Journal: bioRxiv
Article Title: In vitro characterization of the yeast DcpS, a scavenger mRNA decapping enzyme
doi: 10.1101/644492
Figure Lengend Snippet: Biochemical Assays for DcpS. ( a ) Conversion of m 7 GpppA dinucleotide to m 7 GMP. A 100 μL reaction in decapping buffer containing 50 μM m 7 GpppA and either 50 nMy DcpS (blue □) or 85 nM hDcpS (red ○) was incubated at 37 °C. 10 μL samples were withdrawn at the indicated times. The reactions were terminated by the addition of equal volume of phenol/chloroform. The relative quantity of the remaining dinucleotide m 7 GpppA, m 7 GMP, and ADP were determined by LC-MS. ( b ) Decapping of a 25mer Cap 1 RNA by yDcpS. A 30 μL reaction containing 300 ng of total E. coli RNA and 60 ng of 25mer Cap 1 RNA was incubated for 3 hours at 37 °C with 130 ng of yDcpS in 10 mM MES pH 6.5 and 1 mM EDTA. At 0 minutes a 5 μL aliquot was mixed with 2X RNA loading dye stop solution (Lane 1). Likewise 5 μL aliquots were taken at 5, 60, 120, and 180 min (Lanes 2 to 5, respectively). The RNA aliquots were analyzed by 15% TBE-Urea PAGE stained with SYBR Gold. ( c ) Capillary electrophoresis of 3’-FAM-labeled 5’-capped 25mer RNA. Top panel shows a representative example of the decapping reaction progress. The 25mer Cap 0 RNA was incubated with yDcpS for various times indicated on the left. Bottom panel shows the mobility shift for standards of 25mer RNAs containing different 5’ ends. All data were plotted relative to GeneScan™120 LIZ™ Applied Biosystems standards.
Article Snippet: Human DcpS (hDcpS) was purchased from Enzymax LLC, Lexington, KY.
Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, Staining, Electrophoresis, Labeling, Mobility Shift
