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. 2005 Aug 2;33(14):4443-54.
doi: 10.1093/nar/gki758. Print 2005.

Identification and characterization of endogenous small interfering RNAs from rice

Ramanjulu Sunkar  1 , Thomas Girke, Jian-Kang Zhu
Affiliations

Identification and characterization of endogenous small interfering RNAs from rice

Ramanjulu Sunkar et al. Nucleic Acids Res. .

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.

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Figures

Figure 1
Figure 1
Rice endogenous siRNAs. (A) Histogram of the number of siRNAs identified from the libraries of shoots and roots of young seedlings, and inflorescence tissues. (B) Size distribution of endogenous siRNAs. (C) The rice endogenous siRNAs correspond to sequences from ORFs, transposons, introns and intergenic regions. (D) Sequence composition of the 5′ ends of rice siRNAs as a function of length.
Figure 2
Figure 2
Endogenous siRNAs in clusters. The distance between two siRNAs is indicated. 5′ and 3′ flanking genes are shown. siRNAs are indicated by green or red arrows. For siRNAs that overlap the ORF of a gene, the siRNAs are shown on top and bottom of the gene. Location of siRNA on the genome not drawn to scale. A–F are different siRNA clusters.
Figure 3
Figure 3
Expression analysis of some of the rice endogenous siRNAs by northern hybridization. Northern blots of low molecular weight RNA isolated from different tissues were probed with labeled oligonucleotides. The tRNA and 5S rRNA bands were visualized by ethidium bromide staining of polyacrylamide gels and served as loading controls. Labeled RNA oligonucleotide was used as size marker and the position was indicated. A–F are different siRNAs as listed.
Figure 4
Figure 4
PCR-based detection of endogenous siRNAs. PCR amplification was performed using a 5′ primer for the 5′ adapter sequence and a 3′ primer specific for the candidate siRNAs.
Figure 5
Figure 5
Identification of siRNA-guided cleavage products of target mRNAs in rice. (A) mRNA 9639.m00201, (B) mRNA 9636.m02299 and (C) mRNA 9632.m00807. Mapping of cleavage sites was performed by RLM-5′ RACE. Partial mRNA sequences from target genes were aligned with siRNAs. Numbers indicate the fraction of cloned PCR products terminating at different positions.
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