TY - JOUR
T1 - Single-molecule imaging of microRNA-mediated gene silencing in cells
AU - Kobayashi, Hotaka
AU - Singer, Robert H.
N1 - Funding Information:
We are grateful to Yukihide Tomari for providing pAWH-Rluc-let-7-A-N-HhR. We thank Xiuhua Meng and Melissa Lopez-Jones for technical assistance, Hanae Sato for sharing the protocols of smFISH and SINAPS, and Carolina Eliscovich for sharing the protocol for IF-FISH. We are grateful to Keisuke Shoji for supporting the analysis of small RNA-seq data. We thank David P. Bartel and all the members of the Robert H. Singer laboratory for insightful discussion and critical comments. The authors thank Timothy J. Stasevich for sharing preliminary data. This work was supported by NIH grants R01NS083085 and R35GM136296 (to R.H.S.), JSPS Overseas Research Fellowships (to H.K.), JBS Osamu Hayaishi Memorial Scholarship for Study Abroad (to H.K.), and JST PRESTO Grant JPMJPR20E7 (to H.K.). 114 40
Funding Information:
We are grateful to Yukihide Tomari for providing pAWH-Rluc-let-7-A114 -N40 -HhR. We thank Xiuhua Meng and Melissa Lopez-Jones for technical assistance, Hanae Sato for sharing the protocols of smFISH and SINAPS, and Carolina Eliscovich for sharing the protocol for IF-FISH. We are grateful to Keisuke Shoji for supporting the analysis of small RNA-seq data. We thank David P. Bartel and all the members of the Robert H. Singer laboratory for insightful discussion and critical comments. The authors thank Timothy J. Stasevich for sharing preliminary data. This work was supported by NIH grants R01NS083085 and R35GM136296 (to R.H.S.), JSPS Overseas Research Fellowships (to H.K.), JBS Osamu Hayaishi Memorial Scholarship for Study Abroad (to H.K.), and JST PRESTO Grant JPMJPR20E7 (to H.K.).
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - MicroRNAs (miRNAs) are small non-coding RNAs, which regulate the expression of thousands of genes; miRNAs silence gene expression from complementary mRNAs through translational repression and mRNA decay. For decades, the function of miRNAs has been studied primarily by ensemble methods, where a bulk collection of molecules is measured outside cells. Thus, the behavior of individual molecules during miRNA-mediated gene silencing, as well as their spatiotemporal regulation inside cells, remains mostly unknown. Here we report single-molecule methods to visualize each step of miRNA-mediated gene silencing in situ inside cells. Simultaneous visualization of single mRNAs, translation, and miRNA-binding revealed that miRNAs preferentially bind to translated mRNAs rather than untranslated mRNAs. Spatiotemporal analysis based on our methods uncovered that miRNAs bind to mRNAs immediately after nuclear export. Subsequently, miRNAs induced translational repression and mRNA decay within 30 and 60 min, respectively, after the binding to mRNAs. This methodology provides a framework for studying miRNA function at the single-molecule level with spatiotemporal information inside cells.
AB - MicroRNAs (miRNAs) are small non-coding RNAs, which regulate the expression of thousands of genes; miRNAs silence gene expression from complementary mRNAs through translational repression and mRNA decay. For decades, the function of miRNAs has been studied primarily by ensemble methods, where a bulk collection of molecules is measured outside cells. Thus, the behavior of individual molecules during miRNA-mediated gene silencing, as well as their spatiotemporal regulation inside cells, remains mostly unknown. Here we report single-molecule methods to visualize each step of miRNA-mediated gene silencing in situ inside cells. Simultaneous visualization of single mRNAs, translation, and miRNA-binding revealed that miRNAs preferentially bind to translated mRNAs rather than untranslated mRNAs. Spatiotemporal analysis based on our methods uncovered that miRNAs bind to mRNAs immediately after nuclear export. Subsequently, miRNAs induced translational repression and mRNA decay within 30 and 60 min, respectively, after the binding to mRNAs. This methodology provides a framework for studying miRNA function at the single-molecule level with spatiotemporal information inside cells.
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U2 - 10.1038/s41467-022-29046-5
DO - 10.1038/s41467-022-29046-5
M3 - Article
C2 - 35301300
AN - SCOPUS:85126642803
VL - 13
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
IS - 1
M1 - 1435
ER -