Characterization and purification of a mammalian endoribonuclease specific for the alpha -globin mRNA.
The alpha-globin mRNA has previously been shown to be the target of an erythroid-enriched endoribonuclease (ErEN) activity which cleaves the mRNA within the 3'-untranslated region. We have currently undertaken a biochemical approach to purify this enzyme and have begun characterization of the enzyme to determine requirements for substrate recognition as well as optimal cleavage conditions. Through mutational analysis and truncations we show that a 26-nucleotide region of the alpha-globin 3'-untranslated region is an autonomous element that is both necessary and sufficient for cleavage by ErEN. Mutations throughout this region abolish cleavage activity by ErEN suggesting that the entire sequence is important for recognition and cleavage. ErEN is most active under biological salt concentrations and temperature and activity of the enzyme does not require cations. The size for ErEN was estimated by denaturing gel filtration analysis and is approximately 40 kDa. Interestingly, the exquisite specificity of ErEN cleavage became compromised with increased purity of the enzyme suggesting the involvement of other proteins in specificity of ErEN cleavage. Nondenaturing gel filtration of MEL extract demonstrated that ErEN is a component of an approximately 160 kDa complex implying that additional proteins may regulate ErEN activity and provide increased cleavage specificity.
Rodgers
Nancy D
ND
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA.
Wang
Zuoren
Z
Kiledjian
Megerditch
M
D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002412:Cations; D002460:Cell Line; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000737:chemistry* / Q000302:isolation & purification*; D005914:Globins / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012492:Salts / Q000494:pharmacology; D016622:Silver Staining; D013347:Subcellular Fractions; D013696:Temperature
D020413:3' Untranslated Regions; D002412:Cations; D012333:RNA, Messenger; D012492:Salts; D012313:RNA; D005914:Globins; D004722:Endoribonucleases; C447614:erythroid-enriched endoribonuclease protein, mouse