Cloning of a cDNA encoding an RNA binding protein by screening expression libraries using a northwestern strategy.
Successful cloning with cDNA expression libraries involves interaction of ligand molecules (probes) with expressed fusion proteins of interest. So far those ligands leading to successful results fall into three classes: (i) antibodies, (ii) protein ligands that interact with the protein of interest, and (iii) DNA sequences recognized by transcription factors. We have previously identified a 50-kDa protein (called DSEF-1) which interacts with a functionally important 14-base G-rich RNA sequence located downstream of the simian virus 40 late polyadenylation signal. By using small RNAs containing the DSEF-1 binding site as probes, a cDNA clone was isolated whose gene product interacted in a sequence-specific fashion with the DSEF-1 binding site. This RNA binding protein contains three potential RNA recognition motifs. We present here a procedure to obtain cDNA clones of RNA binding proteins using recognition site probes.
Qian
Z
Z
Department of Microbiology & Molecular Genetics, UMD-New Jersey Medical School, Newark 07103.
Wilusz
J
J
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003101:Collodion; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D015347:RNA Probes; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D008567:Membranes, Artificial; D015347:RNA Probes; D016601:RNA-Binding Proteins; D003101:Collodion