PEP444c encoded within the MIR444c gene regulates microRNA444c accumulation in barley

Abstract

MicroRNAs are small, noncoding RNA molecules that regulate the expression of their target genes. The MIR444 gene family is present exclusively in monocotyledons, and microRNAs444 from this family have been shown to target certain MADS‐box transcription factors in rice and barley. We identified three barley MIR444 (MIR444a/b/c) genes and comprehensively characterised their structure and the processing pattern of the primary transcripts (pri‐miRNAs444). Pri‐microRNAs444 undergo extensive alternative splicing, generating functional and nonfunctional pri‐miRNA444 isoforms. We show that barley pri‐miRNAs444 contain numerous open reading frames (ORFs) whose transcripts associate with ribosomes. Using specific antibodies, we provide evidence that selected ORFs encoding PEP444a within MIR444a and PEP444c within MIR444c are expressed in barley plants. Moreover, we demonstrate that CRISPR‐associated endonuclease 9 (Cas9)‐mediated mutagenesis of the PEP444c‐encoding sequence results in a decreased level of PEP444 transcript in barley shoots and roots and a 5‐fold reduced level of mature microRNA444c in roots. Our observations suggest that PEP444c encoded by the MIR444c gene is involved in microRNA444c biogenesis in barley.

Biological Activity of Artificial Plant Peptides Corresponding to the Translational Products of Small ORFs in Primary miRNAs and Other Long “Non-Coding” RNAs

“…CRISPR-cas9-mediated gene editing of Hv-miPEP444c coding sequence has revealed a reduction of root (30%) and shoot (20%) surface area in mutated plants compared to the wild type. CRISPR-cas9 mutagenesis of Hv-miPEP444a gives rise to lethal mutations [ 68 ]. Second, bioinformatic tools have predicted two miPEPs in Zea mays plants.…”

“…PoaceaeOryza sativa Os-miPEP156e 36 aa unknown enhances the transcript level of pri-miR156e and reduces inhibition of seedling growth resulting in an increase in root length and biomass under Cd stress [67] Oryza sativa Os-miPEP172b 9 aa unknown increases the expression of pri-miRNA and improves the resistance of rice seedlings against Cd stress [67] Hordeum vulgare Hv-miPEP444c 119 aa unknown CRISPR-cas9 mediated gene editing of Hv-miPEP444c coding sequence has revealed that a reduction of root and shoot surface area [68] Zea mays Zma-miPEP159d unknown unknown [69] Plant family names and the size of peptides are given in bold.…”