Short versus long double-stranded RNA activation of a post-transcriptional gene knockdown pathway

<p>RNA interference (RNAi) utilizes a conserved cellular autoimmune defense mechanism involving the internalization of dsRNA into cells and the activation of a set of RNAi related genes. Using RNAi, complete sex reversal is achievable in males of the prawn <i>Macrobrachium rosenbergii</i> by knocking down the transcript level of an insulin-like androgenic gland hormone (<i>Mr-IAG</i>) through injections of dsRNA of the entire <i>Mr-IAG</i> ORF sequence (ds<i>Mr-IAG</i> – 518bp). Interestingly, <i>in-vivo</i> knockdown success and ds<i>Mr-IAG</i> lengths seemed to correlate, with long dsRNA being the most effective and short dsRNA fragments showing no effect. However, little is known about the RNAi machinery in <i>M. rosenbergii</i>. We discovered the <i>Mr-Dicer</i> and <i>Mr-Argonaute</i> gene families, associated with the major knockdown pathways, in our <i>M. rosenbergii</i> transcriptomic library. In response to ds<i>Mr-IAG</i> administration, only post-transcriptional pathway-related gene transcript levels were upregulated. In addition, a passive dsRNA channel (a <i>SID1</i> gene ortholog) that allows external dsRNA to enter cells was found. Its function was validated by observing <i>Mr-SID1</i> specific upregulation dependent on dsRNA lengths, while attempted loss-of-function experiments were lethal. Our results, which suggest differential systemic responses to dsRNA lengths, provide evidence that the above RNAi-based manipulation occurs via the post-transcriptional pathway. The temporal nature of the latter pathway supports the safety of using such RNAi-based biotechnologies in aquaculture and environmental applications. Unlike reports of RNAi driven by the administration of small dsRNA fragments <i>in-vitro</i>, the case presented here demonstrates length dependency <i>in-vivo</i>, suggesting further complexity in the context of the entire organism.</p

Development of a new molar tooth during an induced molt cycle in an ecdysone-injected male <i>C</i>. <i>quadricarinatus</i> and spatial and temporal expression patterns of the <i>Cq-M13</i> transcript <i>in vitro</i> and <i>in silico</i>.

<p><b>A</b>. Changes in molt mineralization index (MMI) during the induced molt cycle following repetitive injections of ecdysone. The x axis is normalized to days from ecdysis. A dashed line on an isolated mandible represents a cut through which a transverse plane is visible (A-top). <b>B</b>. Visualization of the new molar tooth assembly process in the crayfish mandible on days -9, -8, -7, -4, -3 and 0, following ecdysone injection by <i>ex vivo</i> micro-computed tomography. The top and bottom series of images show a view of the transverse and posterior planes of the mandibles correspondingly. White arrows point to the newly formed molar tooth. <b>C</b>. <i>In silico</i> transcriptomic analysis of <i>Cq-M13</i> read counts from four different molt stages. Different letters above columns represent statistically significant differences (p < 0.05 ± SE). <b>D</b>. Agarose gels showing RT-PCR products demonstrating spatial and temporal expression patterns of the <i>Cq-M13</i> transcript in cuticle-, molar-, basal segment (B.S.)-, maxillae- and gastrolith-forming tissues, as well as in hepatopancreas (Hepato) and testis.</p

A Novel Chitin Binding Crayfish Molar Tooth Protein with Elasticity Properties

<div><p>The molar tooth of the crayfish <i>Cherax quadricarinatus</i> is part of the mandible, and is covered by a layer of apatite (calcium phosphate). This tooth sheds and is regenerated during each molting cycle together with the rest of the exoskeleton. We discovered that molar calcification occurs at the pre-molt stage, unlike calcification of the rest of the new exoskeleton. We further identified a novel molar protein from <i>C</i>. <i>quadricarinatus</i> and cloned its transcript from the molar-forming epithelium. We termed this protein Cq-M13. The temporal level of transcription of <i>Cq-M13</i> in an NGS library of molar-forming epithelium at different molt stages coincides with the assembly and mineralization pattern of the molar tooth. The predicted protein was found to be related to the pro-resilin family of cuticular proteins. Functionally, <i>in vivo</i> silencing of the transcript caused molt cycle delay and a recombinant version of the protein was found to bind chitin and exhibited elastic properties.</p></div

Chitin-binding ability of rCq-M13.

<p>Following incubation with chitin powder, equal amounts of Negative control—BSA (top), Positive control—GAP 65 (middle, using gastrolith protein extract with known chitin binding abilities [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127871#pone.0127871.ref002" target="_blank">2</a>], and rCq-M13 (bottom) underwent three consecutive washes prior to SDS-PAGE. Lane 1 represent the first wash with DDW, lane 2 represent the second wash with NaCl (0.2 M) and lane 3 represent the third wash with denaturation buffer (DB, containing SDS and 2-mercaptoethanol).</p

<i>Cq-M13</i> silencing effects.

<p><b>A</b>. Levels of <i>Cq-M13</i> transcripts following <i>in vivo</i> dsRNA injections in male crayfish, as assessed by real-time RT-PCR following short-term silencing. Animals were injected with ecdysone and either ds<i>Cq-M13</i> or ds<i>CqVg</i>. Different letters represent significant differences and error bars represent standard error (p < 0.05 ± SE). <b>B</b>. Molt cycle progress elongation of <i>Cq-M13</i>-silenced <i>C</i>. <i>quadricarinatus</i> males. The experimental group was injected with <i>Cq-M13</i> dsRNA and the control group was injected with <i>CqVg</i> dsRNA. Both groups were injected with ecdysone to induce their molt cycle. Asterisk represents a significant difference (p < 0.05± SE).</p