Short interfering RNA induced generation and translation of stable 5' mRNA cleavage intermediates.
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.
The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia.
Richa
R
Singhania
The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia.
Sandra
S
Pavey
The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia.
Elizabeth
E
Payne
The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia.
Wenyi
W
Gu
The John Curtin School of Medical Research, The Australian National University, Canberra, Australia.
Jennifer
J
Clancy
Menzies Health Institute Queensland and School of Medical Science, Griffith University, Southport, Australia.
Luqman
L
Jubair
The John Curtin School of Medical Research, The Australian National University, Canberra, Australia.
Thomas
T
Preiss
The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia.
Nicholas
N
Saunders
The University of Queensland Diamantina Institute, University of Queensland, Brisbane, Australia; Menzies Health Institute Queensland and School of Medical Science, Griffith University, Southport, Australia. Electronic address: n.mcmillan@griffith.edu.au.
Nigel A J
NA
McMillan
D000818:Animals; D045744:Cell Line, Tumor; D002584:Cervix Uteri / Q000378:metabolism / Q000473:pathology / Q000821:virology; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D005260:Female; D020868:Gene Silencing; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D052162:Human papillomavirus 16 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D050725:Papillomavirus E7 Proteins / Q000235:genetics / Q000378:metabolism*; D011132:Polyribosomes / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D020871:RNA Stability; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism
D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C059731:oncogene protein E7, Human papillomavirus type 16
RNAi; mRNA cleavage; mRNA decay; shRNA; siRNA