Nuclear and cytoplasmic RNA exosomes and PELOTA1 prevent miRNA-induced secondary siRNA production in Arabidopsis.

Amplification of short interfering RNA (siRNAs) via RNA-dependent RNA polymerases (RdRPs) is of fundamental importance in RNA silencing. Plant microRNA (miRNA) action generally does not involve engagement of RdRPs, in part thanks to a poorly understood activity of the cytoplasmic exosome adaptor SKI2. Here, we show that inactivation of the exosome subunit RRP45B and SKI2 results in similar patterns of miRNA-induced siRNA production.

Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavage in vivo.

MicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity, in vitro and in silico studies have suggested that RNA structure may influence the accessibility of mRNAs to miRNA-induced silencing complexes (miRISCs), thereby affecting RNA silencing.

Characterization of Promoter Bidirectionality and Antisense RNAs by Inactivation of Nuclear RNA Decay Pathways.

In animals, RNA polymerase II initiates transcription bidirectionally from gene promoters to produce pre-mRNAs on the forward strand and promoter upstream transcripts (PROMPTs) on the reverse strand. PROMPTs are degraded by the nuclear exosome. Previous studies based on nascent RNA approaches concluded that Arabidopsis () does not produce PROMPTs.

Detection of Slicer Activity by Immunopurified Plant ARGONAUTE1.

Small RNA-guided endonucleolysis ("slicing") of target mRNA is the signature biochemical activity underlying many RNA silencing phenomena. The catalytic slicer activity resides in Argonaute (AGO) proteins. Here, we present two protocols to detect microRNA-guided slicer activity of AGO1 immunopurified from Arabidopsis tissues.