ABSTRACT
CPSF73 is the endonuclease that catalyzes the cleavage reaction for 3'-end processing of mRNA precursors (pre-mRNAs) in two distinct machineries, a canonical machinery for the majority of pre-mRNAs and a U7 snRNP (U7 machinery) for replication-dependent histone pre-mRNAs in animal cells. CPSF73 also possesses 5'-3' exonuclease activity in the U7 machinery, degrading the downstream cleavage product after the endonucleolytic cleavage. Recent studies show that CPSF73 is a potential target for developing anticancer, antimalarial, and antiprotozoal drugs, spurring interest in identifying new small-molecule inhibitors against this enzyme. CPSF73 nuclease activity has so far been demonstrated using a gel-based end-point assay, using radiolabeled or fluorescently labeled RNA substrates. By taking advantage of unique properties of the U7 machinery, we have developed a novel, real-time fluorescence assay for the nuclease activity of CPSF73. This assay is facile and high-throughput, and should also be helpful for the discovery of new CPSF73 inhibitors.
Subject(s)
Biological Assay , Cleavage And Polyadenylation Specificity Factor/metabolism , Histones/metabolism , RNA 3' End Processing , RNA Precursors/metabolism , Ribonucleoprotein, U7 Small Nuclear/metabolism , Cell-Free System , Cleavage And Polyadenylation Specificity Factor/chemistry , Cleavage And Polyadenylation Specificity Factor/genetics , Enzyme Assays , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Fluorescence , Fluorescent Dyes/chemistry , Histones/chemistry , Histones/genetics , Humans , Models, Molecular , Phenylalanine/analogs & derivatives , Phenylalanine/chemistry , Phenylalanine/pharmacology , Piperazines/chemistry , Piperazines/pharmacology , Proteolysis , RNA Precursors/chemistry , RNA Precursors/genetics , Rhodamines/chemistry , Ribonucleoprotein, U7 Small Nuclear/chemistry , Ribonucleoprotein, U7 Small Nuclear/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacologyABSTRACT
NMR spectroscopy is a key technique that has significantly advanced our understanding of RNA structure and dynamics. However, determination of large RNA structures by NMR spectroscopy remains a significant technical challenge. In this review, we highlight advances that facilitate NMR studies of large RNAs, including methods for sample preparation, isotope labeling strategies, and data acquisition. In addition, we review hybrid approaches that have been instrumental in the structure determination of large RNAs.
