Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins.
Defects in RNA-binding proteins have been implicated in human genetic disorders. However, efforts in understanding the functions of these proteins have been hampered by the inability to obtain their mRNA substrates. To identify cognate cellular mRNAs associated with an RNA-binding protein, we devised a strategy termed isolation of specific nucleic acids associated with proteins (SNAAP). The SNAAP technique allows isolation and subsequent identification of these mRNAs. To assess the validity of this approach, we utilized cellular mRNA and protein from K562 cells and alphaCP1, a protein implicated in a-globin mRNA stability, as a model system. Immobilization of an RNA-binding protein with the glutathione-S-transferase (GST) domain enables isolation of mRNA within an mRNP context and the identity of the bound mRNAs is determined by the differential display assay. The specificity of protein-RNA interactions was considerably enhanced when the interactions were carried out in the presence of cellular extract rather than purified components. Two of the mRNAs specifically bound by alphaCP1 were mRNAs encoding the transmembrane receptor protein, TAPA-1, and the mitochondrial cytochrome c oxidase subunit II enzyme, coxII. A specific poly(C)-sensitive complex formed on the TAPA-1 and coxII 3' UTRs consistent with the binding of aCP1. Furthermore, direct binding of purified alphaCP proteins to these 3' UTRs was demonstrated and the binding sites determined. These results support the feasibility of the SNAAP technique and suggest a broad applicability for the approach in identifying mRNA targets for clinically relevant RNA-binding proteins that will provide insights into their possible functions.
Trifillis
P
P
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08855, USA.
Day
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N
Kiledjian
M
M
D020413:3' Untranslated Regions / Q000378:metabolism; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods*; D004268:DNA-Binding Proteins; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism; D005821:Genetic Techniques; D005982:Glutathione Transferase / Q000378:metabolism; D006454:Hemoglobins / Q000235:genetics; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D020014:K562 Cells; D008565:Membrane Proteins; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D060225:Tetraspanin 28
D020413:3' Untranslated Regions; D015703:Antigens, CD; C554798:CD81 protein, human; D004268:DNA-Binding Proteins; D006454:Hemoglobins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D008565:Membrane Proteins; C094604:PCBP1 protein, human; D010446:Peptide Fragments; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D060225:Tetraspanin 28; C070077:alpha(A) globin; D003576:Electron Transport Complex IV; D005982:Glutathione Transferase