Characterization of the mRNA ligands bound by the RNA binding protein hnRNP A2 utilizing a novel in vivo technique.
Post-transcriptional regulation is an important mechanism in cellular response to stimuli, allowing for the rapid and discrete expression of relevant proteins. Genes regulated by this mechanism have specific cis -acting elements, frequently in their 3' untranslated regions (UTRs), that have been shown to serve as recognition sites for trans -acting RNA-binding proteins. Unfortunately, the identification of specific mRNA ligands for different RNA binding proteins in vivo has been limited by a lack of adequate methodology. We have developed a novel technique that addresses this shortcoming, using immunoprecipitation of RNA binding proteins from polysomes followed by RT-PCR and library screening to identify the in vivo mRNA ligands of RNA binding proteins. Utilizing this approach, we have identified 32 known and 16 novel mRNAs specifically bound by the heterogeneous nuclear ribonucleoprotein (hnRNP) A2. Of the clones identified, 74% contained AU-rich elements and/or poly-uridine tracts in their 3' UTRs, cis -acting elements that have been established as impacting mRNA stability. The high percentage of clones containing these uridine-rich sequences compares favorably with the high affinity binding of poly-uridine RNA by hnRNP A2 in vitro. These data thus support the representative nature of the technique.
Brooks
S A
SA
Department of Medicine, Dartmouth Medical School, Hanover, NH 03756, USA.
Rigby
W F
WF
D020413:3' Untranslated Regions; D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D015723:Gene Library; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D008024:Ligands; D011132:Polyribosomes; D011233:Precipitin Tests; D011485:Protein Binding; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012261:Ribonucleoproteins / Q000276:immunology / Q000378:metabolism*
D020413:3' Untranslated Regions; D000911:Antibodies, Monoclonal; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D008024:Ligands; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C439768:hnRNP A2