Expression, Purification And Functional Study Of RNA Degrading Enzyme In Arabidopsis Thaliana

The balance of endogenous mi RNA is critical for the normal growth and development of plants and animals. Biosynthesis and degradation are the two main ways to maintain the intracellular levels of mi RNA. The mechanism of mi RNA biosynthesis have been studied extensively, but little is known about the mechanism of mi RNA degradation.Professor Chen Xuemei’s lab has discovered that SDN1(Small RNA Degrading Nuclease1) in Arabidopsis thaliana has mi RNA degradation activity. The objective of this study is to identify more genes other than SDN1 that are involved in mi RNA degradation. Using reverse genetics approach, we found find 14 genes that have phyligenetic relations with SDN1 from Arabidopsis thaliana database, phylogenetic tree analysis show that amone these 14 genes, At5g05540 and At5g25800 colse phyligenetic relations with SDN1, we refer these two genes as SDN2 and SDN5.Exosome is a complex where intracellular m RNA degradation takes place, it is unknown whether exosome is also involved in mi RNA degradation. SOV(Suppressor of Varicose) is one of the exosome proteins, which has the function of m RNA degradation.In this study, we tested mi RNA degradation activity of SDN2, SDN5, and SOV.SDN2, SDN5, and SOV genes were amplified and inserted into prokaryotic expression vector with GST tag, GST-SDN5,GST-SDN2 and GST-SOV fusion proteins were expressed in Escherichia coli. Using affinity chromatography, GST-SDN5, GST-SDN2 and GST-SOV proteins were purified respectively. mi RNA173 and mi RNA173+7U that are biotin-labeled at their 5’ end are used as substrates. mi RNA degradation activities of GST-SDN2, GST-SDN5 and GST-SOV were detected by incubating one of the putative enzymes with one of the substrates at 37 ℃(for GST-SDN2 and GST-SDN5), or 30 ℃( for GST-SOV) in a water bath. 3’-5 ’ mi RNA degradation activities were detected in GST-SDN2, GST-SDN5, but not in GST-SOV.