Ribonomic analysis of human Pum1 in HeLa cells

PUF family proteins are among the best characterized regulatory RNA-binding proteins in non-mammalian species, but relatively little is known about mRNA targets or functions of mammalian PUF proteins. In this study, we used ribonomic analysis to identify and analyze mRNAs associated with ribonucleoproteins containing an endogenous human PUF protein, Pum1. Pum1 associated mRNAs were highly enriched for genes encoding proteins that function in transcriptional regulation and cell cycle/proliferation, results consistent with the post-transcriptional RNA regulon model and the proposed ancestral functions of PUF proteins in stem cell biology. Analysis of 3’UTR sequences of Pum1 associated mRNAs revealed a core Pum1 consensus sequence, UGUAHAUA. Pum1 knockdown demonstrated that Pum1 enhances decay of associated mRNAs, and re-localization of Pum1 to stress granules suggested that Pum1 functions in repression of translation. This study is the first in vivo genome-wide mRNA target identification of a mammalian PUF protein and provides direct evidence that human PUF proteins regulate stability of associated mRNAs. Comparison of Pum1 associated mRNAs to mRNA targets of PUF proteins from S. cerevisiae and Drosophila demonstrates how a well conserved RNA-binding domain and cognate binding sequence have been evolutionarily rewired to regulate the collective expression of different sets of functionally related genes. Keywords: RIP-chip Pum1 IP, negative IP, and total IP samples were analyzed for each of 3 biological replicates. Each IP or total RNA sample was run on a separate array versus a common reference sample