21-nt phasiRNAs direct target mRNA cleavage in rice male germ cells

In grasses, phased small interfering RNAs (phasiRNAs), 21- or 24-nucleotide (nt) in length, are predominantly expressed in anthers and regulate male fertility. However, their targets and mode of action on the targets remain unknown. Here we profile phasiRNA expression in premeiotic and meiotic spikelets as well as in purified male meiocytes at early prophase I, tetrads and microspores in rice. We show that 21-nt phasiRNAs are most abundant in meiocytes at early prophase I while 24-nt phasiRNAs are more abundant in tetrads and microspores. By performing highly sensitive degradome sequencing, we find that 21-nt phasiRNAs direct target mRNA cleavage in male germ cells, especially in meiocytes at early prophase I. The target genes in early prophase I meiocytes show an enrichment for carbohydrate biosynthetic and metabolic pathways. Our study provides strong evidence that 21-nt phasiRNAs act in a target-cleavage mode and may facilitate the progression of meiosis by fine-tuning carbohydrate biosynthesis and metabolism in male germ cells. To profile phasiRNAs and explore the potential targets of phasiRNAs in male germ cells at different stages, we collected highly pure meiocytes at early prophase I tetrads, and microspores, respectively, by micromanipulation. Pre-meiotic spikelets, in which anthers are at stage 2 to stage 3 , meiotic spikelets in which germ cells are mostly at the early prophase of meiosis I, and embryos from seeds germinated 24h at 28℃, were also collected as controls. We then using these samples performed low-input sRNA-seq, sRNA-seq and high sensitive degradome-seq in three biological replicates.

在禾本科植物中，长度为21或24核苷酸（nt）的阶段性小干扰RNA（phased small interfering RNAs，phasiRNAs）主要在花药中表达，调控雄性育性。然而，其靶标基因及作用模式迄今尚未明确。本研究以水稻为材料，对减数分裂前及减数分裂期小穗、纯化获得的减数分裂I早期前期雄性减数分裂细胞、四分体和小孢子中的phasiRNA表达谱进行了分析。研究发现，21 nt长度的phasiRNAs在减数分裂I早期前期的减数分裂细胞中丰度最高，而24 nt长度的phasiRNAs在四分体和小孢子中含量更为丰富。通过高灵敏度降解组测序技术，本研究证实21 nt长度的phasiRNAs可介导雄性生殖细胞中的靶标mRNA剪切，该现象在减数分裂I早期前期的减数分裂细胞中尤为显著。对减数分裂I早期前期减数分裂细胞中的靶标基因进行富集分析后发现，其显著富集于碳水化合物生物合成与代谢通路。本研究提供了强有力的证据，表明21 nt长度的phasiRNAs通过靶标剪切模式发挥作用，并可能通过精细调控雄性生殖细胞中的碳水化合物生物合成与代谢过程，促进减数分裂进程。为解析不同发育阶段雄性生殖细胞中的phasiRNA表达谱并探究其潜在靶标，本研究通过显微操作技术分别分离得到高纯度的减数分裂I早期前期雄性减数分裂细胞、四分体及小孢子。同时，本研究还收集了以下样本作为对照：花药处于2至3期的减数分裂前小穗、生殖细胞大多处于减数分裂I早期前期的减数分裂期小穗，以及在28℃条件下萌发24小时的种子胚。随后，利用上述样本完成了三次生物学重复的低起始量小RNA测序（low-input sRNA-seq）、常规小RNA测序（sRNA-seq）及高灵敏度降解组测序（degradome-seq）。