Right- and left-loop short shRNAs have distinct and unusual mechanisms of gene silencing.
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.
SomaGenics, Inc., 2161 Delaware Avenue, Santa Cruz, CA 95060, USA.
Anne
A
Dallas
Heini
H
Ilves
Qing
Q
Ge
Pavan
P
Kumar
Joshua
J
Shorenstein
Sergei A
SA
Kazakov
Trinna L
TL
Cuellar
Michael T
MT
McManus
Mark A
MA
Behlke
Brian H
BH
Johnston
D060565:Argonaute Proteins / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000737:chemistry / Q000378:metabolism; D059367:RNA Cleavage; D034622:RNA Interference; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D035683:MicroRNAs; D012322:RNA Precursors; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D043244:Ribonuclease III