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Henn, C H, 424001
No full textThis record was harvested from a previous catalogue system and will be withdrawn in 2025. Information in this record may be superseded or incomplete. Visit this record in UMA's new catalogue at: https://archives.library.unimelb.edu.au/nodes/view/391804Surname: HENN. Given Name(s) or Initials: C H. Military Service Number or Last Known Location: 424001. Missing, Wounded and Prisoner of War Enquiry Card Index Number: 53898.209165
Item: [2016.0049.24097] "Henn, C H, 424001
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
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 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
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
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
Methylation of 21U RNAs Requires <i>C. elegans</i> HEN1 Ortholog HENN-1.
No full text<p>A) HENN-1 is required for 21U RNA methylation. Endogenous (<i>xkSi1</i>) and germline-specific (<i>xkSi2</i>) expression of <i>henn-1::gfp</i> rescue 21U RNA methylation in <i>henn-1(tm4477)</i> mutant embryo. Total embryo RNA of the indicated genotypes was β-eliminated (βe +) or control treated (βe −) and probed for piRNA 21UR-4292. <i>prg-1(tm872)</i> lacks 21U RNAs and is included as a negative control. Below, ethidium bromide staining of 5.8S rRNA is shown. Additional 21U RNA northern blots are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002617#pgen.1002617.s003" target="_blank">Figure S3A</a>. B) <i>C. elegans</i> miRNAs are unmethylated. Total embryo RNA was probed for miR-1. Variable intensity of 5.8S rRNA bands in embryo indicates unequal loading.</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
<i>henn-1</i> is required for 22G siR-1 activity.
No full text<p>(A) Images show GFP fluorescence in control- and siRNA sensor-transgenic <i>C. elegans</i> treated with vector or <i>henn-1</i> RNAi. (B) Protein blot assays for GFP from <i>C. elegans</i> containing control or siRNA sensor transgenes and treated with vector, <i>ergo-1</i>, or <i>henn-1</i> RNAi. Actin protein is shown as a loading control. One of three biological replicates is shown. (C) Images show GFP expression in control- and siRNA sensor-transgenic wild type and <i>henn-1</i> mutant <i>C. elegans</i>. Upper panel, GFP fluorescence in whole worms. Lower panel, antibody stained GFP in dissected germlines. (D) Protein blot assay for GFP in wild type or <i>henn-1</i> mutants containing control or siRNA sensor transgenes. Actin protein is shown as a loading control. One of three biological replicates is shown.</p
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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