Brad Hook

PUF proteins bind Pop2p to regulate messenger RNAs

PUF proteins, a family of RNA-binding proteins, interact with the 3′ untranslated regions (UTRs) of specific mRNAs to control their translation and stability. PUF protein action is commonly correlated with removal of the poly(A) tail of target mRNAs. Here, we focus on how PUF proteins enhance deadenylation and mRNA decay. We show that a yeast PUF protein physically binds Pop2p, which is a component of the Ccr4p–Pop2p–Not deadenylase complex, and that Pop2p is required for PUF repression activity. By binding Pop2p, the PUF protein simultaneously recruits the Ccr4p deadenylase and two other enzymes involved in mRNA regulation, Dcp1p and Dhh1p. We reconstitute regulated deadenylation in vitro and demonstrate that the PUF-Pop2p interaction is conserved in yeast, worms and humans. We suggest that the PUF-Pop2p interaction underlies regulated deadenylation, mRNA decay and repression by PUF proteins.

PUF Protein-mediated Deadenylation Is Catalyzed by Ccr4p

PUF proteins control gene expression by binding to the 3′-untranslated regions of specific mRNAs and triggering mRNA decay or translational repression. Here we focus on the mechanism of PUF-mediated regulation. The yeast PUF protein, Mpt5p, regulates HO mRNA and stimulates removal of its poly(A) tail (i.e. deadenylation). Mpt5p repression in vivo is dependent on POP2, a component of the cytoplasmic Ccr4p-Pop2p-Not complex that deadenylates mRNAs. In this study, we elucidate the individual roles of the Ccr4p and Pop2p deadenylases in Mpt5p-regulated deadenylation. Both in vivo and in vitro, Pop2p and Ccr4p proteins are required for Mpt5p-regulated deadenylation of HO. However, the requirements for the two proteins differ dramatically: the enzymatic activity of Ccr4p is essential, whereas that of Pop2p is dispensable. We conclude that Pop2p is a bridge through which the PUF protein recruits the Ccr4p enzyme to the target mRNA, thereby stimulating deadenylation. Our data suggest that PUF proteins may enhance mRNA degradation and repress expression by both deadenylation-dependent and -independent mechanisms, using the same Pop2p bridge to recruit a multifunctional Pop2p complex to the mRNA.

FBF and Its Dual Control of gld-1 Expression in the Caenorhabditis elegans Germline

FBF, a PUF RNA-binding protein, is a key regulator of the mitosis/meiosis decision in the Caenorhabditis elegans germline. Genetically, FBF has a dual role in this decision: it maintains germ cells in mitosis, but it also facilitates entry into meiosis. In this article, we explore the molecular basis of that dual role. Previous work showed that FBF downregulates gld-1 expression to promote mitosis and that the GLD-2 poly(A) polymerase upregulates gld-1 expression to reinforce the decision to enter meiosis. Here we ask whether FBF can act as both a negative regulator and a positive regulator of gld-1 expression and also investigate its molecular mechanisms of control. We first show that FBF co-immunoprecipitates with gld-1 mRNA, a result that complements previous evidence that FBF directly controls gld-1 mRNA. Then we show that FBF represses gld-1 expression, that FBF physically interacts with the CCF-1/Pop2p deadenylase and can stimulate deadenylation in vitro, and that CCF-1 is partially responsible for maintaining low GLD-1 in the mitotic region. Finally, we show that FBF can elevate gld-1 expression, that FBF physically interacts with the GLD-2 poly(A) polymerase, and that FBF can enhance GLD-2 poly(A) polymerase activity in vitro. We propose that FBF can affect polyadenylation either negatively by its CCF-1 interaction or positively by its GLD-2 interaction.

Conserved regulation of MAP kinase expression by PUF RNA-binding proteins.

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.

Two Yeast PUF Proteins Negatively Regulate a Single mRNA

mRNA stability and translation are regulated by protein repressors that bind 3′-untranslated regions. PUF proteins provide a paradigm for these regulatory molecules: like other repressors, they inhibit translation, enhance mRNA decay, and promote poly(A) removal. Here we show that a single mRNA in Saccharomyces cerevisiae, encoding the HO endonuclease, is regulated by two distinct PUF proteins, Puf4p and Mpt5p. These proteins bind to adjacent sites and can co-occupy the mRNA. Both proteins are required for full repression and deadenylation in vivo; their removal dramatically stabilizes the mRNA. The two proteins act through overlapping but non-identical mechanisms: repression by Puf4p is dependent on deadenylation, whereas repression by Mpt5p can occur through additional mechanisms. Combinatorial action of the two regulatory proteins may allow responses to specific environmental cues and be common in 3′-untranslated region-mediated control.

LIP‐1 phosphatase controls the extent of germline proliferation in Caenorhabditis elegans

Caenorhabditis elegans germline cells are maintained in an undifferentiated and mitotically dividing state by Notch signaling and the FBF (for fem‐3 binding factor) RNA‐binding protein. Here, we report that the LIP‐1 phosphatase, a proposed homolog of mitogen‐activated protein (MAP) kinase phosphatases, is required for the normal extent of germline proliferation, and that lip‐1 controls germline proliferation by regulating MAP kinase activity. In wild‐type germ lines, LIP‐1 protein is present in the proximal third of the mitotic region, consistent with its effect on germline proliferation. We provide evidence that lip‐1 expression in the germline mitotic region is controlled by a combination of GLP‐1/Notch signaling and FBF repression. Unexpectedly, FBF controls the accumulation of lip‐1 mRNA, and therefore is likely to control its stability or 3′‐end formation. In a sensitized mutant background, LIP‐1 can function as a pivotal regulator of the decision between proliferation and differentiation. The control of germline proliferation by LIP‐1 has intriguing parallels with the control of stem cells and progenitor cells in vertebrates.

Catalysis of Protein Folding by an Immobilized Small-Molecule Dithiol

The isomerization of non‐native disulfide bonds often limits the rate of protein folding. Small‐molecule dithiols can catalyze this process. Here, a symmetric trithiol, tris(2‐mercaptoacetamidoethyl)amine, is designed on the basis of criteria known to be important for efficient catalysis of oxidative protein folding. The trithiol is synthesized and attached to two distinct solid supports via one of its three sulfhydryl groups. The resulting immobilized dithiol has an apparent disulfide E°′ = –208 mV, which is close to that of protein disulfide isomerase (E°′ = –180 mV). Incubation of the dithiol immobilized on a TentaGel resin with a protein containing non‐native disulfide bonds produced only a 2‐fold increase in native protein. This dithiol appeared to be inaccessible to protein. In contrast, incubation of the dithiol immobilized on styrene‐glycidyl methacrylate microspheres with the non‐native protein produced a 17‐fold increase in native protein. This increase was 1.5‐fold greater than that of a monothiol immobilized on the microspheres. Thus, the choice of both the solid support and thiol can affect catalysis of protein folding. The use of dithiol‐decorated microspheres is an effective new strategy for preparative protein folding in vitro.