Pre-microRNA processing activity in nuclear extracts from Arabidopsis suspension cells.

ABSTRACT

MicroRNAs (miRNAs) play important roles in a variety of biological phenomena, such as development and responses to abiotic and biotic stresses, by regulating complementary target transcripts. Primary MIRNA transcripts (pri-miRNAs) are processed into miRNA/miRNA* duplexes via pre-miRNA intermediates by DICER family proteins. In plants miRNA/miRNA* duplexes are produced from pri-miRNAs exclusively by a complex containing DICER-LIKE1 (DCL1), Hyponastic leaves1 (HYL1) and SERREATE (SE) in the nucleus. Pri-miRNA or pre-miRNA processing activity has been detected using recombinant DCL1, HYL1 and SE proteins expressed in insect cells and immunopurified DCL1-HYL1-SE-containing complexes that transiently co-overexpressed in Nicotiana benthamiana. However, the processing of pre-miRNAs into miRNA/miRNA* using nuclear extracts has not been reported. Here I report the detection of pre-miRNA processing in nuclear extracts prepared from Arabidopsis suspension cells. In the nuclear extracts including DCL1, small RNAs that were 21 nucleotides in length were excised from a part of miRNA/miRNA* regions on pre-miRNAs. This system is potentially useful for in vitro analyses of pre-miRNA processing.