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

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Singhania, R
Pavey, S
Payne, E
Gu, W
Clancy, J
Jubair, L
Preiss, T
Saunders, N
McMillan, NAJ
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2016
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Abstract

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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Biochimica et Biophysica Acta

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1859

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8

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© 2016 Elsevier B.V.. This is the author-manuscript version of this paper. Reproduced in accordance with the copyright policy of the publisher. Please refer to the journal's website for access to the definitive, published version.

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Biochemistry and cell biology not elsewhere classified

Genetics

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