Analysis of mRNA-derived siRNAs in mutants of mRNA maturation and surveillance pathways in Arabidopsis thaliana

Defects in RNA maturation and RNA decay factors may generate substrates for the RNA interference machinery. This phenomenon was observed in plants where mutations in some RNA-related factors lead to the production of RNA-quality control small interfering RNAs and several mutants show enhanced silencing of reporter transgenes. To assess the potential of RNAi activation on endogenous transcripts, we sequenced small RNAs from a set of Arabidopsis thaliana mutants with defects in various RNA metabolism pathways. We observed a global production of siRNAs caused by inefficient pre-mRNA cleavage and polyadenylation leading to read-through transcription into downstream antisense genes. In addition, in the lsm1a lsm1b double mutant, we identified NIA1, SMXL5, and several miRNA-targeted mRNAs as producing siRNAs, a group of transcripts suggested being especially sensitive to deficiencies in RNA metabolism. However, in most cases, RNA metabolism perturbations do not lead to the widespread production of siRNA derived from mRNA molecules. This observation is contrary to multiple studies based on reporter transgenes and suggests that only a very high accumulation of defective mRNA species caused by specific mutations or substantial RNA processing defects trigger RNAi pathways. Founded by National Science Centre Grant, Poland (UMO-2013/08/M/NZ1/00931, UMO-2018/29/B/NZ3/01980). Overall design: Small RNA profiles of ahg2-1, ccr4a, pab2 pab4, upf1-5, upf3-1, lsm1a lsm1b, ncb4, cstf64-2, fy-2 and rsr1-2 mutant plants with Col-0 wild type plants (three biological replicates) in three experimental sets. Set 1: Col0_1, lsm1a lsm1b, ncb4, upf1-5 and upf3-1. Set 2: Col0_2, ahg2-1, ccr4a, pab2 pab4, fy-2 and rsr1-2. Small RNA-seq data for Col-0 plants matching cstf64-2 mutant from set 3 are part of the earlier submission: GSE99600. Samples for small RNA-seq in GSE99600 submission and cstf64-2 small RNA-seq in this experiment were prepared in parallel from plants grown at the same time.