Identification of AUF-1 ligands reveals vast diversity of early response gene mRNAs

Cell activation is associated with diverse and widespread changes in gene e xpression at both the transcriptional and post-transcriptional levels. AUF1 is a recently described cytoplasmic protein which likely participates in t he post-transcriptional regulation (PTR) of AU-rich (ARE) mRNAs including t hose coding for cytokines and proto-oncogenes, Individual mRNAs subject to AUF1-mediated PTR can be predicted if AREs are present or the mRNA in quest ion interacts in vitro or in vivo with AUF1. However, there are few, if any , general approaches for characterizing the overall repertoire of mRNAs sub ject to PTR by AUF1, In an effort to identify these mRNAs, we incubated tot al mRNA from mitogen-activated peripheral blood mononuclear cells (PBMCs) w ith AUF1 in vitro, AUF1-mRNA complexes were retarded on membranes, bound mR NAs eluted with high salt, and either used to generate a cDNA library or re bound to AUF1 a second or third time prior to elution and cDNA library cons truction. We have obtained partial nucleotide sequences from 130 clones whi ch shows that the AUF1 selected libraries are rich in mRNAs containing 3' u ntranslated region AREs including a large number of early response gene cDN As, 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 activ ation.