Involvement of a novel intronic microRNA in cross regulation of N-methyltransferase genes involved in caffeine biosynthesis in Coffea canephora.

There are numerous reports on intronic miRNAs from plants, most of which are involved in the regulation of
unrelated genes. Some of the target genes are antagonistic to the host genes. Intronic miRNAs in animal systems,
however, are known to have synergistic effects. This article is the first to report a similar regulatory effect
of a miRNA originating from an intron in plants. NMT genes involved in caffeine biosynthesis were silenced to
obtain transformants with reduced caffeine. Transcript analysis revealed the accumulation of transcripts for a
related NMT gene (CaMTL1) in transformants bearing either antisense or RNAi constructs. The altered expression
was assumed to relate to the silencing of the NMT genes. Bioinformatics analysis of the genes involved in
biosynthesis revealed the presence of an intronic miRNA originating from the intron of the theobromine
synthase gene targeting CaMTL1. The putative miRNA was cloned and sequenced. Modified 5′-RLM-RACE
mapping of the cleavage site and subsequent Northern blotting experimentally demonstrated the presence
and activity of such a miRNA in Coffea canephora. This novel regulatory mechanism previously unreported
in plants will shed more light onto the evolution of multigene families and the role of introns in this process.