PRJDB2204: Rice germline‐specific Argonaute MEL 1 protein binds to phasiRNA s generated from more than 700 lincRNA s
概要: Small RNA s that interact with Argonaute (AGO ) proteins play central roles in RNA‐mediated silencing. MEIOSIS ARRESTED AT LEPTOTENE1 (MEL 1), a rice AGO , has specific functions in the development of pre‐meiotic germ cells and the progression of meiosis. Here, we show that MEL 1, which is located mostly in the cytoplasm of germ cells, associates preferentially with 21‐nucleotide phased small interfering RNA s (phasiRNA s) that bear a 5′‐terminal cytosine. Most phasiRNA s are derived from 1171 intergenic clusters distributed on all rice chromosomes. From these clusters, over 700 large intergenic, non‐coding RNAs (lincRNA s) that contain the consensus sequence complementary to miR2118 are transcribed specifically in inflorescences, and cleaved within the miR2118 site. Cleaved lincRNA s are processed via DICER ‐LIKE 4 (DCL 4) protein, resulting in production of phasiRNAs. This study provides the evidence that the miR2118‐dependent and the DCL 4‐dependent pathways are both required for biogenesis of 21‐nt phasiRNA s associated with germline‐specific MEL 1 AGO in rice, and over 700 lincRNA s are key factors for induction of this biogenesis during reproductive‐specific stages.
项目整体设计: RNA-seq of wild type Nipponbare's anthers
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All plants were grown in a field in the city of Mishima, Shizuoka, Japan. |
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The rice (Oryza sativa L., subspecies japonica ) cultivar Nipponbare, was used as the wild type plant. mel1‐1 homozygous mutants were obtained by insertion of an endogenous retrotransposon Tos17, and subsequent backcrosses (three times) with Nipponbare (BC3F4). |
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Total RNA was extracted from pre‐meiotic 0.4‐mm anthers (for RNA‐sequencing and qRT‐PCR) with TRIZOL® Reagent (Life Technologies. |
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A total RNA sample was extracted from 0.4‐mm anthers from WT flowers. RNAs (2 μg) purified using oligo(dT) beads were deep sequenced using a Hiseq2000 (Illumina) in accordance with the manufacturer's instructions. |
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poly(A)+ RNA |
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SINGLE |
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ILLUMINA |
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Illumina HiSeq 2000 |
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Unspecific |
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Rice germline-specific Argonaute MEL1 protein binds to phasiRNAs generated from more than 700 lincRNAs.
The Plant journal : for cell and molecular biology . 2014-04-15 [PMID:
24635777]
