Mol Plant. 2015 Mar 11. pii: S1674-2052(15)00170-7.
Using a forward genetic screen, this paper identifies a new component, XCT/CMA33, which seems to affect miRNA, tasiRNA and heterochromatic siRNA levels to some extent. This protein is highly conserved across eukaryotes and was previously shown to be involved in circadian rhythms and ethylene responses in Arabidopsis. Overall, the data suggests that XCT/CMA33 is required for the accumulation of miRNAs, tasiRNAs and heterochromatic siRNAs, through modulating the transcription of DCL1, DCL3, and DCL4 genes, respectively (based on Pol II occupancy assays). Although miRNAs have been found to be associated with circadian rhythms in animals, there are no data yet to suggest a direct link (or indirect link via XCT) between miRNAs and circadian rhythms in plants. It’s important to point out that XCT seems to be specific to only these three DCL genes but not other components of the small RNA biogenesis machinery. Despite the low degree of changes between wild-type and xct mutant, I found this paper interesting, especially because all of the phenotypes were able to be rescued by XCT transgene. Below are my detailed notes about the experiments performed..
They performed forward genetic screen and analyzed one of the mutants, cma33 (compromised miRNA activity 33), which displayed decreased trichome clustering, and plant stature with curled leaves and shorter siliques (Fig. 1A, B). They also observed increased accumulation of some miRNA target transcripts indicating an impairment in miRNA activity (Fig. 1C). Then, they found that cma33 carries an early stop codon in a gene encoding for a nuclear localized protein XAP5 CIRCADIAN TIMEKEEPER (XCT), which is highly conserved across species. XCT was previously shown to be involved in circadian clock and ethylene signaling. Transgenic expression of amiR-trichome causes an increased trichome clustering in amiR-triOX. A cross between amiR-triOX and xct-2 (T-DNA insertion mutant) showed reduced clustering of trichomes, indicating the role of XCT in miRNA activity.
Small RNA Northern blot showed reduced accumulation of amiR-trichome in cma33 compared to amiR-triOX (Fig. 2A). Also endogenous miRNA levels were decreased in xct-2 relative to Col-0 (Fig. 2B). They also observed a decrease in accumulation of DCL4-dependent miRNA, miR822 (Fig. 2C). Small RNA phenotype of xct-2 was fully complemented by introducing wild-type XCT gene. Increase accumulation of pri-miRNAs in xct–2 mutant suggested that XCT/CMA33 is involved in regulating pri-miRNA processing (Fig. 2D).
xct-2/ago1-25 double mutant displayed more severe developmental phenotype compared to single mutants. Also, it showed reduced accumulation of miRNAs while increased target transcript levels (Fig. 3). They further looked at the accumulation levels of tasiRNAs and a several heterochromatic siRNA loci. Reduced tasiRNA levels in correlation with increased target RNA transcripts seems to be an indirect effect since tasiRNAs are dependent on miRNA cleavage (Fig. 4A, B). Reduced accumulation of heterochromatic siRNAs was rescued by the XCT transgene. Similarly, decreased methylation at SIMPLEHAT2 and MEA-ISR loci was also rescued by the XCT transgene (Fig. 4C, D). However, I found the degree of change in both tasiRNA targets and methylation levels at the heterochromatin low.
In order to identify XCT/CMA33-interacting components in the small RNA biogenesis pathway, authors have tried Y2H, BiFC, and CoIP assays but they all failed. Then, they decided to check expression levels of genes involved in the biogenesis of different small RNAs. Accumulation of DCL (both transcript and protein levels) was reduced in the xct-2 mutant, whereas miR168-targeted AGO1 transcript levels were increased, possibly because of decreased miR168 levels (Fig.5 A-C). From the tasiRNA and heterochromatic siRNA biogenesis pathways, only DCL4 and DCL3 levels were reduced in the xct-2 mutant, respectively (Fig. 5D, E). Reduction of DCL3 protein was rescued by XCT transgene (Fig. 5F, G). Overall, data suggests that XCT/CMA33 regulates miRNAs, tasiRNAs and heterochromatic siRNAs through regulating the expression of DCL1, DCL4, and DCL3, respectively.
They ChIP’ed the promoter and coding regions of DCL genes using an antibody against the largest subunit of Pol II. Pol II occupancy seemed to be decreased at all regions in DCL1-3, but not in DCL4 (Fig. 6). Thus, data suggests that XCT/CMA33 affects the accumulation of small RNAs via promoting Pol II occupancy at DCL1, DCL2, and DCL3 genes.