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Up-Regulation of microRNA* Strands by Their Target Transcripts

Kang, Sung-Min, Choi, Ji-Woong, Hong, Su-Hyung, Lee, Heon-Jin·2013·International Journal of Molecular Sciences
DOI 10.3390/ijms140713231PMC PMC3742184
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Abstract

During microRNA (miRNA) biogenesis, one strand of a 21–23 nucleotide RNA duplex is preferentially selected for entry into an RNA-induced silencing complex (RISC). The other strand, known as the miRNA* species, is typically thought to be degraded. Previous studies have provided miRNA* selection models, but it remains unclear how the dominance of one arm arises during the biogenesis of miRNA. Using miRNA sponge-like methods, we cloned four tandem target sequences (artificial target) of miR-7b* and then measured miR-7b* expression levels after transfection of the artificial target. miR-7b* levels were found to significantly increase after transfection of the artificial target. We postulate that the abundance of target transcripts drives miRNA arm selection.

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Figure 1 The expression patterns of miR-7b and miR-7b*. (A) Basal levels of miR-7b and miR-7b* expression in AtT-20 cells were analyzed by qRT-PCR; (B) Design of miRNA artificial targets. Renilla (hRluc) containing miRNA artificial targets were constructed by inserting multiple miRNA binding sites into the 3′ UTR of a hRluc reporter gene driven by a T7 promoter. The figure shows the nucleotide sequence of the miRNA artificial targets: the red letters are a mutated sequence; (C) Luciferase assay using the miRNA artificial target reporter constructs. COS-7 cells were co-transfected with the miR-7b* mimic oligonucleiotide and miRNA artificial target (1. miR-7b, 2. miR-7b*, 3. miR-7b* mutation I, 4. miR-7b* mutation II, 5. miR-338-3p). Renilla luciferase activity was normalized against firefly luciferase activity and fold changes are compared to miR-338-3p. The results shown are the means of three independent transfections (error bars indicate standard deviations).
Figure 1 The expression patterns of miR-7b and miR-7b*. (A) Basal levels of miR-7b and miR-7b* expression in AtT-20 cells were analyzed by qRT-PCR; (B) Design of miRNA artificial targets. Renilla (hRluc) containing miRNA artificial targets were constructed by inserting multiple miRNA binding sites into the 3′ UTR of a hRluc reporter gene driven by a T7 promoter. The figure shows the nucleotide sequence of the miRNA artificial targets: the red letters are a mutated sequence; (C) Luciferase assay using the miRNA artificial target reporter constructs. COS-7 cells were co-transfected with the miR-7b* mimic oligonucleiotide and miRNA artificial target (1. miR-7b, 2. miR-7b*, 3. miR-7b* mutation I, 4. miR-7b* mutation II, 5. miR-338-3p). Renilla luciferase activity was normalized against firefly luciferase activity and fold changes are compared to miR-338-3p. The results shown are the means of three independent transfections (error bars indicate standard deviations).CC BY · PubMed Central
Figure 2 Changed expression levels of miR-7b and miR-7b* after transfection with multi-transcript artificial targets. (A) 24 h after transfection of miRNA artificial targets (1 and 1′. null, 2 and 2′. empty vector, 3 and 3′. miR-7b*, 4 and 4′. miR-7b* mutation I, 5 and 5′. miR-7b* mutation II), AtT-20 total RNA was extracted and analyzed by northern blotting using probes specific for miR-7b and miR-7b*, respectively. Lane 3′ shows that miR-7b* expression was significantly increased following transfection of the miR-7b* artificial target. In contrast, miR-7b expression was similar in non-transfected and miR-7b* artificial targets transfected samples; (B) Twenty-four hours after various artificial targets (1 and 1′. null, 2 and 2′. empty vector, 3 and 3′. miR-7b, 4 and 4′. miR-7b*, 5 and 5′. miR-7b* mutation I, 6 and 6′. miR-7b* mutation II) were transfected, miR-7b (left) and miR-7b* (right) expression levels were analyzed by qRT-PCR. miRNA expression levels were normalize to U6. Fold changes are expressed versus empty target of each (2 or 2′); (C) Twenty-four hours after various artificial targets (1 and 1′. empty vector, 2 and 2′. miR-7b*, 3 and 3′. miR-7b* mutation I, 4 and 4′. miR-7b* mutation II) were transfected, AtT-20 lysates were immunoprecipitated (IP) using anti-Ago2 antibody. IPs were analyzed by immunoblotting with the same anti-Ago2 antibody (upper panel). Co-immunoprecipitated RNA was extracted and analyzed by qRT-PCR (lower panel). Fold changes are expressed versus the empty target for each (1 or 1′). Error bars in graphs indicate standard deviations; p-values are also indicated.
Figure 2 Changed expression levels of miR-7b and miR-7b* after transfection with multi-transcript artificial targets. (A) 24 h after transfection of miRNA artificial targets (1 and 1′. null, 2 and 2′. empty vector, 3 and 3′. miR-7b*, 4 and 4′. miR-7b* mutation I, 5 and 5′. miR-7b* mutation II), AtT-20 total RNA was extracted and analyzed by northern blotting using probes specific for miR-7b and miR-7b*, respectively. Lane 3′ shows that miR-7b* expression was significantly increased following transfection of the miR-7b* artificial target. In contrast, miR-7b expression was similar in non-transfected and miR-7b* artificial targets transfected samples; (B) Twenty-four hours after various artificial targets (1 and 1′. null, 2 and 2′. empty vector, 3 and 3′. miR-7b, 4 and 4′. miR-7b*, 5 and 5′. miR-7b* mutation I, 6 and 6′. miR-7b* mutation II) were transfected, miR-7b (left) and miR-7b* (right) expression levels were analyzed by qRT-PCR. miRNA expression levels were normalize to U6. Fold changes are expressed versus empty target of each (2 or 2′); (C) Twenty-four hours after various artificial targets (1 and 1′. empty vector, 2 and 2′. miR-7b*, 3 and 3′. miR-7b* mutation I, 4 and 4′. miR-7b* mutation II) were transfected, AtT-20 lysates were immunoprecipitated (IP) using anti-Ago2 antibody. IPs were analyzed by immunoblotting with the same anti-Ago2 antibody (upper panel). Co-immunoprecipitated RNA was extracted and analyzed by qRT-PCR (lower panel). Fold changes are expressed versus the empty target for each (1 or 1′). Error bars in graphs indicate standard deviations; p-values are also indicated.CC BY · PubMed Central
Figure 3 miR-338-3p and miR-338-5p expressions. (A) miR-338-3p and miR-338-5p expression were analyzed by qRT-PCR of the total RNA extracted from AtT-20 cells. miR-338-3p levels were about twice as high as miR-338-5p levels in AtT-20 cells; (B) Schematic representation of miRNA artificial targets. Construct map of the miR-338-3p and miR-338-5p artificial targets with miR-338-3p and miR-338-5p artificial target sequences below; (C) Both miR-338-3p and miR-338-5p expression was up-regulated by each of artificial targets. However, the change of miR-338-3p was not as great as miR-338-5p (miR-338*) by their artificial target. The expression levels of miR-338-5p were significantly elevated by miR-338-5p artificial target transfection but not by transfection of the miR-338-3p artificial target or empty vector (1 and 1′. null, 2 and 2′. empty vector, 3 and 3′. miR-338-3p, 4 and 4′. miR-338-5p). Fold changes are expressed versus the empty target for each (2 or 2′). Error bars in graphs indicate standard deviations; p-values are also indicated.
Figure 3 miR-338-3p and miR-338-5p expressions. (A) miR-338-3p and miR-338-5p expression were analyzed by qRT-PCR of the total RNA extracted from AtT-20 cells. miR-338-3p levels were about twice as high as miR-338-5p levels in AtT-20 cells; (B) Schematic representation of miRNA artificial targets. Construct map of the miR-338-3p and miR-338-5p artificial targets with miR-338-3p and miR-338-5p artificial target sequences below; (C) Both miR-338-3p and miR-338-5p expression was up-regulated by each of artificial targets. However, the change of miR-338-3p was not as great as miR-338-5p (miR-338*) by their artificial target. The expression levels of miR-338-5p were significantly elevated by miR-338-5p artificial target transfection but not by transfection of the miR-338-3p artificial target or empty vector (1 and 1′. null, 2 and 2′. empty vector, 3 and 3′. miR-338-3p, 4 and 4′. miR-338-5p). Fold changes are expressed versus the empty target for each (2 or 2′). Error bars in graphs indicate standard deviations; p-values are also indicated.CC BY · PubMed Central
Figure 4 Schematic diagram of miRNA arm selection. Pri-miRNA is transcribed by RNA polymerase II and processed into pre-miRNA by Drosha/DGCR8 in the nucleus. Then pre-miRNA is processed into a duplex form of mRNA that is unwound during RISC assembly in the cytoplasm. Selection of the miRNA* sequence is determined by target transcript abundances. When miRNA* target transcripts are sufficiently abundant the miRNA* sequence is stabilized and acts on target transcripts in the same manner as miRNA.
Figure 4 Schematic diagram of miRNA arm selection. Pri-miRNA is transcribed by RNA polymerase II and processed into pre-miRNA by Drosha/DGCR8 in the nucleus. Then pre-miRNA is processed into a duplex form of mRNA that is unwound during RISC assembly in the cytoplasm. Selection of the miRNA* sequence is determined by target transcript abundances. When miRNA* target transcripts are sufficiently abundant the miRNA* sequence is stabilized and acts on target transcripts in the same manner as miRNA.CC BY · PubMed Central

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Research works printed but not linked: 26

  1. Bartel, 2004, MicroRNAs: Genomics, biogenesis, mechanism, and function, Cell, 116, 281, 10.1016/S0092-8674(04)00045-5 · DOI
  2. Kim, 2010, Modifications of small RNAs and their associated proteins, Cell, 143, 703, 10.1016/j.cell.2010.11.018 · DOI
  3. Lewis, 2005, Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets, Cell, 120, 15, 10.1016/j.cell.2004.12.035 · DOI · Paper page
  4. Bushati, 2007, microRNA Functions, Annu. Rev. Cell Dev. Biol, 23, 175, 10.1146/annurev.cellbio.23.090506.123406 · DOI · Paper page
  5. Lee, 2002, MicroRNA maturation: Stepwise processing and subcellular localization, EMBO J, 21, 4663, 10.1093/emboj/cdf476 · DOI
  6. Rand, 2005, Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation, Cell, 123, 621, 10.1016/j.cell.2005.10.020 · DOI
  7. Matranga, 2005, Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes, Cell, 123, 607, 10.1016/j.cell.2005.08.044 · DOI · Paper page
  8. Wang, 2009, Structural insights into RNA processing by the human RISC-loading complex, Nat. Struct. Mol. Biol, 16, 1148, 10.1038/nsmb.1673 · DOI
  9. Leuschner, 2006, Cleavage of the siRNA passenger strand during RISC assembly in human cells, EMBO Rep, 7, 314, 10.1038/sj.embor.7400637 · DOI
  10. Iwasaki, 2009, Drosophila argonaute1 and argonaute2 employ distinct mechanisms for translational repression, Mol. Cell, 34, 58, 10.1016/j.molcel.2009.02.010 · DOI
  11. Diederichs, 2007, Dual role for argonautes in microRNA processing and posttranscriptional regulation of microRNA expression, Cell, 131, 1097, 10.1016/j.cell.2007.10.032 · DOI
  12. Bhayani, 2012, Functional relevance of miRNA sequences in human disease, Mutat. Res, 731, 14, 10.1016/j.mrfmmm.2011.10.014 · DOI
  13. Khvorova, 2003, Functional siRNAs and miRNAs exhibit strand bias, Cell, 115, 209, 10.1016/S0092-8674(03)00801-8 · DOI · Paper page
  14. Schwarz, 2003, Asymmetry in the assembly of the RNAi enzyme complex, Cell, 115, 199, 10.1016/S0092-8674(03)00759-1 · DOI · Paper page
  15. Hui, 2011, MicroRNA evolution by arm switching, EMBO Rep, 12, 172, 10.1038/embor.2010.191 · DOI · Paper page
  16. Li, 2012, miRNA arm selection and isomiR distribution in gastric cancer, BMC Genomics Electron. Resour, 13, S13, 10.1186/1471-2164-13-S1-S13 · DOI · Paper page
  17. Okamura, 2008, The regulatory activity of microRNA* species has substantial influence on microRNA and 3′ UTR evolution, Nat. Struct. Mol. Biol, 15, 354, 10.1038/nsmb.1409 · DOI · Paper page
  18. Arvey, 2010, Target mRNA abundance dilutes microRNA and siRNA activity, Mol. Syst. Biol, 6, 363, 10.1038/msb.2010.24 · DOI
  19. Larsson, 2010, mRNA turnover rate limits siRNA and microRNA efficacy, Mol. Syst. Biol, 6, 433, 10.1038/msb.2010.89 · DOI
  20. Chatterjee, 2011, Target-mediated protection of endogenous microRNAs in C. elegans, Dev. Cell, 20, 388, 10.1016/j.devcel.2011.02.008 · DOI · Paper page
  21. Ebert, 2007, MicroRNA sponges: Competitive inhibitors of small RNAs in mammalian cells, Nat. Methods, 4, 721, 10.1038/nmeth1079 · DOI · Paper page
  22. Lee, 2006, miR-7b, a microRNA up-regulated in the hypothalamus after chronic hyperosmolar stimulation, inhibits Fos translation, Proc. Natl. Acad. Sci. USA, 103, 15669, 10.1073/pnas.0605781103 · DOI
  23. Hutvagner, 2008, Argonaute proteins: Key players in RNA silencing, Nat. Rev. Mol. Cell Biol, 9, 22, 10.1038/nrm2321 · DOI
  24. Meister, 2004, Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs, Mol. Cell, 15, 185, 10.1016/j.molcel.2004.07.007 · DOI · Paper page
  25. Liu, 2004, Argonaute2 is the catalytic engine of mammalian RNAi, Science, 305, 1437, 10.1126/science.1102513 · DOI · Paper page
  26. Kwak, 2012, The N domain of Argonaute drives duplex unwinding during RISC assembly, Nat. Struct. Mol. Biol, 19, 145, 10.1038/nsmb.2232 · DOI

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Metadata

Authors
Kang, S.-M., Choi, J.-W., Hong, S.-H., & Lee, H.-J.
Affiliations
Department of Oral Microbiology, School of Dentistry, Kyungpook National University, Daegu 700-412, Korea
ORCID
Lee, Heon-Jin
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2013-06-26
Volume
14
Issue
7
Pages
13231-13240
ISSN
1422-0067
DOI
10.3390/ijms140713231
PMC
PMC3742184
Cited by
20
References
0
Language
en
Licence
creativecommons.org/licenses/by/3.0/ (vor)

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