PUF proteins: RNA binding, protein interactions, and RNA regulation

In eukaryotic cells, protein expression from mRNAs is highly controlled, often through regulatory elements in the mRNA 3' untranslated regions (3'UTRs). Regulatory proteins bind these sites and control mRNA stability, translation and localization. The PUF family of 3' UTR binding proteins modulates mRNA expression in a wide variety of eukaryotic species. They do so either by enhancing turnover or repressing translation, and act combinatorially with other regulatory proteins. PUF proteins function in a variety of developmental processes: embryonic development, stem cell maintenance, germline differentiation, and neuronal development.; I show that two Caenorhabditis elegans PUF proteins, FBF and PUF-8, differ in RNA-binding specificity. FBF activity requires the presence of a single 'extra' nucleotide in the middle of an eight-nucleotide site, whereas PUF-8 requires its absence. I also show FBF-1 and its close relative, FBF-2 (collectively called FBF), bind with similar affinity to multiple RNA sites. I discover that the binding affinity of FBF for these sites can be increased when bound to CPB-1.; The yeast PUF protein, Puf5p/Mpt5p, negatively regulates expression of the HO gene by binding to the 3' UTR of HO mRNA. Here I report that another PUF protein, Puf4p, also binds to and regulates expression of the same mRNA. Puf4p represses expression of an mRNA reporter that contains a HO 3' UTR. A third RNA binding protein, Ngr1p, also binds the same 3'UTR and represses expression of the mRNA. I propose that these three proteins - Mpt5p, Ngr1p, and Puf4p - work together to regulate HO mRNA in yeast.; Mpt5p physically interacts with Pop2p, which is a component of the Ccr4p/Pop2p/Not deadenylase complex, and Pop2p is required for PUF repression activity. Reconstituted deadenylation in vitro using purified components demonstrated that the PUF-Pop2p mechanism was conserved in yeast, worms and humans. I suggest that the PUF-Pop2p interaction underlies regulated deadenylation, repression, and mRNA decay by PUF proteins.