Identification of mRNA bound to RNA binding proteins by differential display.
A large number of RNA binding proteins have recently been identified that influence various human genetic disorders. However, the specific function of many of these proteins and what role they may play in a particular disease remains unclear. Identification of the substrate mRNA bound by an RNA binding protein will provide insights into the function of that protein and how its aberrant expression could lead to a disease phenotype. We have developed a technique termed SNAAP, for isolation of specific nucleic acids associated with proteins, to identify natural mRNA substrates for an RNA binding protein. The technique couples affinity purification of specific mRNAs bound by an RNA binding protein, with the identification of that mRNA using differential display (DD). Methods are described herein for the isolation and identification of endogenous mRNAs bound by any RNA binding protein, as well as methodology to validate the specificity of the binding. The availability of technologies to isolate the cognate substrate mRNAs potentially bound and regulated by an RNA binding protein involved in genetic disorders will greatly expedite our etiological understanding of the disorder and provide modalities for intervention.
Carr-Schmid
Anne
A
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, USA.
Jiao
Xinfu
X
Kiledjian
Megerditch
M
D020413:3' Untranslated Regions; D000818:Animals; D001665:Binding Sites; D002021:Buffers; D018076:DNA, Complementary / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D008297:Male; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry; D013737:Testis / Q000378:metabolism; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D020413:3' Untranslated Regions; D002021:Buffers; C101358:DAZL protein, human; C508515:DAZL protein, mouse; D018076:DNA, Complementary; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012313:RNA; D012685:Sepharose; D005982:Glutathione Transferase; D005978:Glutathione