Developmentally regulated expression and complex processing of barley pri-microRNAs

Abstract

BackgroundMicroRNAs (miRNAs) regulate gene expression via mRNA cleavage or translation inhibition. In spite of barley being a cereal of great economic importance, very little data is available concerning its miRNA biogenesis. There are 69 barley miRNA and 67 pre-miRNA sequences available in the miRBase (release 19). However, no barley pri-miRNA and MIR gene structures have been shown experimentally. In the present paper, we examine the biogenesis of selected barley miRNAs and the developmental regulation of their pri-miRNA processing to learn more about miRNA maturation in barely.ResultsTo investigate the organization of barley microRNA genes, nine microRNAs - 156g, 159b, 166n, 168a-5p/168a-3p, 171e, 397b-3p, 1120, and 1126 - were selected. Two of the studied miRNAs originate from one MIR168a-5p/168a-3p gene. The presence of all miRNAs was confirmed using a Northern blot approach. The miRNAs are encoded by genes with diverse organizations, representing mostly independent transcription units with or without introns. The intron-containing miRNA transcripts undergo complex splicing events to generate various spliced isoforms. We identified miRNAs that were encoded within introns of the noncoding genes MIR156g and MIR1126. Interestingly, the intron that encodes miR156g is spliced less efficiently than the intron encoding miR1126 from their specific precursors. miR397b-3p was detected in barley as a most probable functional miRNA, in contrast to rice where it has been identified as a complementary partner miRNA*. In the case of miR168a-5p/168a-3p, we found the generation of stable, mature molecules from both pre-miRNA arms, confirming evolutionary conservation of the stability of both species, as shown in rice and maize. We suggest that miR1120, located within the 3′ UTR of a protein-coding gene and described as a functional miRNA in wheat, may represent a siRNA generated from a mariner-like transposable element.ConclusionsSeven of the eight barley miRNA genes characterized in this study contain introns with their respective transcripts undergoing developmentally specific processing events prior to the dicing out of pre-miRNA species from their pri-miRNA precursors. The observed tendency to maintain the intron encoding miR156g within the transcript, and preferences in splicing the miR1126-harboring intron, may suggest the existence of specific regulation of the levels of intron-derived miRNAs in barley.

Biogenesis, Turnover, and Mode of Action of Plant MicroRNAs

“…Pri-miRNAs contain introns, and widespread alternative splicing of pri-miRNA transcripts has been detected (Mica et al, 2009;Szarzynska et al, 2009;Kruszka et al, 2013). In Arabidopsis, alternative splicing of pri-miR162a (Hirsch et al, 2006) or the pri-miR842-miR846 dicistron (Jia and Rock, 2013) disrupts miRNA-containing stem-loops and reduces the accumulation of processed miRNAs.…”

Salt Stress Reveals a New Role for ARGONAUTE1 in miRNA Biogenesis at the Transcriptional and Posttranscriptional Levels

“…The 39 end of pri-mRNA is modified by the cleavage and polyadenylation machinery that adds a poly(A) tail to the 39 end of all pri-mRNAs. Moreover, many plant pri-miRNAs, like most mRNA precursors (pre-mRNA), contain introns that are excised by the spliceosome (Szarzynska et al, 2009;Kruszka et al, 2013;Zielezinski et al, 2015). We have recently shown that the splicing of such introns from pri-miRNAs, or more precisely their active 59 splice sites, stimulates miRNA maturation (Bielewicz et al, 2013;Schwab et al, 2013).…”

A stable tRNA-like molecule is generated from the long noncoding RNA GUT15 in Arabidopsis

“…For the small RNA library construction and stem-loop reverse transcription, total RNA enriched for small RNAs was isolated from 100 mg of 14-day-old A . thaliana seedlings using a previously described protocol [46]. The integrity and quality of the NGS-dedicated RNA were verified using an Agilent RNA 6000 Nano Kit (Agilent Technologies).…”

“…The RNA electrophoresis, blot transfer and hybridization were performed as previously described [46,50]. All hybridizations were performed with three biological replicates.…”