Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis.
Bottom Line: AtGRP7 overexpression caused a significant reduction in the level of 30 miRNAs and an increase for 14 miRNAs with a minimum log2 fold change of ± 0.5.Mutation of an arginine in the RNA recognition motif abrogated in vivo binding and the effect on miRNA and pri-miRNA levels, indicating that AtGRP7 inhibits processing of these pri-miRNAs by direct binding.Thus, AtGRP7 is an hnRNP-like protein with a role in processing of pri-miRNAs in addition to its role in pre-mRNA splicing.
Affiliation: Molecular Cell Physiology, Bielefeld University.Show MeSH
Mentions: Because AtGRP7 did not appear to affect miRNA levels indirectly via a global effect on processing factors, we tested whether it directly interacts with pri-miRNAs in vivo. We performed RIP on transgenic plants expressing AtGRP7:GFP under control of its own promoter in the atgrp7–1 background. Pri-miR398b and pri-miR398c were enriched in RNP complexes precipitated with GFP-Trap® beads (IP+) from these AtGRP7::AtGRP7:GFP plants relative to mock precipitates with RFP-Trap® beads (IP−) (Figure 5A). PP2A which served as a negative control was not significantly enriched. Both pri-miR398b and pri-miR398c were not enriched in precipitates from plants expressing GFP alone. Notably, they were also not recovered from plants expressing AtGRP7::AtGRP7-R49Q:GFP (Figure 5B). The pri-miR398a level was too low to allow reliable quantification, in line with its weak expression (68). Furthermore, pri-miR172b and pri-miR159a were enriched in IP+ relative to IP− and not present in precipitates from plants expressing AtGRP7::AtGRP7-R49Q:GFP or GFP only. For pri-miR390b, the expression level was also too low to allow reliable quantification.