Identification of AUF-1 ligands reveals vast diversity of early response gene mRNAs.
Cell activation is associated with diverse and widespread changes in gene expression at both the transcriptional and post-transcriptional levels. AUF1 is a recently described cytoplasmic protein which likely participates in the post-transcriptional regulation (PTR) of AU-rich (ARE) mRNAs including those coding for cytokines and proto-oncogenes. Individual mRNAs subject to AUF1-mediated PTR can be predicted if AREs are present or the mRNA in question interacts in vitro or in vivo with AUF1. However, there are few, if any, general approaches for characterizing the overall repertoire of mRNAs subject to PTR by AUF1. In an effort to identify these mRNAs, we incubated total mRNA from mitogen-activated peripheral blood mono-nuclear cells (PBMCs) with AUF1 in vitro. AUF1-mRNA complexes were retarded on membranes, bound mRNAs eluted with high salt, and either used to generate a cDNA library or rebound to AUF1 a second or third time prior to elution and cDNA library construction. We have obtained partial nucleotide sequences from 130 clones which shows that the AUF1 selected libraries are rich in mRNAs containing 3' untranslated region AREs including a large number of early response gene cDNAs. As a test of the validity of this method, we also show that a randomly selected, novel mRNA contained in the library is stabilized upon cell activation.
Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, WI 53792, USA.
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D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D017781:Genes, Immediate-Early; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D008024:Ligands; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D008024:Ligands; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor