Feed backwards model for microRNA processing and splicing in plants.

MicroRNAs (miRNAs) are a class of small RNAs, ranging in size from 19–24 nucleotides that mediate gene silencing at the post-transcriptional level. As in animals, plant primary miRNA transcripts (pri-miRNAs) contain imperfect, self-complementary stem–loop regions. Yet, there are some fundamental differences between animal and plant miRNAs including (i) genomic location of the primary miRNA transcripts—animal pri-miRNAs are generally encoded within introns of proteincoding genes whilst plant primiRNAs are more often intergenic and comprise independent protein-non-coding transcription units; and (ii) miRNA biogenesis—animal pri-miRNAs are first processed by Drosha in the nucleus and then exported from the nucleus and cleaved in the cytoplasm by Dicer [1], but plants have no Drosha homologue. The plant Dicer homologue Dicer-like 1 (DCL1) carries out processing events in the nucleus, typically resulting in an approximately 21nucleotide mature mRNA/ miRNA passenger strand duplex [2]. Plant pri-miRNA genes have a typical exon–intron structure resembling a protein-coding gene. Most plant primiRNA stem–loops are exonic, but surrounded by introns that more often occur towards the 3ʹ adjacent side. It has been thought that the region outside the stem– loop is largely dispensable and lacks any regulatory role. However, in this issue of EMBO reports, two back-to-back articles from the groups of Voinnet and Jarmolowski indicate a clear connection between dicing and splicing. The Voinnet group [3] investigated two representative miRNA loci: MIR163 and MIR172a, both are expressed in Arabidopsis thaliana and have 3ʹ end-located introns. To determine the influence of intronic sequences on miRNA expression, they expressed several transgene variants either transiently in Nicotiana benthamania leaves or as stable transgenic lines in an Arabidopsis MIR T-DNA knockout background using the viral CaMV 35S promoter. In detail, four MIR163 isoforms were tested: with and without an intron. Consistently, higher levels of mature miR163 were generated from the intron containing forms as compared with the intron-less ones. Adding more generality, MIR172a was investigated and found to harbour two introns interrupted by an exon downstream from the miRNA stem–loop. Consistently, introncontaining isoforms produced higher levels of miRNA compared with their intron-less counterparts. Overall, this points to a positive correlation between the presence of an intron in the primary transcript and increased miRNA production. To test if this behaviour is specific to introns of miRNA genes, unrelated intron sequences of various lengths—Petunia chsA and potato LS1—were placed at the 3ʹ end of intron-less MIR163‐4 and MIR172b transgenes. Interestingly, these exogenous introns have a similar activating effect to native introns, which is position-dependent as placing them 5ʹ to the miRNA stem–loop is less effective. To better understand the crosstalk between splicing and dicing, first point mutations that inactivate splicing were introduced at the 5ʹ splice site in both the MIR163 and MIR172a genes. Only a slight reduction in miRNA levels was observed, suggesting that miRNA accumulation does not require splicing of introns, but rather is mediated by as yet unidentified intronic elements. The elusive phenomenon of intron-mediated enhancement (IME) of gene expression was investigated. To test this, a clever trick of introducing MIR163 transgenes with or without an intron into dicer-like 1 (dcl-1) mutants was carried out. Precursor processing in this situation will not reduce primary transcript levels, which can therefore be measured directly. Only a slight increase in transcript level from the intron-containing construct was observed, C B 5’