21-nt phasiRNAs direct target mRNA cleavage in rice male germ cells
Ontology highlight
ABSTRACT: 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.
Project description:Plants do not specify their germline until late in their life cycle. Hence, the plant germline is normally specified from terminally differentiated somatic cells, though the precise mechanism(s) are unknown. We have found that male gametogenesis in maize is associated with the accumulation of distinct 21-nt phased small-interfering RNAs (phasiRNAs) generated by meiosis-associated argonaute (MAGO) proteins. MAGO1 accumulates in the epidermis of pre-meiotic anthers while MAGO2 is found in developing meiocytes. We have found that MAGO proteins are required for meiocyte development as mutants display chromosomal defects and male infertility. Furthermore, we detect the heat stress-induced activation of a distinct class of Long terminal repeat retrotransposons in the male germline of MAGO mutants. Our data suggests that MAGO proteins and the reproductive phasiRNAs play important roles protecting the germline from transposable elements during environmental stress conditions.
Project description:In maize, 24-nt phased, secondary small interfering RNAs (phasiRNAs) are abundant in meiotic stage anthers, but their distribution and functions are not precisely known. Using laser capture microdissection we analyzed tapetal cells, meiocytes, and other somatic cells at several stages of anther development to establish the timing of 24-PHAS precursor transcripts and the 24-nt phasiRNA products. By integrating RNA and small RNA (sRNA) profiling plus single-molecule and sRNA FISH (smFISH or sRNA-FISH) spatial detection, we demonstrate that the tapetum is the primary site of 24-PHAS precursor and Dcl5 transcripts and the resulting 24-nt phasiRNAs. Interestingly, 24-nt phasiRNAs accumulate in all cell types, with the highest levels in meiocytes, followed by tapetum. Our data support the conclusion that 24-nt phasiRNAs are mobile from tapetum to meiocytes and to other somatic cells. We discuss possible roles for 24-nt phasiRNAs in anther cell types.
Project description:Maize anthers, the male reproductive floral organs, express two classes of phased, small interfering RNAs (phasiRNAs). RNA profiling from ten sequential cohorts of staged maize anthers plus mature pollen revealed that 21-nt phased siRNAs (21-phasiRNAs) from 463 loci appear abruptly after germinal and initial somatic cell fate specification and then diminish, while 24-nt phased siRNAs (24-phasiRNAs) from 176 loci coordinately accumulate during meiosis and persist as haploid gametophytes differentiate into pollen. RNA sequencing of anther developmental mutants, together with in situ RNA hybridization detection of phasiRNA biogenesis factors, demonstrated that 21-phasiRNAs and 24-phasiRNAs are independently regulated. Furthermore, 21-phasiRNAs require epidermal cells while 24-phasiRNAs require functional tapetal cells. Maize phasiRNAs and mammalian PIWI-interacting RNAs (piRNAs) illustrate convergent evolution of small RNAs to support male reproduction.
Project description:Maize anthers, the male reproductive floral organs, express two classes of phased, small interfering RNAs (phasiRNAs). RNA profiling from ten sequential cohorts of staged maize anthers plus mature pollen revealed that 21-nt phased siRNAs (21-phasiRNAs) from 463 loci appear abruptly after germinal and initial somatic cell fate specification and then diminish, while 24-nt phased siRNAs (24-phasiRNAs) from 176 loci coordinately accumulate during meiosis and persist as haploid gametophytes differentiate into pollen. RNA sequencing of anther developmental mutants, together with in situ RNA hybridization detection of phasiRNA biogenesis factors, demonstrated that 21-phasiRNAs and 24-phasiRNAs are independently regulated. Furthermore, 21-phasiRNAs require epidermal cells while 24-phasiRNAs require functional tapetal cells. Maize phasiRNAs and mammalian PIWI-interacting RNAs (piRNAs) illustrate convergent evolution of small RNAs to support male reproduction.
Project description:Maize anthers, the male reproductive floral organs, express two classes of phased, small interfering RNAs (phasiRNAs). RNA profiling from ten sequential cohorts of staged maize anthers plus mature pollen revealed that 21-nt phased siRNAs (21-phasiRNAs) from 463 loci appear abruptly after germinal and initial somatic cell fate specification and then diminish, while 24-nt phased siRNAs (24-phasiRNAs) from 176 loci coordinately accumulate during meiosis and persist as haploid gametophytes differentiate into pollen. RNA sequencing of anther developmental mutants, together with in situ RNA hybridization detection of phasiRNA biogenesis factors, demonstrated that 21-phasiRNAs and 24-phasiRNAs are independently regulated. Furthermore, 21-phasiRNAs require epidermal cells while 24-phasiRNAs require functional tapetal cells. Maize phasiRNAs and mammalian PIWI-interacting RNAs (piRNAs) illustrate convergent evolution of small RNAs to support male reproduction.
Project description:Maize anthers, the male reproductive floral organs, express two classes of phased, small interfering RNAs (phasiRNAs). RNA profiling from ten sequential cohorts of staged maize anthers plus mature pollen revealed that 21-nt phased siRNAs (21-phasiRNAs) from 463 loci appear abruptly after germinal and initial somatic cell fate specification and then diminish, while 24-nt phased siRNAs (24-phasiRNAs) from 176 loci coordinately accumulate during meiosis and persist as haploid gametophytes differentiate into pollen. RNA sequencing of anther developmental mutants, together with in situ RNA hybridization detection of phasiRNA biogenesis factors, demonstrated that 21-phasiRNAs and 24-phasiRNAs are independently regulated. Furthermore, 21-phasiRNAs require epidermal cells while 24-phasiRNAs require functional tapetal cells. Maize phasiRNAs and mammalian PIWI-interacting RNAs (piRNAs) illustrate convergent evolution of small RNAs to support male reproduction.
Project description:In maize, 24-nt phased, secondary small interfering RNAs (phasiRNAs) are abundant in meiotic stage anthers, but their distribution and functions are not precisely known. Using laser capture microdissection (LCM), we analyzed tapetal cells, meiocytes, and other somatic cells at several stages of anther development to establish the timing of 24-PHAS precursor transcripts and the 24-nt phasiRNA products. This dataset includes 24-nt phasiRNA part of data. 24-nt phasiRNAs are found to accumulate in all cell types, with the highest levels in meiocytes, followed by tapetum.
Project description:Temperature-sensitive male sterility is one of the core components for hybrid rice breeding based on two-line system. We previously found that knockout of ARGONAUTE 1d (AGO1d) caused temperature-sensitive male sterility in rice by influencing phased small interfering RNA (phasiRNA) biogenesis and function. However, the specific phasiRNAs and their targets underlying the temperature-sensitive male sterility in ago1d mutant remain to be determined. Here, we demonstrate that the ago1d mutant displays normal female fertility but complete male sterility at low temperature. Through a multi-omics analysis of small RNA, degradome, and transcriptome, we found that 21-nt phasiRNAs account for the greatest proportion of the 21-nt small RNA (sRNA) species in rice anthers and are sensitive to low temperature and markedly down-regulated in ago1d mutant. Moreover, we found that 21-nt phasiRNAs are essential for the mRNA cleavage of a set of fertility- and cold tolerance-associated genes, such as EDT1, TDR, OsPCF5 and OsTCP21, directly or indirectly determined by AGO1d-mediated gene silencing. The loss-of-function of 21-nt phasiRNAs can result in up-regulation of their targets and causes varying degrees of defects in male fertility and grain setting. Our results highlight the essential functions of 21-nt phasiRNAs in temperature-sensitive male sterility in rice and suggest their promising application in two-line hybrid rice breeding in the future.
Project description:Maize anthers, the male reproductive floral organs, express two classes of phased, small interfering RNAs (phasiRNAs). RNA profiling from ten sequential cohorts of staged maize anthers plus mature pollen revealed that 21-nt phased siRNAs (21-phasiRNAs) from 463 loci appear abruptly after germinal and initial somatic cell fate specification and then diminish, while 24-nt phased siRNAs (24-phasiRNAs) from 176 loci coordinately accumulate during meiosis and persist as haploid gametophytes differentiate into pollen. RNA sequencing of anther developmental mutants, together with in situ RNA hybridization detection of phasiRNA biogenesis factors, demonstrated that 21-phasiRNAs and 24-phasiRNAs are independently regulated. Furthermore, 21-phasiRNAs require epidermal cells while 24-phasiRNAs require functional tapetal cells. Maize phasiRNAs and mammalian PIWI-interacting RNAs (piRNAs) illustrate convergent evolution of small RNAs to support male reproduction. Examination of maize phasiRNAs by high throughput sequencing for RNA-seq, small RNA, and PARE profiling
Project description:Maize anthers, the male reproductive floral organs, express two classes of phased, small interfering RNAs (phasiRNAs). RNA profiling from ten sequential cohorts of staged maize anthers plus mature pollen revealed that 21-nt phased siRNAs (21-phasiRNAs) from 463 loci appear abruptly after germinal and initial somatic cell fate specification and then diminish, while 24-nt phased siRNAs (24-phasiRNAs) from 176 loci coordinately accumulate during meiosis and persist as haploid gametophytes differentiate into pollen. RNA sequencing of anther developmental mutants, together with in situ RNA hybridization detection of phasiRNA biogenesis factors, demonstrated that 21-phasiRNAs and 24-phasiRNAs are independently regulated. Furthermore, 21-phasiRNAs require epidermal cells while 24-phasiRNAs require functional tapetal cells. Maize phasiRNAs and mammalian PIWI-interacting RNAs (piRNAs) illustrate convergent evolution of small RNAs to support male reproduction. Examination of maize phasiRNAs by high throughput sequencing for RNA-seq, small RNA and PARE profiling.