 108-residue stretch, whereas the N-terminal regions of the two proteins are highly divergent. Disruption of rpa17 ⁺ shows that the gene is essential for cell growth. Sequence comparison with other α-related subunits from different species showed that RPA17 contains an 81-amino acid block that is evolutionarily conserved. Deletion analysis of the N- and C-terminal regions of RPA17 and AC19 confirms that the 81-amino acid block is important for the function of the α-related subunits. Received: 1 October 1998 / Accepted: 3 December 1998     

The master role of siRNAs in plant immunity

Gene silencing mediated by small noncoding RNAs (sRNAs) is a fundamental gene regulation mechanism in eukaryotes that broadly governs cellular processes. It has been established that sRNAs are critical regulators of plant growth, development, and antiviral defence, while accumulating studies support positive roles of sRNAs in plant defence against bacteria and eukaryotic pathogens such as fungi and oomycetes. Emerging evidence suggests that plant sRNAs move between species and function as antimicrobial agents against nonviral parasites. Multiple plant pathosystems have been shown to involve a similar exchange of small RNAs between species. Recent analysis about extracellular sRNAs shed light on the understanding of the selection and transportation of sRNAs moving from plant to parasites. In this review, we summarize current advances regarding the function and regulatory mechanism of plant endogenous small interfering RNAs (siRNAs) in mediating plant defence against pathogen intruders including viruses, bacteria, fungi, oomycetes, and parasitic plants. Beyond that, we propose potential mechanisms behind the sorting of sRNAs moving between species and the idea that engineering siRNA‐producing loci could be a useful strategy to improve disease resistance of crops.     

Milligram scale production of potent recombinant small interfering RNAs in Escherichia coli

Small interfering RNAs (siRNAs) are invaluable research tools for studying gene functions in mammalian cells. siRNAs are mainly produced by chemical synthesis or by enzymatic digestion of double‐stranded RNA (dsRNA) produced in vitro. Recently, bacterial cells, engineered with ectopic plant viral siRNA binding protein p19, have enabled the production of “recombinant” siRNAs (pro‐siRNAs). Here, we describe an optimized methodology for the production of milligram amount of highly potent recombinant pro‐siRNAs from Escherichia coli cells. We first optimized bacterial culture medium and tested new designs of pro‐siRNA production plasmid. Through the exploration of multiple pro‐siRNA related factors, including the expression of p19 protein, (dsRNA) generation method, and the level of RNase III, we developed an optimal pro‐siRNA production plasmid. Together with a high–cell density fed‐batch fermentation method in a bioreactor, we have achieved a yield of ~10 mg purified pro‐siRNA per liter of bacterial culture. The pro‐siRNAs produced by the optimized method can achieve high efficiency of gene silencing when used at low nanomolar concentrations. This new method enables fast, economical, and renewable production of pure and highly potent bioengineered pro‐siRNAs at the milligram level. Our study also provides important insights into the strategies for optimizing the production of RNA products in bacteria, which is an under‐explored field.     

A simplified vector system for visualization of localized RNAs in Schizosaccharomyces pombe

RNA localization is an important event that is essential for the polarization and differentiation of a cell. Although several methods are currently used to detect localized RNAs, a simplified detection system has not yet been developed for Schizosaccharomyces pombe. In the present study, we describe a new vector system for the visualization of localized RNAs in S. pombe using a U1A-tag-GFP system. A pREP1-U1A-tag vector plasmid to express U1A-tagged RNA and a pREP2-U1AGFP plasmid to produce a U1A-GFP fusion protein were constructed for this system. Since the U1A-GFP protein binds U1A-tagged RNA, fluorescence is observed at the location of U1A-tagged RNA in cells expressing both of these. The nucleolar localization of U3 snoRNA was successfully detected using this system, and a novel RNA localized at the DNA region of the nucleus was found by screening localized RNAs. This system will accelerate the study of localized RNAs in S. pombe.     

Identification of maize lethal necrosis disease causal viruses in maize and suspected alternative hosts through small RNA profiling

Maize lethal necrosis disease (MLND) is a devastating viral disease of maize caused by double infection with Maize chlorotic mottle virus (MCMV) and any one of the Potyviridae family members. Management of MLND requires effective resistance screening and surveillance tools. In this study, we report the use of small RNA (sRNA) profiling to detect MLND causal viruses and further the development of alternative detection markers for use in routine surveillance of the disease-causing viruses. Small RNAs (sRNAs) originating from five viruses namely MCMV, Sugarcane mosaic virus (SCMV), Maize streak virus (MSV), Maize-associated totivirus (MATV) and Maize yellow mosaic virus (MYMV) were assembled from infected maize samples collected from MLND hot spots in Kenya. The expression of the identified viral domains was further validated using quantitative real-time PCR. New markers for the detection of some of the MLND causal viruses were also developed from the highly expressed domains and used to detect the MLND-causative viruses in maize and alternative hosts. These findings further demonstrate the potential of using sRNAs especially from highly expressed viral motifs in the detection of MLND causal viruses. We report the validation of new sets of primers for use in detection of the most common MLND causal viruses MCMV and SCMV in East Africa.