88,197 research outputs found
HENN-1 Is Broadly Expressed in <i>C. elegans</i> Germline.
No full text<p>A) The <i>henn-1</i> mRNA expression profile is consistent with germline enrichment. Levels of <i>henn-1</i> mRNA were assayed throughout development and normalized to <i>eft-2</i> mRNA. Standard deviation is shown for biological triplicates. Non-normalized levels are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002617#pgen.1002617.s013" target="_blank">Figure S13A</a>. B) HENN-1 is detected at all stages of development and in male. Lysates from animals of the indicated stages were probed with anti-HENN-1 rabbit polyclonal antibody. C) HENN-1 is abundant in hermaphrodite proximal germline and enriched in proximal oocyte nucleoplasm (inset). Extruded gonads of <i>xkSi1; henn-1(tm4477)</i> adult hermaphrodites were stained with anti-GFP mouse monoclonal and anti-HENN-1 rabbit polyclonal antibodies. D) HENN-1 is detectable in male proximal and distal gonad, with enrichment in residual bodies during spermatid maturation (inset). Extruded gonads of <i>xkSi1; henn-1(tm4477)</i> adult males were stained with anti-GFP and anti-HENN-1 antibodies. E) Expression of endogenous HENN-1 mirrors expression of HENN-1::GFP from transgene <i>xkSi1</i>. Extruded gonads of wild-type animals were stained with anti-HENN-1 antibody. F) Detection of HENN-1 proteins by immunostaining is specific. Extruded gonads of <i>henn-1(tm4477)</i> mutant animals were stained with anti-GFP and anti-HENN-1 antibodies. E, embryo.</p
HENN-1 expression analysis.
No full text<p>(A) Western blot analysis for HENN-1 expression in different mutant backgrounds. <i>Glp-4(bn2)</i> animals contain almost no germ cells. Tubulin is shown as loading control. (B) Western blot analysis for HENN-1 expression at different time points during development of <i>C. elegans</i>. <i>Glp-4(bn2)</i> animals contain almost no germ cells. Tubulin is shown as loading control. (C) Confocal images (single z-plane) of HENN-1::GFP expressing animals at different developmental stages. Nuclei of embryonic germ cells are outlined in white boxes. Blastomere identities in the four-cell stage embryos are indicated. Scale bars are 10 µm. (D) Immuno-fluorescence with anti-PGL-1 and anti-GFP antibodies. The white arrowhead indicates a site of co-localization. Scale bars are 10 µm. (E) Western blots for HENN-1 (top) and PRG-1 (bottom) on fractions obtained after gel filtration.</p
Global effects of <i>henn-1</i> on 21U RNAs.
No full text<p>(A) Bar diagram displaying the different annotated small RNA reads obtained after deep-sequencing. Reads from structural RNAs were removed before analysis. (B) The expression level of 21U RNAs in wild-type and <i>henn-1</i> mutant backgrounds. Total 21U reads are normalized to total miRNA reads. The differences between wild-type and <i>henn-1</i> mutant samples are significant (Chi-squared test; p<10<sup>−10</sup>). (C) Length distribution plot of 21U RNAs. (D) Scatter plot displaying the fraction of 20-mer species of individual 21U RNA loci that were represented by at least 250 raw reads (20+21-mers) in each of the libraries used for this analysis (<i>henn-1(pk2452)</i> and <i>henn-1(pk2295)</i>). (E) Scatter plot displaying individual 21U loci represented by at least 250 raw reads (20+21-mers) in each of the libraries used for this analysis (wild-type and <i>henn-1(pk2295)</i>). X-axis: fold loss of reads in the <i>henn-1(pk2295)</i> background relative to wild-type. Y-axis: fold increase of 20-mer species in the <i>henn-1(pk2295)</i> background relative to wild-type. (F) Bar diagram displaying the frequencies of non-templated base additions found on 21U reads, as a percentage of the total 21U read count.</p
RNA silencing defects in <i>henn-1</i> mutants.
No full text<p>(A) RNA blot assays of small RNAs in wild type and <i>henn-1</i> mutant embryos and adults. For embryos, one of three biological replicates is shown. EtBr stained tRNAs are shown as a loading control. (B) qRT-PCR assay of small RNA levels in wild type and <i>henn-1</i> mutant embryos. Wild type = 1.0. Error bars display standard deviation from the mean for three biological replicates. P values are for comparisons to wild type. (C) qRT-PCR assay of small RNA target mRNA levels in wild type and <i>henn-1</i> mutant embryos. Wild type = 1.0. Error bars display standard deviation from the mean for three biological replicates. P values are for comparisons to wild type. (D) RNA blot assays for miRNAs in wild type and <i>henn-1</i> mutant L4 larvae. EtBr stained tRNAs are shown as a loading control. (E) qRT-PCR assay of small RNA levels in wild type and <i>henn-1</i> mutant L4 larvae. Wild type = 1.0. (F) qRT-PCR assay of ALG-3/4 target mRNA levels in wild type and <i>henn-1</i> mutant embryos. Wild type = 1.0. Data shown for two independent experiments. (G) Box plots display brood size per individual wild type or <i>henn-1</i> mutant grown at either 20°C or 25°C. n = 20 (20°C) or n = 30 (25°C) individuals per strain. P values are for comparisons to wild type.</p
HENN-1 Stabilizes 21U RNAs.
No full text<p>A) Loss of <i>henn-1</i> impairs 21U RNA accumulation in adult, embryo, and early larva. Levels of 21UR-1848 were assayed by Taqman qPCR in embryo and every four hours across development of wild-type and <i>henn-1(tm4477)</i> mutant animals at 25°C. Standard deviation is shown for biological triplicates. Taqman qPCR data for eight additional 21U RNAs are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002617#pgen.1002617.s004" target="_blank">Figure S4</a>. B) Effects of loss of <i>henn-1</i> are restricted to its small RNA substrates. Levels of miR-1 across development were assayed by Taqman qPCR. Standard deviation is shown for biological triplicates. Additional Taqman qPCR data for miRNAs are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002617#pgen.1002617.s005" target="_blank">Figure S5</a>. C) Loss of <i>henn-1</i> impairs <i>Tc3</i> transposase silencing primarily in early L1 larva. <i>Tc3</i> transposase mRNA levels were assayed by qPCR across development and normalized to mRNA levels of <i>eft-2</i>, an abundantly expressed housekeeping gene. <i>prg-1(tm872)</i> lacks 21U RNAs and is included as a positive control for <i>Tc3</i> upregulation. Significant zero and four hour time points are expanded at right (*: P = 0.0251; **: P = 0.0250, two-tailed <i>t</i>-test). Standard deviation is shown for biological triplicates. E, embryo; hr, hour.</p
HENN-1 is the <i>C. elegans</i> homolog of Hen1.
No full text<p>(A) Left panel: protein gel stained with PageBlue shows purified GST, GST-HENN-1 and GST-HENN-1(D151N) proteins used to perform methyltransferase assays as shown in the right panel and B. Right panel: <i>in vitro</i> methyltransferase activity assay. RNA oligos were incubated with indicated proteins and 14C-labelled SAM. Reaction products were run on a 12% acryl-amide gel. (B) <i>In vitro</i> methyltransferase assay using different RNA substrates each differing in the identity of the most 3′ nucleotide. (C) Northern blot analysis using RNA from wild-type, <i>henn-1(pk2452)</i>, <i>henn-1(pk2295)</i> and <i>henn-1(pk2295); pgl-3:HENN-1::GFP</i> animals. Blots were probed for 21UR1 and 26G species siR26-263. Probing for <i>let-7</i> serves as loading and as oxidation-β-elimination control. (D) Response of wild type (N2), <i>henn-1(pk2295)</i> and <i>henn-1(pk2295); pgl-3:henn-1:GFP</i> to <i>dpy-13</i> RNAi. <i>Henn-1(pk2295)</i> sensitivity is significantly higher than both controls (two-tailed t-test, n = 5: p<0.05 for both). (E) Response of wild type (N2), <i>henn-1(pk2295)</i> and <i>henn-1(pk2295); pgl-3:HENN-1::GFP</i> to <i>pos-1</i> RNAi, delivered at three different dosages: undiluted (100%), diluted one to one (50%) and diluted one to four (25%). At 50% <i>pos-1</i> RNAi, <i>henn-1(pk2295); pgl-3:HENN-1::GFP</i> animals display significant rescue (p = 0.01) of the <i>henn-1(pk2295)</i> RNAi defect (p<0.0005). The p-values at 25% <i>pos-1</i> RNAi are p = 0.04 for both the <i>henn-1(pk2295)</i> RNAi defect and the rescue. P-values were calculated with a two-tailed t-test, n = 10.</p
Chemical interpretation of molecular electron density distributions
No full textIn this study, the two small molecules HS(CH)(CH2), 1, and F(CH)(4)F, 2, are presented, which yield different chemical interpretations when one and the same density is interpreted either by means of Natural Bond Orbital and subsequent Natural Resonance Theory application or by the Quantum Theory of Atoms In Molecules. The first exhibits a S-C bond in the orbital based approach, whereas the density based Quantum Theory of Atoms In Molecules detects no corresponding bond. In F(CH)(4)F a F center dot center dot center dot F bond is detected in the density based approach, whereas in the orbital based approach no corresponding bond is found. Geometrical reasons for the presence of unexpected and the absence of expected bond critical points are discussed
The Caenorhabditis elegans HEN1 ortholog, HENN-1, methylates and stabilizes select subclasses of germline small RNAs.
No full textSmall RNAs regulate diverse biological processes by directing effector proteins called Argonautes to silence complementary mRNAs. Maturation of some classes of small RNAs involves terminal 2'-O-methylation to prevent degradation. This modification is catalyzed by members of the conserved HEN1 RNA methyltransferase family. In animals, Piwi-interacting RNAs (piRNAs) and some endogenous and exogenous small interfering RNAs (siRNAs) are methylated, whereas microRNAs are not. However, the mechanisms that determine animal HEN1 substrate specificity have yet to be fully resolved. In Caenorhabditis elegans, a HEN1 ortholog has not been studied, but there is evidence for methylation of piRNAs and some endogenous siRNAs. Here, we report that the worm HEN1 ortholog, HENN-1 (HEN of Nematode), is required for methylation of C. elegans small RNAs. Our results indicate that piRNAs are universally methylated by HENN-1. In contrast, 26G RNAs, a class of primary endogenous siRNAs, are methylated in female germline and embryo, but not in male germline. Intriguingly, the methylation pattern of 26G RNAs correlates with the expression of distinct male and female germline Argonautes. Moreover, loss of the female germline Argonaute results in loss of 26G RNA methylation altogether. These findings support a model wherein methylation status of a metazoan small RNA is dictated by the Argonaute to which it binds. Loss of henn-1 results in phenotypes that reflect destabilization of substrate small RNAs: dysregulation of target mRNAs, impaired fertility, and enhanced somatic RNAi. Additionally, the henn-1 mutant shows a weakened response to RNAi knockdown of germline genes, suggesting that HENN-1 may also function in canonical RNAi. Together, our results indicate a broad role for HENN-1 in both endogenous and exogenous gene silencing pathways and provide further insight into the mechanisms of HEN1 substrate discrimination and the diversity within the Argonaute family
Effects of <i>henn-1</i> 22G and 26G RNAs.
No full text<p>(A) Length distribution plots of ‘siRNA’ category, containing both 22G and 26G RNAs, in diverse libraries. (B) Bar diagram displaying the frequencies of non-templated base additions found on 26G reads, as a percentage of the total 26G read count. P<0.0001 for <i>henn-1(pk2452)</i> and <i>henn-1(pk2295)</i> relative to wild-type (Chi-squared test). (C) Ratio of ALG-3/4 and ERGO-1 bound 26G RNAs as derived by previously described annotation (see main text), in diverse libraries. P-values of all differences <0.001 (Chi-squared test). (D) The 22G counts, in rpm, for all genes in total, ERGO-1, ALG-3/4 and CSR-1 target genes. 22G count for ‘Total’ was divided by 100 for better visualization.</p
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