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Identification and characterization of endogenous small interfering RNAs from rice.

ABSTRACT: RNA silencing-mediated small interfering RNAs (siRNAs) and microRNAs (miRNAs) have diverse natural roles, ranging from regulation of gene expression and heterochromatin formation to genome defense against transposons and viruses. Unlike miRNAs, endogenous siRNAs are generally not conserved between species; consequently, their identification requires experimental approaches. Thus far, endogenous siRNAs have not been reported from rice, which is a model species for monocotyledonous plants. We identified a large set of putative endogenous siRNAs from root, shoot and inflorescence small RNA cDNA libraries of rice. Most of these siRNAs are from intergenic regions, although a substantial proportion (22%) originates from the introns and exons of protein-coding genes. Northern and RT-PCR analysis revealed that the expression of some of the siRNAs is tissue specific or developmental stage specific. A total of 25 transposons and 21 protein-coding genes were predicted to be cis-targets of some of the siRNAs. Based on sequence homology, we also predicted 111 putative trans-targets for 44 of the siRNAs. Interestingly, approximately 46% of the predicted trans-targets are transposable elements, which suggests that endogenous siRNAs may play an important role in the suppression of transposon proliferation. Using RNA ligase-mediated-5' rapid amplification of cDNA end assays, we validated three of the predicted targets and provided evidence for both cis- and trans-silencing of target genes by siRNAs-guided mRNA cleavage.

SUBMITTER: Sunkar R

PROVIDER: S-EPMC1182700 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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Identification and characterization of endogenous small interfering RNAs from rice.

Sunkar Ramanjulu R Girke Thomas T Zhu Jian-Kang JK

Nucleic acids research 20050802 14

RNA silencing-mediated small interfering RNAs (siRNAs) and microRNAs (miRNAs) have diverse natural roles, ranging from regulation of gene expression and heterochromatin formation to genome defense against transposons and viruses. Unlike miRNAs, endogenous siRNAs are generally not conserved between species; consequently, their identification requires experimental approaches. Thus far, endogenous siRNAs have not been reported from rice, which is a model species for monocotyledonous plants. We iden ...[more]

PMID: 16077027

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Project description:BackgroundSmall RNA (sRNA)-guided RNA silencing is a critical antiviral defense mechanism employed by a variety of eukaryotic organisms. Although the induction of RNA silencing by bipartite and monopartite begomoviruses has been described in plants, the nature of begomovirus/betasatellite complexes remains undefined.Methodology/principal findingsSolanum lycopersicum plant leaves systemically infected with Tomato yellow leaf curl China virus (TYLCCNV) alone or together with its associated betasatellite (TYLCCNB), and Nicotiana benthamiana plant leaves systemically infected with TYLCCNV alone, or together with TYLCCNB or with mutant TYLCCNB were harvested for RNA extraction; sRNA cDNA libraries were then constructed and submitted to Solexa-based deep sequencing. Both sense and anti-sense TYLCCNV and TYLCCNB-derived sRNAs (V-sRNAs and S-sRNAs) accumulated preferentially as 22 nucleotide species in infected S. lycopersicum and N. benthamiana plants. High resolution mapping of V-sRNAs and S-sRNAs revealed heterogeneous distribution of V-sRNA and S-sRNA sequences across the TYLCCNV and TYLCCNB genomes. In TYLCCNV-infected S. lycopersicum or N. benthamiana and TYLCCNV and βC1-mutant TYLCCNB co-infected N. benthamiana plants, the primary TYLCCNV targets were AV2 and the 5' terminus of AV1. In TYLCCNV and betasatellite-infected plants, the number of V-sRNAs targeting this region decreased and the production of V-sRNAs increased corresponding to the overlapping regions of AC2 and AC3, as well as the 3' terminal of AC1. βC1 is the primary determinant mediating symptom induction and also the primary silencing target of the TYLCCNB genome even in its mutated form.Conclusions/significanceWe report the first high-resolution sRNA map for a monopartite begomovirus and its associated betasatellite using Solexa-based deep sequencing. Our results suggest that viral transcript might act as RDR substrates resulting in dsRNA and secondary siRNA production. In addition, the betasatellite affected the amount of V-sRNAs detected in S. lycopersicum and N. benthamiana plants.

Dataset Information

Identification and characterization of endogenous small interfering RNAs from rice.

Publications

Identification and characterization of endogenous small interfering RNAs from rice.

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