Journal: Nucleic Acids Research

Article Title: Systematic screening of archaeal MazF homologs reveals Tth-MazF1, a versatile, sequence-specific ribonuclease from Thermococcus thioreducens

doi: 10.1093/nar/gkag338

Figure Lengend Snippet: Direct measurement of 5′ Cap and poly(A) tail incorporation with Tth-MazF1. ( A ) A schematic of the 5′ Cap and poly(A) tail characterization in Tth-MazF1 mRNA digests. ( B ) Heat map depicting the fraction of Cap incorporation in the initial Tth-MazF1 18-nt nucleotide cleavage product of a FLuc mRNA treated with FCE or untreated (NE). Relative quantification of individual species was performed by comparison of the intensity of corresponding monoisotopic masses. ( C ) (Top) A schematic of the predicted Tth-MazF1 cleavage product from the 3′ end of a GFP mRNA with a 60 nt encoded poly(A) tail. (Bottom) Spectra of observed monoisotopic masses between 15 and 28 kDa and corresponding intensities following cleavage of a GFP mRNA with a 60 nt encoded poly(A) tail. Numbers above each peak indicate the annotated length of each oligonucleotide derived from the poly(A) tail. ( D ) Intensity-weighted density plot of the inferred length of poly(A) tails, which were detected in Tth-MazF1 digests of a GFP mRNA with a 60 nt encoded poly(A) tail.

Article Snippet: Briefly, 50 μg of FLuc mRNA was diluted to a final volume of 50 μl in FCE capping buffer (50 mM Tris–HCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM DTT, pH 8.0) containing 200 μM S-adenosylmethionine (SAM), 500 μM GTP, 50 units of FCE, and 200 units of mRNA Cap 2′-O-methyltransferase (NEB, M0366).

Techniques: Quantitative Proteomics, Comparison, Derivative Assay