Detection of siRNA induced mRNA silencing by RT-qPCR: considerations for experimental design

Holmes, Katherine; Williams, Catrin M; Chapman, Elinor A; Cross, Michael

doi:10.1186/1756-0500-3-53

Cited by 44 publications

(41 citation statements)

References 12 publications

(12 reference statements)

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“…S5). Similar variable qPCR results were previously reported for silenced genes and could be caused by incomplete degradation of mRNA fragments (Shepard et al, 2005;Holmes et al, 2010). Expression analysis in subsequent generations was further complicated by the limited survival and fertility of these seedlings, and by the reversion of the surviving lines to the characteristic DEX-dependent overexpression phenotypes in the subsequent generation.…”

Section: Cosuppression Of Hdg Expression Results In Overproliferationsupporting

confidence: 63%

AIL and HDG proteins act antagonistically to control cell proliferation

et al. 2015

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AINTEGUMENTA-LIKE (AIL) transcription factors are key regulators of cell proliferation and meristem identity. Although AIL functions have been well described, the direct signalling components of this pathway are largely unknown. We show that BABY BOOM (BBM) and other AIL proteins physically interact with multiple members of the L1-expressed HOMEODOMAIN GLABROUS (HDG) transcription factor family, including HDG1, HDG11 and HDG12. Overexpression of HDG1, HDG11 and HDG12 restricts growth due to root and shoot meristem arrest, which is associated with reduced expression of genes involved in meristem development and cell proliferation pathways, whereas downregulation of multiple HDG genes promotes cell overproliferation. These results suggest a role for HDG proteins in promoting cell differentiation. We also reveal a transcriptional network in which BBM and HDG1 regulate several common target genes, and where BBM/AIL and HDG regulate the expression of each other. Taken together, these results suggest opposite roles for AIL and HDG proteins, with AILs promoting cell proliferation and HDGs stimulating cell differentiation, and that these functions are mediated at both the protein-protein interaction and transcriptional level.

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Section: Cosuppression Of Hdg Expression Results In Overproliferationsupporting

confidence: 63%

AIL and HDG proteins act antagonistically to control cell proliferation

et al. 2015

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“…In accordance with our expectations, we found that the injection of hsp83 dsRNA into A. pisum reduced the lifespan, fecundity, and number of viviparous offspring, even though the attenuation of HSP83 expression was not significant. The confirmation of gene knockdown in RNAi experiments is sometimes difficult, particularly if the target gene is expressed at a low level, because it depends on the selected reference genes (Holmes et al 2010; Baumann et al 2015). …”

Section: Discussionmentioning

confidence: 99%

Heat shock protein 83 plays pleiotropic roles in embryogenesis, longevity, and fecundity of the pea aphid Acyrthosiphon pisum

et al. 2016

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Heat shock protein 83 (HSP83) is homologous to the chaperone HSP90. It has pleiotropic functions in Drosophila melanogaster, including the control of longevity and fecundity, and facilitates morphological evolution by buffering cryptic deleterious mutations in wild populations. In the pea aphid Acyrthosiphon pisum, HSP83 expression is moderately induced by bacterial infection but upregulated more strongly in response to heat stress and fungal infection. Stress-inducible heat shock proteins are of considerable evolutionary and ecological importance because they are known to buffer environmental variation and to influence fitness under non-optimal conditions. To investigate the functions of HSP83 in viviparous aphids, we used RNA interference to attenuate its expression and studied the impact on complex parameters. The RNA interference (RNAi)-mediated depletion of HSP83 expression in A. pisum reduced both longevity and fecundity, suggesting this chaperone has an evolutionarily conserved function in insects. Surprisingly, HSP83 depletion reduced the number of viviparous offspring while simultaneously increasing the number of premature nymphs developing in the ovaries, suggesting an unexpected role in aphid embryogenesis and eclosion. The present study indicates that reduced HSP83 expression in A. pisum reveals both functional similarities and differences compared with its reported roles in holometabolous insects. Its impact on aphid lifespan, fecundity, and embryogenesis suggests a function that determines their fitness. This could be achieved by targeting different client proteins, recruiting distinct co-chaperones or transposon activation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00427-016-0564-1) contains supplementary material, which is available to authorized users.

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“…siRNA cleavage fragments persist after cleavage, although it was observed that this does not occur for all mRNAs for reasons not yet clear (18,20).…”

Section: Discussionmentioning

confidence: 86%

“…In mammalian cells mRNA degradation has been observed to be neither rapid nor complete with siRNA-mediated cleavage reported to result in the generation of partially stable 5' or 3' mRNA fragments (17)(18)(19)(20). Indeed, we have previously shown that truncated proteins can be translated from the 5' end of shRNA-targeted mRNA (21).…”

Section: Introductionmentioning

confidence: 95%

Short interfering RNA induced generation and translation of stable 5′ mRNA cleavage intermediates

Singhania

Pavey

Payne

et al. 2016

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Sequence-specific degradation of homologous mRNA is the main mechanism by which short-interfering RNAs (siRNAs) suppress gene expression. Generally, it is assumed that the mRNA fragments resulting from Ago2 cleavage are rapidly degraded, thus making the transcript translation-incompetent. However, the molecular mechanisms involved in the post-cleavage mRNA decay are not completely understood and the fate of cleavage intermediates has been poorly studied. Using specific siRNAs and short-hairpin RNAs (shRNAs) we show that the 5' and 3' mRNA cleavage fragments of human papilloma virus type 16 (HPV-16) E6/7 mRNA, over-expressed in cervical malignancies, are unevenly degraded. Intriguingly, the 5' mRNA fragment was more abundant and displayed a greater stability than the corresponding 3' mRNA fragment in RNAi-treated cells. Further analysis revealed that the 5' mRNA fragment was polysome-associated, indicating its active translation, and this was further confirmed by using tagged E7 protein to show that C-terminally truncated proteins were produced in treated cells. Overall, our findings provide new insight into the degradation of siRNA-targeted transcripts and show that RNAi can alter protein expression in cells as a result of preferential stabilization and translation of the 5' cleavage fragment. These results challenge the current model of siRNA-mediated RNAi and provide a significant step forward towards understanding non-canonical pathways of siRNA gene silencing.

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Detection of siRNA induced mRNA silencing by RT-qPCR: considerations for experimental design

Cited by 44 publications

References 12 publications

AIL and HDG proteins act antagonistically to control cell proliferation

AIL and HDG proteins act antagonistically to control cell proliferation

Heat shock protein 83 plays pleiotropic roles in embryogenesis, longevity, and fecundity of the pea aphid Acyrthosiphon pisum

Short interfering RNA induced generation and translation of stable 5′ mRNA cleavage intermediates

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