Distinct Argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline

Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that in the germ-line, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germ-line nuage structures called P-granules. WAGO-1 silences certain genes, transposons, pseudogenes and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest novel regulatory functions for small RNAs

A Machine Learning Approach for Identifying Novel Cell Type–Specific Transcriptional Regulators of Myogenesis

Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, we combined phylogenetic profiling with a DNA–based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. We first assembled a small number of enhancers that are active in Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, we increased the number of enhancers by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. We then built and trained a classifier using this enhancer set and identified additional related enhancers based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, we found extensive diversity in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type–specific developmental gene expression patterns

Electric field driven torque in ATP synthase.

FO-ATP synthase (FO) is a rotary motor that converts potential energy from ions, usually protons, moving from high- to low-potential sides of a membrane into torque and rotary motion. Here we propose a mechanism whereby electric fields emanating from the proton entry and exit channels act on asymmetric charge distributions in the c-ring, due to protonated and deprotonated sites, and drive it to rotate. The model predicts a scaling between time-averaged torque and proton motive force, which can be hindered by mutations that adversely affect the channels. The torque created by the c-ring of FO drives the γ-subunit to rotate within the ATP-producing complex (F1) overcoming, with the aid of thermal fluctuations, an opposing torque that rises and falls with angular position. Using the analogy with thermal Brownian motion of a particle in a tilted washboard potential, we compute ATP production rates vs. proton motive force. The latter shows a minimum, needed to drive ATP production, which scales inversely with the number of proton binding sites on the c-ring

Top view of the <i>c</i>-ring (yellow) and stator <i>a</i>-subunit (green) of F_O, showing equipotential surface cross-sections (curved lines) perpendicular to the electric field emanating from the half-channels (blue and red circles) in the <i>a</i>-subunit.

<p>Black arrows represent forces due to tangential field components (red arrows) acting on protonated (blue circles) and deprotonated (light circle) sites on the <i>c</i>-ring. (Equipotentials computed using QuickField.)</p

Plots show the predicted <i>c</i>-ring rotation rates vs. proton motive force for various values of <i>n</i>, <i>τ</i>_c, and <i>τ</i>₁. (a)

<p>Predicted rotation rate <i>f</i> (see text) vs. pmf, Δ<i>p</i>, taking <i>τ</i>_c = 40 pN-nm and <i>τ</i>₁ = 20 pN-nm, for various numbers <i>n</i> of proton binding sites on the <i>c</i>-ring. Positive and negative rotation rates correspond to ATP synthesis and ATP hydrolysis, respectively. <b>(b)</b> Theoretical rotation rate <i>f</i> vs. Δ<i>p</i>, assuming <i>τ</i>_c = 40 pN-nm and <i>n</i> = 8, for various values of <i>τ</i>₁. <b>(c)</b> Predicted rotation rate <i>f</i> vs. Δ<i>p</i>, assuming <i>τ</i>₁ = 20 pN·nm and <i>n</i> = 8, for two values of <i>τ</i>_c. <b>(d)</b> Theoretical ATP production rate (dashed line) vs. Δ<i>p</i> (using <i>τ</i>_c = 31 pN·nm and <i>τ</i>₁ = 17 pN·nm), as compared to maximum intracellular ATP/ADP ratios (squares) reported by Nicholls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074978#pone.0074978-Nicholls2" target="_blank">[38]</a>.</p

Side view of F₁F_O ATP synthase, oriented with high-potential side on top, showing the half-channels in the stator <i>a</i>-subunit, <i>c</i>-ring rotor of F_O (<i>c₁₀</i> for <i>E. coli</i>), and F₁ complex, within which the <i>γ</i>-subunit rotates to release three ATP molecules per cycle [adapted from Fillingame [<b>5</b>] with permission].

<p>Side view of F₁F_O ATP synthase, oriented with high-potential side on top, showing the half-channels in the stator <i>a</i>-subunit, <i>c</i>-ring rotor of F_O (<i>c₁₀</i> for <i>E. coli</i>), and F₁ complex, within which the <i>γ</i>-subunit rotates to release three ATP molecules per cycle [adapted from Fillingame <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074978#pone.0074978-Fillingame1" target="_blank">[<b>5</b>]</a> with permission].</p