cDNMT3B 3'UTR and cDNMT3B 3'UTR mutants for each miRNA binding sites generated by point mutation cloned into a pcDNA3 plasmid encoding eGFP and cDNMT3B 3'UTR-specific miRNAs including gga-miR-15c, gga-miR-29b, gga-miR-383, and gga-miR-222 cloned into a pDsRed2-N1 plasmid, respectively, encoding RFP were cotransfected into 293 FT cells.
The use of mutants in the small RNA biogenesis pathway may yet prove to be helpful in miRNA identification in rice, as some of these mutants are enriched for miRNAs, and analyses with high-throughput sequencing can be quite informative, as demonstrated recently in Arabidopsis [46].
Additionally, the expression data for miRNA biogenesis mutants for any particular molecule can be accessed from the corresponding miRNA record.
We surveyed a collection of miRNA mutants for maternal effects on PGC numbers at S10.
We surveyed over 25 miRNA mutants for effects on lifespan and identified only 1 (mir-71) that differed dramatically from wild type.
In this study, in order to dissect miRNA-guided gene regulation in the WT and GMS mutant anthers, a degradome library suitable for miRNA target identification was constructed as described previously [ 29].
Analysis of the miRNA mutant transcriptomes also showed that the pattern of transcriptional changes amongst mutants was not always consistent with the current model for miRNA biogenesis/transport [1], and that dcl3 had little effect on the transcriptome.
To identify mutants affecting miRNA silencing, we screened mutant eyes for variegated GFP fluorescence, which was caused by altered GMR > eGFP:: Brd expression in homozygous mutant clones.
The 2- to 3-fold differences in cellular protein levels for the DGCR8 WT and mutants would be expected to alter global levels of mature miRNAs if DGCR8 were limiting for miRNA biogenesis.
To discover new genes required for miRNA silencing, we used a genetic mosaic strategy in which we examined clones of homozygous mutant eye cells in otherwise heterozygous animals.
