miR-34 activity is modulated through 5'-end phosphorylation in response to DNA damage.

MicroRNA (miRNA) expression is tightly regulated by several mechanisms, including transcription and cleavage of the miRNA precursor RNAs, to generate a mature miRNA, which is thought to be directly correlated with activity. MiR-34 is a tumour-suppressor miRNA important in cell survival, that is transcriptionally upregulated by p53 in response to DNA damage. Here, we show for the first time that there is a pool of mature miR-34 in cells that lacks a 5'-phosphate and is inactive. Following exposure to a DNA-damaging stimulus, the inactive pool of miR-34 is rapidly activated through 5'-end phosphorylation in an ATM- and Clp1-dependent manner, enabling loading into Ago2. Importantly, this mechanism of miR-34 activation occurs faster than, and independently of, de novo p53-mediated transcription and processing. Our study reveals a novel mechanism of rapid miRNA activation in response to environmental stimuli occurring at the mature miRNA level

miR-34 activity is modulated through 5′-end phosphorylation in response to DNA damage

MicroRNA (miRNA) expression is tightly regulated by several mechanisms, including transcription and cleavage of the miRNA precursor RNAs, to generate a mature miRNA, which is thought to be directly correlated with activity. MiR-34 is a tumour-suppressor miRNA important in cell survival, that is transcriptionally upregulated by p53 in response to DNA damage. Here, we show for the first time that there is a pool of mature miR-34 in cells that lacks a 5′-phosphate and is inactive. Following exposure to a DNA-damaging stimulus, the inactive pool of miR-34 is rapidly activated through 5′-end phosphorylation in an ATM- and Clp1-dependent manner, enabling loading into Ago2. Importantly, this mechanism of miR-34 activation occurs faster than, and independently of, de novo p53-mediated transcription and processing. Our study reveals a novel mechanism of rapid miRNA activation in response to environmental stimuli occurring at the mature miRNA level

Fluoride export (FEX) proteins from fungi, plants and animals are 'single barreled' channels containing one functional and one vestigial ion pore

<div><p>The fluoride export protein (FEX) in yeast and other fungi provides tolerance to fluoride (F^-), an environmentally ubiquitous anion. FEX efficiently eliminates intracellular fluoride that otherwise would accumulate at toxic concentrations. The FEX homolog in bacteria, Fluc, is a ‘double-barreled’ channel formed by dimerization of two identical or similar subunits. FEX in yeast and other eukaryotes is a monomer resulting from covalent fusion of the two subunits. As a result, both potential fluoride pores are created from different parts of the same protein. Here we identify FEX proteins from two multicellular eukaryotes, a plant <i>Arabidopsis thaliana</i> and an animal <i>Amphimedon queenslandica</i>, by demonstrating significant fluoride tolerance when these proteins are heterologously expressed in the yeast <i>Saccharomyces cerevisiae</i>. Residues important for eukaryotic FEX function were determined by phylogenetic sequence alignment and functional analysis using a yeast growth assay. Key residues of the fluoride channel are conserved in only one of the two potential fluoride-transporting pores. FEX activity is abolished upon mutation of residues in this conserved pore, suggesting that only one of the pores is functional. The same topology is conserved for the newly identified FEX proteins from plant and animal. These data suggest that FEX family of fluoride channels in eukaryotes are ‘single-barreled’ transporters containing one functional pore and a second non-functional vestigial remnant of a homologous gene fusion event.</p></div

Structural arrangement of fluoride export proteins.

<p>(A) Topology model of bacterial Fluc protein. (B) Tertiary structure of Fluc-<i>Bp</i> (PDB ID: 5FXB) showing two pores with fluoride (dark grey) ions. Sodium ion (purple) is in the middle of the protein dimer. Two monomers are colored brown and light grey. (C) Arrangement of transmembrane helices (top view). Helices TM3b from different monomers (yellow and green) separate two pores and have residues belonging to two different pores but located on the same helix. (D) Topology model of eukaryotic FEX protein with proposed interactions between TM3 and TM8 based on Fluc crystal structure.</p

Effect of mutations at conserved residues in two domains of Fex1p.

<p>Effect of mutations at conserved residues in two domains of Fex1p.</p

Effect of mutations to residues in the hypothetical pores of Fex1p.

<p>Effect of mutations to residues in the hypothetical pores of Fex1p.</p

Alignment of protein sequences from six eukaryotic and two bacterial fluoride channels.

<p>Transmembrane regions were predicted using TMHMM and TMpred servers. Conserved residues for all eukaryotic FEX proteins are highlighted with asterisks. Amino acid numbering corresponds to the protein sequence of Fex1p from <i>S</i>. <i>cerevisiae</i>. The residues highlighted in green are present within both pores of Fluc.</p

Model of Fex1p from <i>S</i>. <i>cerevisiae</i>.

<p>Comparison of the residues in two pores for Fex1p-<i>Sc</i> (red residues are in N-terminal domain and blue are in C-terminal domain) and Fluc-<i>Bp</i> (white). Residues in Pore II and not Pore I of FEX are similar to bacterial Fluc.</p

The helices from both domains that form functional and vestigial pores.

<p>The helices from both domains that form functional and vestigial pores.</p

Functional analysis of conserved fragments in FEX.

<p><i>A</i>, mutations to pore-lining Phe located in TM3 and TM8. <i>B</i>, mutations to PxGTxxxN motif located in TM7. <i>C</i>, mutations to YxxxS fragment located in TM9. The <i>fex1Δfex2Δ</i> strain was transformed with wild-type pRS416-<i>FEX1</i> (WT), empty vector pRS416 (No FEX) or Fex1p mutants in pRS416-<i>FEX1</i>. Serial dilutions of yeast cultures expressing the indicated mutants were incubated on YPD media with different concentrations of NaF.</p