[RNase III-prepared short interfering RNAs induce degradation of SARS-coronavirus mRNAs in human cells].
Identifieur interne : 004F22 ( Main/Exploration ); précédent : 004F21; suivant : 004F23[RNase III-prepared short interfering RNAs induce degradation of SARS-coronavirus mRNAs in human cells].
Auteurs : Xu-Dong Zhu [République populaire de Chine] ; Ying Dang ; Yi Feng ; Tao Li ; Pei-Tang HuangSource :
- Sheng wu gong cheng xue bao = Chinese journal of biotechnology [ 1000-3061 ] ; 2004.
Descripteurs français
- KwdFr :
- MESH :
- génétique : Petit ARN interférent, Virus du SRAS.
- métabolisme : ARN messager, ARN viral.
- physiologie : Ribonuclease III.
- Cellules cultivées, Humains, Interférence par ARN, Plasmides.
English descriptors
- KwdEn :
- MESH :
- chemical , genetics : RNA, Small Interfering.
- chemical , metabolism : RNA, Messenger, RNA, Viral.
- chemical , physiology : Ribonuclease III.
- genetics : SARS Virus.
- Cells, Cultured, Humans, Plasmids, RNA Interference.
Abstract
SARS-associated coronavirus has been identified for the cause of Severe Acute Respiratory Syndrome, for which there is no efficacious drugs or vaccines. RNA interference (RNAi) is a process in cell to degradation specific target mRNA by double-stranded RNA. In mammalian cells, RNAi can be triggered by short interfering RNA (siRNA). RNA interference of virus-specific genes has emerged as a potential antiviral mechanism. This work evaluated if RNase III-prepared short interfering RNAs can induce specific degradation of SARS-coronavirus mRNAs in human cells. Three of SARS genes, RNA dependent RNA polymerase (RdRp), spike and nucleocapsid, were amplified with T7 promoter-flanked primers. Long length double-stranded RNA of these genes were transcribed in vitro and then were cleaved to <30bp length short interfering RNA with E. coli RNase III. These siRNAs were termed esiRNA-R, esiRNA-S and esiRNA-N respectively. RdRp, spike and nucleocapsid DNA fragments were inserted into the plasmid pGL3-Control, obtained plasmids pGL-R, pGL-S and pGL-N can express hybrid mRNAs luciferase-RdRp, spike and -nucleocapsid in cells. Above plasmids and esiRNAs were co-transfected to HEK293F cells with reference plasmid pRL-TK. Firefly luciferase and Renilla luciferase activity were measured. Hybrid mRNAs' abundance was measured using reverse transcription real-time PCR. Firefly luciferase expression of pGL-R was reduced to 13% by esiRNA-R. Expression of pGLS was reduced to 11% by esiRNA-S. Expression of pGL-N was reduced to 40% by esiRNA-N. Control esiRNAs didn't affect luciferase expression; Hybrid mRNAs' abundance was dramatically reduced by corresponding esiRNAs. RNase III-prepared short interfering RNAs induce robust and specific degradation of SARS-coronavirus mRNAs in HEK293F cells. These siRNAs could be used to inhibit SARS-coronavirus in future research.
PubMed: 15968975
Affiliations:
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Le document en format XML
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<term>RNA, Small Interfering (genetics)</term>
<term>RNA, Viral (metabolism)</term>
<term>Ribonuclease III (physiology)</term>
<term>SARS Virus (genetics)</term>
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<term>Interférence par ARN</term>
<term>Petit ARN interférent (génétique)</term>
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<term>Ribonuclease III (physiologie)</term>
<term>Virus du SRAS (génétique)</term>
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<front><div type="abstract" xml:lang="en">SARS-associated coronavirus has been identified for the cause of Severe Acute Respiratory Syndrome, for which there is no efficacious drugs or vaccines. RNA interference (RNAi) is a process in cell to degradation specific target mRNA by double-stranded RNA. In mammalian cells, RNAi can be triggered by short interfering RNA (siRNA). RNA interference of virus-specific genes has emerged as a potential antiviral mechanism. This work evaluated if RNase III-prepared short interfering RNAs can induce specific degradation of SARS-coronavirus mRNAs in human cells. Three of SARS genes, RNA dependent RNA polymerase (RdRp), spike and nucleocapsid, were amplified with T7 promoter-flanked primers. Long length double-stranded RNA of these genes were transcribed in vitro and then were cleaved to <30bp length short interfering RNA with E. coli RNase III. These siRNAs were termed esiRNA-R, esiRNA-S and esiRNA-N respectively. RdRp, spike and nucleocapsid DNA fragments were inserted into the plasmid pGL3-Control, obtained plasmids pGL-R, pGL-S and pGL-N can express hybrid mRNAs luciferase-RdRp, spike and -nucleocapsid in cells. Above plasmids and esiRNAs were co-transfected to HEK293F cells with reference plasmid pRL-TK. Firefly luciferase and Renilla luciferase activity were measured. Hybrid mRNAs' abundance was measured using reverse transcription real-time PCR. Firefly luciferase expression of pGL-R was reduced to 13% by esiRNA-R. Expression of pGLS was reduced to 11% by esiRNA-S. Expression of pGL-N was reduced to 40% by esiRNA-N. Control esiRNAs didn't affect luciferase expression; Hybrid mRNAs' abundance was dramatically reduced by corresponding esiRNAs. RNase III-prepared short interfering RNAs induce robust and specific degradation of SARS-coronavirus mRNAs in HEK293F cells. These siRNAs could be used to inhibit SARS-coronavirus in future research.</div>
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