MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5′ region

Abstract

MicroRNAs (miRNAs) are B22-nucleotide noncoding RNAs that can regulate gene expression by directing mRNA degradation or inhibiting productive translation. Dominant mutations in PHABULOSA (PHB) and PHAVOLUTA (PHV) map to a miR165/166 complementary site and impair miRNA-guided cleavage of these mRNAs in vitro. Here, we confirm that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb-d mutants. In planta, disrupting miRNA pairing near the center of the miRNA complementary site had far milder developmental consequences than more distal mismatches. These differences correlated with differences in miRNA-directed cleavage efficiency in vitro, where mismatch scanning revealed more tolerance for mismatches at the center and 3 0 end of the miRNA compared to mismatches to the miRNA 5 0 region. In this respect, miR165/ 166 resembles animal miRNAs in its pairing requirements. Pairing to the 5 0 portion of the small silencing RNA appears crucial regardless of the mode of post-transcriptional repression or whether it occurs in plants or animals, supporting a model in which this region of the silencing RNA nucleates pairing to its target.

Plant MicroRNAs Display Differential 3' Truncation and Tailing Modifications That Are ARGONAUTE1 Dependent and Conserved Across Species

“…miR165/6 (21 nucleotides) family members play important roles in plant development by targeting type III HD-Zip genes REVOLUTA, PHABULOSA, and PHAVOLUTA to regulate leaf polarity and stem cell identity (Mallory et al, 2004;Williams et al, 2005). In hen1 mutants, we observed that miR166 is first truncated to ;17 nucleotides without any tailing and then is uridylated to ;21 nucleotides, its original length ( Figure 1B).…”

Principles of micro-RNA production and maturation

“…7 positions (No. 1 being overrepresented by a U), are critical for target recognition (Doench and Sharp, 2004;Mallory et al, 2004;Brennecke et al, 2005;Krek et al, 2005;Lai et al, 2005;Lewis et al, 2005). Prediction of miRNAs by current computational methods or detection of miRNAs by Northern blot and microarray analyses cannot pinpoint the ends of miRNAs.…”

Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana

“…Perfect complimentarity at this 5′ region of miRNAs has been previously shown to be critical for miRNA function (Mallory et al 2004b). If NbAP2 also has such a mismatch to Arabidopsis miR172, it seems possible that Arabidopsis miR172 may not be having a repressive effect on NbAP2 but instead could be sequestering NbAP2 mRNA from being regulated by NbmiR172 to result in increased NbAP2 activity.…”