Understanding the structure/sequence preference of plant miRNA biogenesis machinery

MicroRNAs (miRNAs) are ~21nt long endogenous non-coding RNAs that are involved in post-transcriptional silencing of target mRNAs. miRNAs are processed from precursors having a stem-loop structure that are recognized and cleaved by Dicer-like 1 (DCL1) with the help of dsRNA binding protein Hyponastic leaves 1 (HYL1) and a zinc finger protein named Serrate (SE). Plant miRNA stem-loops are very diverse in length and structure than animal miRNAs. Hence, mechanism of miRNA biogenesis from their precursors is poorly understood. Our bioinformatic analysis predicted that plant miRNAs have unique sequence signature. An artificial precursor with different predicted sequence signatures was constructed. This construct was expressed transiently in tobacco leaves and small RNAs were sequenced. Our analysis revealed that indeed the predicted sequence signature is prefered by plant miRNA biogenesis machinery. An artificial miRNA precursor with four stem loops, varying only in miRNA-miRNA* sequences, was infiltrated along with vector control in N. tabacum leaves. Small RNAs were sequenced from both vector and artificial miRNA precursor infiltrated tobacco leaves collected at 4dpi.