• Call for Physical Sciences Papers
  • Sign-up for PNAS eTOC Alerts

RNA stem structure governs coupling of dicing and gene silencing in RNA interference

  1. Sua Myonga,c,d,1
  1. aDepartment of Biophysics, Johns Hopkins University, Baltimore, MD 21218;
  2. bDepartment of Chemistry, Chung-Ang University, Seoul 06974, Korea;
  3. cInstitute for Genomic Biology, University of Illinois, Urbana, IL 61801;
  4. dCenter for Physics of Living Cells, University of Illinois, Urbana, IL 61801
  1. Edited by Brenda L. Bass, University of Utah School of Medicine, Salt Lake City, UT, and approved October 13, 2017 (received for review June 8, 2017)

Significance

RNAi is an RNA-induced gene-silencing pathway that is shared among various organisms. Better understanding of RNAi is urgently needed to improve our knowledge of RNA-mediated gene regulation and to advance the field of functional genomics and its application to gene therapy. We counted with high precision the number of transcripts in each cell’s nucleus and cytoplasm as a function of silencing time to investigate the role of small RNA secondary structures such as loop length and stem mismatches. We screened various structural features of small RNAs and discovered a distinct role of each structural element that contributes to gene-silencing kinetics. We provide a helpful guideline for designing small RNAs for more efficient gene silencing.

Abstract

PremicroRNAs (premiRNAs) possess secondary structures consisting of a loop and a stem with multiple mismatches. Despite the well-characterized RNAi pathway, how the structural features of premiRNA contribute to dicing and subsequent gene-silencing efficiency remains unclear. Using single-molecule FISH, we demonstrate that cytoplasmic mRNA, but not nuclear mRNA, is reduced during RNAi. The dicing rate and silencing efficiency both increase in a correlated manner as a function of the loop length. In contrast, mismatches in the stem drastically diminish the silencing efficiency without impacting the dicing rate. We show that this decoupling effect is not due to the loading to the RNA-induced silencing complex, RNA uptake, or cellular dicing. We postulate that the stem mismatches perturb the handover of the cleaved miRNAs from Dicer to Argonaute, leading to poor strand selection. Our results imply that the stem structures prevalent in cellular miRNAs have suboptimal silencing efficiency.

Footnotes

  • ?1To whom correspondence may be addressed. Email: hrankoh{at}gmail.com or smyong{at}jhu.edu.

This is an open access article distributed under the PNAS license.

Online Impact

                                      1. 99132880 2018-01-23
                                      2. 802899879 2018-01-23
                                      3. 295573878 2018-01-23
                                      4. 352668877 2018-01-23
                                      5. 984633876 2018-01-23
                                      6. 545928875 2018-01-23
                                      7. 976569874 2018-01-23
                                      8. 871324873 2018-01-23
                                      9. 263462872 2018-01-23
                                      10. 577161871 2018-01-23
                                      11. 255603870 2018-01-23
                                      12. 117346869 2018-01-23
                                      13. 90982868 2018-01-23
                                      14. 663415867 2018-01-23
                                      15. 793874866 2018-01-23
                                      16. 843582865 2018-01-23
                                      17. 864971864 2018-01-22
                                      18. 258841863 2018-01-22
                                      19. 957295862 2018-01-22
                                      20. 553518861 2018-01-22