Munc18 and Munc13 regulate early neurite outgrowth

Background information. During development, growth cones of outgrowing neurons express proteins involved in vesicular secretion, such as SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins, Munc13 and Munc18. Vesicles are known to fuse in growth cones prior to synapse formation, which may contribute to outgrowth

Hen1 is required for oocyte development and piRNA stability in zebrafish

Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defence and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. piRNAs carry a 2′-O-methyl modification at their 3′ end, which is added by the Hen1 enzyme. We show that zebrafish hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line, whereas it is dispensable in the testis. Hen1 protein localizes to nuage through its C-terminal domain, but is not required for nuage formation. In hen1 mutant testes, piRNAs become uridylated and adenylated. Uridylation frequency is highest on retro-transposon-derived piRNAs and is accompanied by decreased piRNA levels and mild derepression of transposon transcripts. Altogether, our data suggest the existence of a uridylation-mediated 3′–5′ exonuclease activity acting on piRNAs in zebrafish germ cells, which is counteracted by nuage-bound Hen1 protein. This system discriminates between piRNA targets and is required for ovary development and fully efficient transposon silencing

Hen1 is required for oocyte development and piRNA stability in zebrafish

Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defence and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. piRNAs carry a 2′-O-methyl modification at their 3′ end, which is added by the Hen1 enzyme. We show that zebrafish hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line, whereas it is dispensable in the testis. Hen1 protein localizes to nuage through its C-terminal domain, but is not required for nuage formation. In hen1 mutant testes, piRNAs become uridylated and adenylated. Uridylation frequency is highest on retro-transposon-derived piRNAs and is accompanied by decreased piRNA levels and mild derepression of transposon transcripts. Altogether, our data suggest the existence of a uridylation-mediated 3′–5′ exonuclease activity acting on piRNAs in zebrafish germ cells, which is counteracted by nuage-bound Hen1 protein. This system discriminates between piRNA targets and is required for ovary development and fully efficient transposon silencing

PID-1 is a novel factor that operates during 21U-RNA biogenesis in Caenorhabditis elegans

The Piwi-piRNA pathway represents a small RNA-based mechanism responsible for the recognition and silencing of invading DNA. Biogenesis of piRNAs (21U-RNAs) is poorly understood. In Caenorhabditis elegans, the piRNA-binding Argonaute protein PRG-1 is the only known player acting downstream from precursor transcription. From a screen aimed at the isolation of piRNA-induced silencing-defective (Pid) mutations, we identified, among known Piwi pathway components, PID-1 as a novel player. PID-1 is a mostly cytoplasmic, germline-specific factor essential for 21U-RNA biogenesis, affecting an early step in the processing or transport of 21U precursor transcripts. We also show that maternal 21U-RNAs are essential to initiate silencing

HENN-1 is the <i>C. elegans</i> homolog of Hen1.

<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

<i>henn-1</i> affects 21UR1 sensor activity.

<p>(A) Activity of a transgene expressing GFP (21UR1 sensor), silenced by 21U species 21UR1 in wild-type, <i>prg-1(pk2298)</i> and <i>henn-1(pk2295)</i> mutant backgrounds. The gonads are outlined with a white dashed line. (B) Northern blot analysis of 21UR1 in young adult animals of the indicated genotypes. “Sensor” refers to the 21UR1 sensor also shown in panel A. Signal intensities are related to <i>let-7</i>, and the 21UR1:<i>let-7</i> ratio in N2 is set at one. The 21-mer and 20-mer signals of the 21UR1 probe have also been quantified separately. The signal intensity of the 20-mer relative to the total 21UR1 signal is presented. Repetition of this blot with two independent biological samples has shown that the apparent differences are not reproducible. The 22G-sensor blot shows the signals obtained after hybridizing probes homologous to the 21UR1 sensor.</p