Secondary siRNAs result from unprimed RNA synthesis and form a distinct class
Authors: Karen L Thijssen
Titia Sijen
Ronald H A Plasterk
Florian A Steiner
Journal: Science (2007)
Discipline: Multidisciplinary
Keywords: RNA interference; Caenorhabditis elegans; secondary siRNAs; RNA-directed RNA polymerases; RISC
Affiliations: Hubrecht Laboratory (NIOB-KNAW), Uppsalalaan 8, 3584 CT, the Netherlands.

Abstract:  In Caenorhabditis elegans, an effective RNA interference (RNAi) response requires the production of secondary short interfering RNAs (siRNAs) by RNA-directed RNA polymerases (RdRPs). We cloned secondary siRNAs from transgenic C. elegans lines expressing a single 22-nucleotide primary siRNA. Several secondary siRNAs start a few nucleotides downstream of the primary siRNA, indicating that non-RISC (RNA-induced silencing complex)-cleaved mRNAs are substrates for secondary siRNA production. In lines expressing primary siRNAs with single-nucleotide mismatches, secondary siRNAs do not carry the mismatch but contain the nucleotide complementary to the mRNA. We infer that RdRPs perform unprimed RNA synthesis. Secondary siRNAs are only of antisense polarity, carry 5' di- or triphosphates, and are only in the minority associated with RDE-1, the RNAi-specific Argonaute protein. Therefore, secondary siRNAs represent a distinct class of small RNAs. Their biogenesis depends on RdRPs, and we propose that each secondary siRNA is an individual RdRP product. 

Retracted: Yes

PubPeer comments:
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 Reposting two figures that were eaten by the PubPeer upgrade. Supplemental Figure S1C. * Blue boxes. In the right most panel,  22si-mm-as , the first and third lanes look very similar. There appears to be a sharp vertical background transition, suggestive of a splicing, between the first and second lane. See illustration here: ![file] , 
 Supplemental Figure S3C * Blue, green, and orange boxes: Several parts of both blots appear to be visible multiple times. ![file] , 
 Maikel Peppelenbosch Figure 2D : It seems that sec I and sec I, II, III, IV = VII represent the aggregate electronic signal of various exposure times? Sec I is shown to show that the blot is not overloaded while the right panel shows the biological panel shows the biological effect , 
 Maikel Peppelenbosch: Figure 2D : It seems that sec I and sec I, II, III, IV + VII represent the aggregate electronic signal of various exposure times? Sec I is shown to show that the blot is not overloaded while the right panel shows the biological effect (that secondary siRNA have an absence of a 5u2032-monophosphate? In that case nothing wrong with that figure? , 
 Maikel Peppelenbosch So I mean to say, nothing wrong with figure 2D? It just shows that 5u2032-monophosphate; the 5u2032linker successfully ligated to miR-58 and 22siRNA but failed to ligate to secondary siRNAs and uses two (electronically added) exposure times to show this? I do not see the problem. , 
 RE #7 : * It seems that sec I and sec I, II, III, IV = VII represent the aggregate electronic signal of various exposure times * RE #9 : * ...and uses two (electronically added) exposure times to show this * What makes you think that two different exposure times are being shown? I see no evidence to suggest this. If you believe they are, please explain your reasoning. What is unclear to me is whether the the sec I and sec I,II,III,IV,+VIII panels show the products of separate reactions (using distinct pools of secondary siRNAs as substrates), or whether they are the same set of reaction products that have been detected with different probes. Maybe someone else can clarify... , 
 If the two panels in Figure 2D would be two different exposures or incubations with two different probes of the same blot, then the two marker lanes ( M ) are expected to look similar. However, the two M lanes look very different from each other. The shapes and widths of the M bands look different between the two panels, suggesting that these are two different blots, rather than different probes on the same blot. Here is Figure 2D again, shown in false color to highlight similarities between the two panels. Green arrows have been added to point out some possible sharp horizontal transitions in the right two lanes of the right panel. ![file] Of course, sometimes labeling of figures is not immediately clear so it can be hard for others to understand if images actually represent the same experiments, and if they are supposed to look similar or not. A quick explanation by the authors often can resolve such concerns. It would be very helpful if the authors could take away any concerns by explaining why parts of these blots look so similar. , 
 #11 RE: * It would be very helpful if the authors could take away any concerns... * Agreed. Unfortunately, however, authors might not receive e-mail alerts. The senior last author is no longer in science (though appears to be a tweeter), and a 2019 paper by the first author (PMID: 31212207) suggests a different e-mail address host (@nfi.nl) than the one entered on this page. , 
 Retracted [20 November 2020] .  The authors of the Report u201cSecondary siRNAs result from unprimed RNA synthesis and form a distinct classu201d (1) have been made aware of duplications in Fig. 2D and supplementary figures S1C and S3C. We attempted to find the original data from 2007, but all authors have left the Hubrecht Institute, where the work was performed, and the data could not be located. Although other data in the paper were consistent with the conclusions supported by Fig. 2D and figs. S1C and S3C, all data were taken into account in the review of the paper, and the loss of these figures weakens the conclusions. Therefore, in the spirit of full transparency, we have decided to retract the paper. ,