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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)

  1. Fig. 2.

    RNAi reduces cytoplasmic mRNA without changing nuclear mRNA. (A) Visualization of nuclear mRNA by using dual-color smFISH including green-labeled probes designed against the exon and the red probes against the intron. Yellow spots are produced by the overlap of green and red represent active transcription sites, whereas green spots are nuclear mRNA. (B) Representative dual-color smFISH image displaying the exon and intron of lamin A mRNA taken at 0, 3, and 6 h after iRNA treatment. (C) The histogram shows nuclear (blue bars) and cytoplasmic (white bars) mRNAs as a function of silencing time. (D) Count of nuclear (blue) vs. cytoplasmic mRNA (black) per cell. (E) The number of nuclear mRNA per cell plotted against number of cytoplasmic mRNA per cell for 1, 2, 3, and 6 h postsilencing.

  2. Fig. 3.

    Dicing kinetics is correlated with gene-silencing efficiency. (A) Schematic of the RNAi pathway, which involves dicing, Dicer-to-Ago handover, strand selection, target searching, and mRNA silencing. (B) Dicing results on loop length variants of U1, U5, U15, U27, and U27 with TT overhang. (C) Quantification of dicing rate. (D) mRNA histogram of loop length variants after 4 h of silencing. (E) Silencing efficiency plotted against dicing rate for loop length variants. The peak size (blue symbols) is similar for all RNAs, but the peak shift (black symbols) and shift × size (red symbols) show a linear correlation between the dicing rate and silencing efficiency. See Fig. 1 E and F for fitting parameters.

  3. Fig. 4.

    Stem mismatch breaks the correlation between dicing and silencing. (A) Schematic of the RNAi pathway. Small RNAs with varying numbers of stem mismatches are applied to dicing and silencing measurement. (B) Dicing result of U27 with no mismatch and U27_4M with four mismatches in the stem. (C) Quantification of dicing kinetics. (D) mRNA histogram of silencing induced by different stem-mismatched RNA substrates. (E) Analysis of the histogram in D for the four stem-mismatch variants. (F) Silencing efficiency plotted against dicing rate for stem-mismatch variants.

  4. Fig. 5.

    Stem mismatch does not impact downstream of AGO. (A) Schematic of the RNAi pathway. A Dicer product bearing the same mismatch variation as the Dicer substrate is applied to the silencing assay. (B) mRNA histogram for Dicer product with varying mismatches. (C) Silencing efficiency induced by the Dicer product with the stem mismatches. (D) Silencing efficiency of the Dicer product with varying mismatches plotted against the silencing efficiency of the Dicer substrate with the same mismatches.

  5. Fig. 6.

    Summary: Mismatches in the stem of dsRNA do not interfere with dicing but drastically diminish silencing efficiency. Our result reveals that this is likely due to perturbed Dicer-to-Ago handover in which mismatches in the stem disrupt the A-form helical structure of dsRNA, making it difficult to fit into Ago pocket.

  6. Fig. 7.

    Silencing efficiency of structured RNAs. RNA substrates tested in this study are ordered from the lowest to highest efficiency of gene silencing from left to right. (Upper) Dicer substrate. (Lower) Dicer product. Each panel is subdivided into structural variants.

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