Model for how m6A may stimulate pri-miRNA processing.

<p>m6A is deposited on pri-miRNA by METTL3 and is thought to stimulate the recruitment of Drosha/DGCR8 for co-transcriptional processing of pri-miRNA to pre-miRNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006139#pgen.1006139.ref027" target="_blank">27</a>]. The question mark is to highlight that the identity of the full set of m6A readers in pri-miRNAs is unknown. In yellow is shown RNAP II on DNA surrounded by nucleosomes.</p

Model for the mode of action of the BCDIN3D RNA methyltransferase on the biogenesis of specific miRNAs.

<p>BCDIN3D’s enzymatic activity consists in the methylation of the two available oxygen moieties of the 5′ monophosphate, which removes the 5′ monophosphate charge and makes it bulkier. This methylation blocks the processing of specific miRNAs [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006139#pgen.1006139.ref008" target="_blank">8</a>], possibly through perturbing the interaction of the 5′ monophosphate with its binding pocket in Dicer [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006139#pgen.1006139.ref008" target="_blank">8</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006139#pgen.1006139.ref014" target="_blank">14</a>].</p

List of known messenger or miRNA modifications, with chemical structures, abbreviation, writers, erasers, and readers.

<p><b>*</b>The <i>Drosophila</i> dTET enzyme has recently been shown to hydroxylate 5meC on RNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006139#pgen.1006139.ref011" target="_blank">11</a>].</p

Simplified schematic of (A) the miRNA biogenesis pathway and (B) the interaction between the mRNA and miRNA.

<p><b>(A)</b> The primary miRNA precursor (pri-miRNA) is synthesized by RNAP II. The pri-miRNA is first cleaved by Drosha to release a hairpin loop–shaped RNA called pre-miRNA. The loop of this pre-miRNA is further cleaved by Dicer to generate a miRNA duplex. The miRNA duplex is dissociated and the passenger strand (dashed line) is discarded while the guide strand is loaded onto the Argonaute protein to form an active RISC complex. <b>(B)</b> Example of miRNA–target mRNA interaction by base-pairing mainly at the seed region of miRNA (nt 2–8) but also on other downstream regions of the miRNA. In the mRNA, the coding region is represented as a double line. <b>N.B.</b> The asterisks indicate the m7G cap (7-methyl-guanosine with 5′, 5′-triphosphate linkage).</p

3D-Printed Microfluidic Microdissector for High-Throughput Studies of Cellular Aging

Due to their short lifespan, rapid division, and ease of genetic manipulation, yeasts are popular model organisms for studying aging in actively dividing cells. To study replicative aging over many cell divisions, individual cells must be continuously separated from their progeny via a laborious manual microdissection procedure. Microfluidics-based soft-lithography devices have recently been used to automate microdissection of the budding yeast <i>Saccharomyces cerevisiae</i>. However, little is known about replicative aging in <i>Schizosaccharomyces pombe</i>, a rod-shaped yeast that divides by binary fission and shares many conserved biological functions with higher eukaryotes. In this report, we develop a versatile multiphoton lithography method that enables rapid fabrication of three-dimensional master structures for polydimethylsiloxane (PDMS)-based microfluidics. We exploit the rapid prototyping capabilities of multiphoton lithography to create and characterize a cell-capture device that is capable of high-resolution microscopic observation of hundreds of individual <i>S. pombe</i> cells. By continuously removing the progeny cells, we demonstrate that cell growth and protein aggregation can be tracked in individual cells for over ∼100 h. Thus, the fission yeast lifespan microdissector (FYLM) provides a powerful on-chip microdissection platform that will enable high-throughput studies of aging in rod-shaped cells

3D-Printed Microfluidic Microdissector for High-Throughput Studies of Cellular Aging

Due to their short lifespan, rapid division, and ease of genetic manipulation, yeasts are popular model organisms for studying aging in actively dividing cells. To study replicative aging over many cell divisions, individual cells must be continuously separated from their progeny via a laborious manual microdissection procedure. Microfluidics-based soft-lithography devices have recently been used to automate microdissection of the budding yeast <i>Saccharomyces cerevisiae</i>. However, little is known about replicative aging in <i>Schizosaccharomyces pombe</i>, a rod-shaped yeast that divides by binary fission and shares many conserved biological functions with higher eukaryotes. In this report, we develop a versatile multiphoton lithography method that enables rapid fabrication of three-dimensional master structures for polydimethylsiloxane (PDMS)-based microfluidics. We exploit the rapid prototyping capabilities of multiphoton lithography to create and characterize a cell-capture device that is capable of high-resolution microscopic observation of hundreds of individual <i>S. pombe</i> cells. By continuously removing the progeny cells, we demonstrate that cell growth and protein aggregation can be tracked in individual cells for over ∼100 h. Thus, the fission yeast lifespan microdissector (FYLM) provides a powerful on-chip microdissection platform that will enable high-throughput studies of aging in rod-shaped cells

Click Quantitative Mass Spectrometry Identifies PIWIL3 as a Mechanistic Target of RNA Interference Activator Enoxacin in Cancer Cells

Enoxacin is a small molecule that stimulates RNA interference (RNAi) and acts as a growth inhibitor selectively in cancer but not in untransformed cells. Here, we used alkenox, a clickable enoxacin surrogate, coupled with quantitative mass spectrometry, to identify PIWIL3 as a mechanistic target of enoxacin. PIWIL3 is an Argonaute protein of the PIWI subfamily that is mainly expressed in the germline and that mediates RNAi through piRNAs. Our results suggest that cancer cells re-express PIWIL3 to repress RNAi through miRNAs and thus open a new opportunity for cancer-specific targeting