Extensive association of HuR with hnRNP proteins within immunoselected hnRNP and mRNP complexes

Abstract

Regulated gene expression at the post-transcriptional level in higher eukaryotes is based on a network of interactions among RNA-binding proteins (RBPs) operating within multifactorial ribonucleoprotein (RNP) complexes, notably heterogeneous nuclear ribonucleoprotein (hnRNP) and mRNP complexes. We are interested in interactions involving hnRNP proteins participating in several steps of mRNA processing (mainly pre-mRNA splicing) and HuR with an established role in stability/translation of associated mRNAs. hnRNP and HuR proteins have a major nucleoplasmic localization and ability to shuttle between nucleus and cytoplasm. We report here on interactions between hnRNP and HuR proteins that were identified in the context of isolated hnRNP and mRNP complexes. This was done by the application of immunoprecipitation and pull-down assays on different sub-cellular fractions prepared from cells of human and mouse origin, as well as in vivo localization studies. A range of specific associations of HuR with the shuttling hnRNP A1 and A3 and the non-shuttling hnRNP C1/C2 was identified and ascribed discrete properties with respect to stability to RNase A and increasing salt, as well as to cellular distribution. The likelihood of a biological relevance of these associations was tested under heat shock conditions in growing cells, which appeared to affect both the sub-nuclear distribution and interaction of HuR with hnRNPs. The establishment of an extensive association of HuR with hnRNP components of nuclear hnRNP/mRNP and cytoplasmic mRNP complexes supports its broader participation in mRNA processing events than initially anticipated.