Right- and left-loop short shRNAs have distinct and unusual mechanisms of gene silencing.
Right- and left-loop short shRNAs have distinct and unusual mechanisms of gene silencing.
复制标题
右环和左环短 shRNA 具有独特且不寻常的基因沉默机制。
DOI：
10.1093/nar/gks662
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发表时间：
2012-10
影响因子：
14.9
通讯作者：
Johnston BH
中科院分区：
文献类型：
--
作者：
Dallas A;Ilves H;Ge Q;Kumar P;Shorenstein J;Kazakov SA;Cuellar TL;McManus MT;Behlke MA;Johnston BH
Small hairpin RNAs (shRNAs) having duplex lengths of 25–29 bp are normally processed by Dicer into short interfering RNAs (siRNAs) before incorporation into the RNA-induced silencing complex (RISC). However, shRNAs of ≤19 bp [short shRNAs (sshRNAs)] are too short for Dicer to excise their loops, raising questions about their mechanism of action. sshRNAs are designated as L-type or R-type according to whether the loop is positioned 3′ or 5′ to the guide sequence, respectively. Using nucleotide modifications that inhibit RNA cleavage, we show that R- but not L-sshRNAs require loop cleavage for optimum activity. Passenger-arm slicing was found to be important for optimal functioning of L-sshRNAs but much less important for R-sshRNAs that have a cleavable loop. R-sshRNAs could be immunoprecipitated by antibodies to Argonaute-1 (Ago1); complexes with Ago1 contained both intact and loop-cleaved sshRNAs. In contrast, L-sshRNAs were immunoprecipitated with either Ago1 or Ago2 and were predominantly sliced in the passenger arm of the hairpin. However, ‘pre-sliced’ L-sshRNAs were inactive. We conclude that active L-sshRNAs depend on slicing of the passenger arm to facilitate opening of the duplex, whereas R-sshRNAs primarily act via loop cleavage to generate a 5′-phosphate at the 5′-end of the guide strand.
