PEP444c encoded within the MIR444c gene regulates microRNA444c accumulation in barley

Abstract

MicroRNAs are small, non-coding RNA molecules that regulate 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 characterized their structure and the processing pattern of the primary transcripts (pri-miRNAs444). Pri-microRNAs444 undergo extensive alternative splicing, by which functional and non-functional pri-miRNA444 isoforms are generated. 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. Taken together, our observations suggest that PEP444c encoded by the MIR444c gene is involved in microRNA444c biogenesis in barley.