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ABSTRACT: Small RNAs are key molecules in RNA silencing pathways that exert sequence-specific regulation of gene expression and chromatin modifications in many eukaryotes. In plants, endogenous small RNAs, including microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) play an important role in biological processes such as development and stress responses. In addition, viral genome-derived siRNAs are produced during viral infection, and they exhibit anti-viral defense by an RNA silencing pathway. These endogenous and exogenous small RNAs are mainly 21-24 nucleotides in length. Here, we describe a method to identify small RNA sequences from plant tissues. Small RNAs are purified by column fractionation and gel excision from total RNAs. These small RNAs are ligated at both termini to DNA/RNA chimeric adapters and reverse-transcribed to produce cDNAs. By the following PCR amplification, BanI restriction sites are added to cDNAs, which enables directional concatamerization. Concatamerized-fragments are cloned and sequenced. This method could be applied to identify small RNA sequences from many sources, e.g., mutant plants, plants in various stress environments, and virus-infected plants.
Methods in molecular biology (Clifton, N.J.) 01/2010; 631:123-38.
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ABSTRACT: RNA silencing is a broadly conserved machinery and is involved in many biological events. Small RNAs are key molecules in RNA silencing pathway that guide sequence-specific gene regulations and chromatin modifications. The silencing machinery works as an anti-viral defense in virus-infected plants. It is generally accepted that virus-specific small interfering (si) RNAs bind to the viral genome and trigger its cleavage. Previously, we have cloned and obtained sequences of small RNAs from Arabidopsis thaliana infected or uninfected with crucifer Tobacco mosaic virus. MicroRNAs (miRNAs) accumulated to a higher percentage of total small RNAs in the virus-infected plants. This was partly because the viral replication protein binds to the miRNA/miRNA* duplexes. In the present study, we mapped the sequences of small RNAs other than virus-derived siRNAs to the Arabidopsis genome and assigned each small RNA. It was demonstrated that only miRNAs increased as a result of viral infection. Furthermore, some newly identified miRNAs and miRNA candidates were found from the virus-infected plants despite a limited number of examined sequences. We propose that it is advantageous to use virus-infected plants as a source for cloning and identifying new miRNAs.
DNA Research 11/2007; 14(5):227-33. · 5.16 Impact Factor
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ABSTRACT: The sequence profiles of small interfering RNAs (siRNAs) in Arabidopsis infected with the crucifer tobamovirus tobacco mosaic virus (TMV)-Cg were determined by using a small RNA cloning technique. The majority of TMV-derived siRNAs were 21 nt in length. The size of the most abundant endogenous small RNAs in TMV-infected plants was 21 nt, whilst in mock-inoculated plants, it was 24 nt. Northern blot analysis revealed that some microRNAs (miRNAs) accumulated more in TMV-infected plants than in mock-inoculated plants. The question of whether the TMV-Cg-encoded 126K replication protein, an RNA-silencing suppressor, caused small RNA enrichment was examined. Transient expression of the replication protein did not change the pattern of miRNA processing. However, miRNA, miRNA* (the opposite strand of the miRNA duplex) and hairpin-derived siRNA all co-immunoprecipitated with the replication protein. Gel mobility-shift assays indicated that the replication protein binds small RNA duplexes. These results suggest that the tobamovirus replication protein functions as a silencing suppressor by binding small RNA duplexes, changing the small RNA profile in infected plants.
Journal of General Virology 09/2007; 88(Pt 8):2347-52. · 3.36 Impact Factor
