Abstract

Plant microRNAs (miRNAs) guide cytosolic post‐transcriptional gene silencing of sequence‐complementary transcripts within the producing cells, as well as in distant cells and tissues. Here, we used an artificial miRNA‐based system (amiRSUL) in Arabidopsis thaliana to explore the still elusive mechanisms of inter‐cellular miRNA movement via forward genetics. This screen identified many mutant alleles of HASTY (HST), the ortholog of mammalian EXPORTIN5 (XPO5) with a recently reported role in miRNA biogenesis in Arabidopsis. In both epidermis‐peeling and grafting assays, amiRSUL levels were reduced much more substantially in miRNA‐recipient tissues than in silencing‐emitting tissues. We ascribe this effect to HST controlling cell‐to‐cell and phloem‐mediated movement of the processed amiRSUL, in addition to regulating its biogenesis. While HST is not required for the movement of free GFP or siRNAs, its cell‐autonomous expression in amiRSUL‐emitting tissues suffices to restore amiRSUL movement independently of its nucleo‐cytosolic shuttling activity. By contrast, HST is dispensable for the movement and activity of amiRSUL within recipient tissues. Finally, HST enables movement of endogenous miRNAs that display mostly unaltered steady‐state levels in hst mutant tissues. We discuss a role for HST as a hitherto unrecognized regulator of miRNA movement in relation to its recently assigned nuclear function at the nexus of MIRNA transcription and miRNA processing.

Highlights

  • In RNA silencing, small (s)RNAs, 20–32-nt in size, incorporate into ARGONAUTE(AGO)-like proteins to guide RNA-induced silencing complexes (RISCs) to sequence-complementary target RNAs (Ghildiyal & Zamore, 2009)

  • MiRNA-target interactions may be accompanied by the cytosolic de novo conversion of target transcripts into dsRNA, which is processed by DCL4 into populations of 21-nt small interfering RNAs (siRNAs) known as trans-acting siRNAs (Vazquez et al, 2004b; Gasciolli et al, 2005) and phased siRNAs (Fei et al, 2013)

  • Arabidopsis pri-miR319 was re-engineered into 5’U-terminal amiRSUL predicted to target, in an AGO1-dependent manner, the magnesium chelatase subunit CHLORINA42 (CH42 or SUL) mRNA, which is required for chlorophyll accumulation (Fig 1A; Appendix Fig S1A). pri-amiRSUL was mobilized under the phloem companion-cell (CC)-specific pSUC2 promoter to produce several pSUC2::amiRSUL single-locus transgenic lines

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Summary

Introduction

In RNA silencing, small (s)RNAs, 20–32-nt in size, incorporate into ARGONAUTE(AGO)-like proteins to guide RNA-induced silencing complexes (RISCs) to sequence-complementary target RNAs (Ghildiyal & Zamore, 2009). MiRNA-target interactions may be accompanied by the cytosolic de novo conversion of target transcripts into dsRNA, which is processed by DCL4 into populations of 21-nt siRNAs known as trans-acting siRNAs (tasiRNAs) (Vazquez et al, 2004b; Gasciolli et al, 2005) and phased siRNAs (phasiRNAs) (Fei et al, 2013). Both types of siRNAs endow a 2021 The Authors.

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