Wickens M et al. (2001) Cold Spring Harb Symp Quant Biol "PUF proteins and 3'UTR regulation in the Caenorhabditis elegans germ line."

Bernstein D, Crittenden S, Kimble J, Luitjens C, Wickens M

[Cold Spring Harb Symp Quant Biol, 2001]

In a simple and too common view, mRNAs are a dull milepost between DNA and protein in the Central Dogma. They acquire middling interest as guides for the ribosome, tRNAs, and translation factors. Yet mRNAs have lives of their own, lives that the cell cares about enormously. mRNAs are born, leave home, mate with ribosomes, produce proteins, and die, all as the cell sees fit. Powerful forces ? in the form of specific mRNA-binding proteins and their cohorts ? guide individual mRNAs into unique variations on this common path. These proteins govern mRNA stability, localization, and translation.

Kwak JE et al. (2007) RNA "A family of poly(U) polymerases."

Kwak JE, Wickens M

[RNA, 2007]

The GLD-2 family of poly(A) polymerases add successive AMP monomers to the 3'' end of specific RNAs, forming a poly(A) tail. Here, we identify a new group of GLD-2-related nucleotidyl transferases from Arabidopsis, Schizosaccharomyces pombe, Caenorhabditis elegans, and humans. Like GLD-2, these enzymes are template independent and add nucleotides to the 3'' end of an RNA substrate. However, these new enzymes, which we refer to as poly(U) polymerases, add poly(U) rather than poly(A) to their RNA substrates.

Chritton JJ et al. (2011) J Biol Chem "A role for the poly(A)-binding protein Pab1p in PUF protein-mediated repression."

Chritton JJ, Wickens M

[J Biol Chem, 2011]

PUF proteins regulate translation and mRNA stability throughout eukaryotes. Using a cell-free translation assay, we examined the mechanisms of translational repression of PUF proteins in the budding yeast Saccharomyces cerevisiae. We demonstrate that the poly(A)-binding protein Pab1p is required for PUF-mediated translational repression for two distantly related PUF proteins: S. cerevisiae Puf5p and Caenorhabditis elegans FBF-2. Pab1p interacts with oligo(A) tracts in the HO 3'-UTR, a target of Puf5p, to dramatically enhance the efficiency of Puf5p repression. Both the Pab1p ability to activate translation and interact with eukaryotic initiation factor 4G (eIF4G) were required to observe maximal repression by Puf5p. Repression was also more efficient when Pab1p was bound in close proximity to Puf5p. Puf5p may disrupt translation initiation by interfering with the interaction between Pab1p and eIF4G. Finally, we demonstrate two separable mechanisms of translational repression employed by Puf5p: a Pab1p-dependent mechanism and a Pab1p-independent mechanism.

Chritton JJ et al. (2010) RNA "Translational repression by PUF proteins in vitro."

Wickens M, Chritton JJ

[RNA, 2010]

PUF (Pumilio and FBF) proteins provide a paradigm for mRNA regulatory proteins. They interact with specific sequences in the 3' untranslated regions (UTRs) of target mRNAs and cause changes in RNA stability or translational activity. Here we describe an in vitro translation assay that reconstitutes the translational repression activity of canonical PUF proteins. In this system, recombinant PUF proteins were added to yeast cell lysates to repress reporter mRNAs bearing the 3'UTRs of specific target mRNAs. PUF proteins from Saccharomyces cerevisiae and Caenorhabditis elegans were active in the assay and were specific by multiple criteria. Puf5p, a yeast PUF protein, repressed translation of four target RNAs. Repression mediated by the HO 3'UTR was particularly efficient, due to a specific sequence in that 3'UTR. The sequence lies downstream from the PUF binding site and does not affect PUF protein binding. PUF-mediated repression was sensitive to the distance between the ORF and the regulatory elements in the 3'UTR: excessive distance decreased repression activity. Our data demonstrate that PUF proteins function in vitro across species, that different mRNA targets are regulated differentially, and that specific ancillary sequences distinguish one yeast mRNA target from another. We suggest a model in which PUF proteins can control translation termination or elongation.

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[Biochemistry, 2012]

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

O'Connell EM et al. (2015) J Infect Dis "Targeting Filarial Abl-like Kinases: Orally Available, Food and Drug Administration-Approved Tyrosine Kinase Inhibitors Are Microfilaricidal and Macrofilaricidal."

Nutman TB, O'Connell EM, Bennuru S, Steel C, Dolan MA

[J Infect Dis, 2015]

BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and >100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.

Stumpf CR et al. (2008) RNA "A Caenorhabditis elegans PUF protein family with distinct RNA binding specificity."

Stumpf CR, Kimble J, Wickens M

[RNA, 2008]

PUF proteins comprise a highly conserved family of sequence-specific RNA binding proteins that regulate target mRNAs via binding directly to their 3''UTRs. The Caenorhabditis elegans genome encodes several PUF proteins, which cluster into four groups based on sequence similarity; all share amino acids that interact with the RNA in the cocrystal of human Pumilio with RNA. Members of the FBF and the PUF-8/9 groups bind different but related RNA sequences. We focus here on the binding specificity of representatives of a third cluster, comprising PUF-5, -6, and -7. We performed in vivo selection experiments using the yeast three-hybrid system to identify RNA sequences that bind PUF-5 and PUF-6, and we confirmed binding to optimal sites in vitro. The consensus sequences derived from the screens are similar for PUF-5 and PUF-6 but differ from those of the FBF or PUF-8/-9 groups. Similarly, neither PUF-5 nor PUF-6 bind the recognition sites preferred by the other clusters. Mutagenesis studies confirmed the unique RNA specificity of PUF-5/-6. Using the PUF-5 consensus derived from our experiments, we searched a database of C. elegans 3''UTRs to identify potential targets of PUF-5, several of which indeed bind PUF-5. Therefore the consensus has predictive value and provides a route to finding genuine targets of these proteins.

Wang L et al. (2004) RNA "Tissue-specific modification of gld-2 mRNA in C. elegans: likely C-to-U editing."

Kimble J, Wickens M, Wang L

[RNA, 2004]

Seventeen years after the discovery of tissue-specific apoB mRNA editing, only three nucleus-encoded mRNAs have been shown to undergo C-to-U editing. All three mRNAs occur in mammals. apoB mRNA editing is tissue-specific and occurs normally, whereas NF1 and NAT1 mRNA editing is found largely in tumors. Here we report the first example of C-to-U RNA editing in Caenorhabditis elegans. The gld-2 gene encodes an atypical poly(A) polymerase that governs the mitosis/meiosis decision in the germ line as well as progression through meiosis and early embryogenesis. At least two of its alternatively spliced transcripts are germline-specific. We find that most and perhaps all germline-specific transcripts generated by the gld-2 gene undergo C-to-U editing, but that somatic transcripts show no detectable editing. The gld-2 C-to-U editing event changes the codon from CCG to CUG, which is predicted to cause a proline to leucine substitution in the protein sequence. Our findings suggest the presence of a sequence- and tissue-specific cytidine deaminase acting on RNA, or CDAR. This CDAR modifies a specific base in gld-2 mRNA, and acts only in the germline.

Lemire BD et al. (2009) Mech Ageing Dev "C. elegans longevity pathways converge to decrease mitochondrial membrane potential."

Behrendt M, Decorby A, Lemire BD, Gaskova D

[Mech Ageing Dev, 2009]

Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (DeltaPsi(m)) across the inner membrane. In this work, we show that a lower DeltaPsi(m) is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants daf-2(e1370), age-1(hx546), clk-1(qm30), isp-1(qm150) and eat-2(ad465) all have a lower DeltaPsi(m) than wild type animals. The lower DeltaPsi(m) of daf-2(e1370) is daf-16 dependent, indicating that the insulin-like signaling pathway not only regulates lifespan but also mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease DeltaPsi(m). Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates DeltaPsi(m). We conclude that longevity pathways converge on the mitochondria and lead to a decreased DeltaPsi(m). Our results are consistent with the 'uncoupling to survive' hypothesis, which states that dissipation of the DeltaPsi(m) will extend lifespan.

Seo J et al. (2005) Arch Environ Contam Toxicol "Biodegradation of the insecticide N,N-diethyl-m-toluamide by fungi: identification and toxicity of metabolites."

Kim SD, Cha CJ, Hur HG, Lee YG, Seo J, Ahn JH

[Arch Environ Contam Toxicol, 2005]

Fungi (Cunninghamella elegans ATCC 9245, Mucor ramannianus R-56, Aspergillus niger VKMF-1119, and Phanerochaete chrysosporium BKMF-1767) were tested to elucidate the biologic fate of the topical insect repellent N,N-diethyl-m-toluamide (DEET). The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column, showed that three peaks occurred after incubation of C. elegans, with which 1 mM DEET was combined as a final concentration. The peaks were not detected in the control experiments with either DEET alone or tested fungus alone. The metabolites produced by C. elegans exhibited a molecular mass of 207 with a fragment ion (m/z) at 135, a molecular mass of 179 with an m/z at 135, and a molecular mass of 163 with an m/z at 119, all of which correspond to N,N-diethyl-m-toluamide-N-oxide, N-ethyl-m-toluamide-N-oxide, and N-ethyl-m-toluamide, respectively. M. ramannianus R-56 also produced N, N-diethyl-m-toluamide-N-oxide and N-ethyl-m-toluamide but did not produce N-ethyl-m-toluamide-N-oxide. For the biologic toxicity test with DEET and its metabolites, the freshwater zooplankton Daphnia magna was used. The biologic sensitivity in decreasing order was DEET > N-ethyl-m-toluamide > N,N-diethyl-m-toluamide-N-oxide. Although DEET and its fungal metabolites showed relatively low mortality compared with other insecticides, the toxicity was increased at longer exposure periods. These are the first reports of the metabolism of DEET by fungi and of the biologic toxicity of DEET and its fungal metabolites to the freshwater zooplankton D. magna.