Artificial mirtron-mediated gene knockdown: functional DMPK silencing in mammalian cells.
Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs. Here we present a design algorithm for artificial mirtrons and demonstrate, for the first time, efficient gene knockdown of myotonic dystrophy protein kinase (DMPK) target sequences in Renilla luciferase 3' UTR and subsequently pathogenic DMPK mRNA, causative of Type I myotonic dystrophy, using artificial mirtrons cloned as eGFP introns. Deep sequencing of artificial mirtrons suggests that functional mature transcripts corresponding to the designed sequence were produced in high abundance. They were further shown to be splicing-dependent, Drosha-independent, and partially dependent on exportin-5, resulting in the precise generation of pre-miRNAs. In a murine myoblast line containing a pathogenic copy of human DMPK with more than 500 CUG repeats, the DMPK artificial mirtron corrected DM1-associated splicing abnormalities of the Serca-1 mRNA, demonstrating the therapeutic potential of mirtron-mediated RNAi. Thus, further development and exploitation of the unique properties of mirtrons will benefit future research and therapeutic RNAi applications as an alternative to conventional RNAi strategies.
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom.
Yiqi
Y
Seow
Christopher R
CR
Sibley
Matthew J A
MJ
Wood
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D006801:Humans; D007438:Introns; D028884:Karyopherins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D032446:Myoblasts / Q000378:metabolism; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D043244:Ribonuclease III / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D014158:Transcription, Genetic
C585517:DMPK protein, human; C585516:DMPK protein, mouse; D028884:Karyopherins; C446511:XPO5 protein, human; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases; C517918:DROSHA protein, human; D043244:Ribonuclease III; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506943:ATP2A1 protein, human