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Time-resolved small RNA sequencing unravels molecular principles of microRNA homeostasis [WT s4U time-course miRNA-seq]

ABSTRACT: Argonaute (Ago)-bound microRNAs (miRNAs) silence mRNA expression in a dynamic and regulated manner to control organismal development, physiology and disease. Here, we employed metabolic sequencing of small RNAs for a comprehensive view on intracellular miRNA kinetics in Drosophila. Based on absolute biogenesis and decay rates, miRNAs rank among the fastest produced and most long-lived cellular transcripts, enabling them to reach >105 copies per cell at steady-state. Tight coupling of steps in biogenesis produces mature miRNAs within minutes and is effectively disrupted by pre-miRNA uridylation. In contrast, control over Ago protein homeostasis generates a kinetic bottleneck that cooperates with ncRNA surveillance to ensure faithful miRNA loading. Finally, regulated small RNA decay enables the rapid turnover of specific Ago1-bound miRNAs but not of Ago2-bound siRNAs, reflecting key differences in the robustness of small RNA silencing pathways. Our work opens new experimental avenues to deconvolute the timescales, molecular features, and regulation of small RNA silencing pathways in living cells.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE129550 | GEO | 2019/08/06

REPOSITORIES: GEO

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Project description:MicroRNAs (miRNAs) are a class of small RNAs which typically function by guiding cleavage of target messenger RNAs. They have been shown to play major roles in a variety of plant processes including development, and responses to pathogens and environmental stresses. To identify new miRNAs and regulation in Arabidopsis thaliana, 27 small RNA libraries were constructed and sequenced from various tissues, stresses and small RNA biogenesis mutants, resulting in 95 million genome-matched sequences. The use of rdr2 to enrich the miRNA population greatly enhanced this analysis and led to the discovery of 44 new miRNAs arising from both known and new precursors. Parallel Analysis of RNA Ends (PARE) data provide evidence that the majority guide target cleavage. The inclusion of novel stress/tissue conditions, such as submergence-stressed flowers, enabled identification of new stress regulation of both miRNAs and their targets, all of which were validated in wild type plants. By combining small RNA expression analysis with ARGONAUTE (AGO) immunoprecipitation data and global target cleavage data from PARE, a much more complete picture of Arabidopsis miRNAs was obtained. This combinatorial approach led to the discovery of AGO loading and target cleavage biases, which gave important insights into tissue-specific expression patterns, pathogen responses and the role of sequence variation among closely related miRNA family members. Examination of various tissues, stresses and small RNA biogenesis mutants in Arabidopsis by high-throughput sequencing for small RNA profiling. We have used AGO-IP and PARE data from the published data, which were downloaded from NCBI GEO with the following accession number. AGO-IP from GSM253622, GSM707682, GSM642335, GSM642336, GSM512703, GSM512702, GSM707683, GSM707684, GSM707685, GSM149080, GSM253623, GSM304283, GSM642337, GSM642338, GSM253624, GSM415788, GSM707686, GSM707687, GSM707688, GSM707689, GSM415787, GSM149081, GSM253625, GSM415789, GSM415790, GSM304285, GSM415791, GSM415792 PARE sequencing data from xrn4 flowers were obtained from Gene Expression Omnibus with accession number GSM280227.

Project description:It is largely unknown how microRNAs (miRNAs) are degraded in vivo or whether active miRNA turnover is critical to specific developmental processes. Arabidopsis ARGONAUTE10 (AGO10) maintains stem cell homeostasis in meristems by repression of miR165/6, a conserved miRNA acting through AGO1. Here, we report that AGO10, through its binding to miR165/6, reduces miR165/6 accumulation by enhancing its decay. We show that AGO10 promotes the 3’ truncation of miR165/6 by SMALL RNA DEGRADING NUCLEASES (SDNs) and that AGO10-bound miR165/6 is more susceptible to degradation by SDN1 than AGO1-bound miR165/6. Our work uncovers a mechanism that governs the stability of miRNAs, establishes the importance of spatially controlled miRNA decay in stem cell homeostasis, and sheds light on the diversification and specialization of Argonaute proteins. Both biogenesis and degradation contribute to the steady-state levels of microRNAs (miRNAs). miRNA degradation in both plants and animals is associated with 3’ truncation and 3’ uridylation, and in Arabidopsis, the nucleotidyl transferase HEN1 SUPPRESSOR1 is responsible for 3’ uridylation. The SMALL RNA DEGRADING NUCLEASE (SDN) family of 3’ to 5 exonucleases degrades short RNAs in vitro and limits the accumulation of miRNAs in vivo, but it is not known whether SDNs are responsible for the accumulation of 3’ truncated miRNA species in vivo. In addition, there has not been an example of active miRNA turnover being critical to specific developmental processes. Arabidopsis ARGONAUTE10 (AGO10) maintains stem cell homeostasis in meristems by sequestration of miR165/6, a conserved miRNA acting through AGO1. Here, we report that AGO10, through binding to miR165/6, reduces miR165/6 accumulation by enhancing its degradation. We show that SDNs are responsible for the generation of 3’ truncated miRNAs in vivo and AGO10 promotes the 3’ truncation of miR165/6 by SDNs. Our work uncovers a novel mechanism of miRNA degradation mediated by an argonaute protein, which contrasts the known stabilizing effects of most AGO proteins on their associated miRNAs. This work, together with previous studies on AGO10, demonstrates that spatially regulated miRNA degradation underlies stem cell maintenance in plants.

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Time-resolved small RNA sequencing unravels molecular principles of microRNA homeostasis [WT s4U time-course miRNA-seq]

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