Identification of Mobile RNA Based on the MS2 Protein System

doi:10.7668/hbnxb.20192131

Abstract

Abstract: In order to conveniently and quickly characterize RNA mobility, explore the intercellular transport mechanism of RNA involved in plant development and physiological response and reduce the experimental cost, this study successfully constructed the expression vector pSL24Ubik by adding the 24 tandem stem-loop repeats(SL24) to the binary vector pUbiK.The recombinant vector pSL24Ubik-FT and pSL24Ubik-GUS were sequentially constructed by homologous recombination of mobile RNA FT and non-mobile RNA GUS fragments into expression vector pSL24Ubik.pSL24UbiK-FT and p1390-35S-MS2_FD-GFP, pSL24UbiK-GUS and p1390-35S-MS2_FD-GFP were collectively transient expressed in tobacco leaves by Agrobacterium-infiltration method.MS2/SL24 system in which 24 tandem stem-loop repeats fused with target RNA were driven by ubiquitin promoter, and phage coat protein MS2 was fused with nucleus-localized GFP. Since MS2 could specifically recognize SL24 RNA sequence, the localization of fusion RNA could be revealed by the MS2-GFP fluorescent signal under the confocal scanning microscope. The result showed that mobile RNA FT had a spotty signal near the cytoplasm, while non-mobile RNA GUS had no fluorescence signal near the cytoplasm, but rather in the nucleus. The result indicated that the target RNA was linked to the expression vector pSL24UbiK. By co-infecting tobacco leaves with p1390-35S-MS2_FD-GFP, the cell localization of GFP could be observed under confocal microscope to determine whether the RNA could move in the cell. This study provides a new technical method for the future study of RNA migration.

Key words: RNA mobility, SL24, MS2, MS2-GFP

CLC Number:

Q78
Q943.2

LI Luping, DENG Zhuying, WANG Zhipeng, LIANG Dacheng. Identification of Mobile RNA Based on the MS2 Protein System[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(4): 31-36. doi: 10.7668/hbnxb.20192131.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20192131

http://www.hbnxb.net/EN/Y2021/V36/I4/31

References

[1] Banerjee A K, Chatterjee M, Yu Y, Suh S G, Miller W A, Hannapel D J. Dynamics of a mobile RNA of potato involved in a long-distance signaling pathway[J]. The Plant Cell, 2006, 18(12):3443-3457.doi:10.1105/tpc.106.042473.
[2] Lu K J, Huang N C, Liu Y S, Lu C G, Yu T S. Long-distance movement of Arabidopsis FLOWERING LOCUS T RNA participates in systemic floral regulation[J]. RNA Biology, 2012, 9(5):653-662.doi:10.4161/rna.19965.
[3] Huang N C, Jane W N, Chen J, Yu T S. Arabidopsis thaliana CENTRORADIALIS homologue(ATC) acts systemically to inhibit floral initiation in Arabidopsis[J]. The Plant Journal, 2012, 72(2):175-184.doi:10.1111/j.1365-313x.2012.05076.x.
[4] Lough T J, Lucas W J. Integrative plant biology:Role of phloem long-distance macromolecular trafficking[J]. Annual Review of Plant Biology, 2006, 57:203-232.doi:10.1146/annurev.arplant.56.032604.144145.
[5] Kehr J, Buhtz A. Long distance transport and movement of RNA through the phloem[J]. Journal of Experimental Botany, 2008, 59(1):85-92.doi:10.1093/jxb/erm176.
[6] Jorgensen R A, Atkinson R G, Forster R L S, Lucas W J. An RNA-based information superhighway in plants[J]. Science, 1998, 279(5356):1486-1487.doi:10.1126/science.279.5356.1486.
[7] Gaupels F, Buhtz A, Knauer T, Deshmukh S, Waller F, van Bel A J E, Kogel K H, Kehr J. Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem Sap[J]. Journal of Experimental Botany, 2008, 59(12):3297-3306.doi:10.1093/jxb/ern181.
[8] Deeken R, Ache P, Kajahn I, Klinkenberg J, Bringmann G, Hedrich R. Identification of Arabidopsis thaliana phloem RNAs provides a search criterion for phloem-based transcripts hidden in complex datasets of microarray experiments[J]. The Plant Journal, 2008, 55(5):746-759.doi:10.1111/j.1365-313X.2008.03555.x.
[9] Guo S G, Zhang J G, Sun H H, Salse J, Lucas W J, Zhang H Y, et al. The draft genome of watermelon(Citrulluslanatus) and resequencing of 20 diverse accessions[J]. Nature Genetics, 2013, 45(1):51-58.doi:10.1038/ng.2470.
[10] Thieme C J, Rojas-Triana M, Stecyk E, Schudoma C, Zhang W N, Yang L, Minambres M, Walther D, Schulze W X, Paz-Ares J, Scheible W R, Kragler F. Endogenous Arabidopsis messenger RNAs transported to distant tissues[J]. Nature Plants, 2015, 1(4):15025.doi:10.1038/nplants.2015.25.
[11] Yang Y Z, Mao L Y, Jittayasothorn Y, Kang Y M, Jiao C, Fei Z J, Zhong G Y. Messenger RNA exchange between scions and rootstocks in grafted grapevines[J]. BMC Plant Biology, 2015, 15(1):251.doi:10.1186/s12870-015-0626-y.
[12] Zhang W N, Thieme C J, Kollwig G, Apelt F, Yang L, Winter N, Andresen N, Walther D, Kragler F. tRNA-related sequences trigger systemic mRNA transport in plants[J]. The Plant Cell, 2016, 28(6):1237-1249.doi:10.1105/tpc.15.01056.
[13] Li S, Wang X T, Xu W Y, Liu T, Cai C M, Chen L Y, Clark C B, Ma J X.Unidirectional movement of small RNAs from shoots to roots in interspecific heterografts[J]. Nature Plants, 2021, 7:50-59.doi:10.1038/s41477-020-00829-2.
[14] Keryer-Bibens C, Barreau C, Osborne H B. Tethering of proteins to RNAs by bacteriophage proteins[J]. Biology of the Cell, 2008, 100(2):125-138.doi:10.1042/BC20070067.
[15] Urbanek M O, Galka-Marciniak P, Olejniczak M, Krzyzosiak W J. RNA imaging in living cells-methods and applications[J]. RNA Biology, 2014, 11(8):1083-1095.doi:10.4161/rna.35506.
[16] Bertrand E, Chartrand P, Schaefer M, Shenoy S M, Singer R H, Long R M. Localization of ASH1 mRNA particles in living yeast[J]. Molecular Cell, 1998, 2(4):437-445.doi:10.1016/s1097-2765(00) 80143-4.
[17] Rook M S, Lu M, Kosik K S. CaMKIIalpha 3'untranslated region-directed mRNA translocation in living neurons:Visualization by GFP linkage[J]. The Journal of Neuroscience, 2000, 20(17):6385-6393.doi:10.1523/jneurosci.20-17-06385.2000.
[18] Forrest K M, Gavis E R. Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for Nanos mRNA localization in Drosophila[J]. Current Biology, 2003, 13(14):1159-1168.doi:10.1016/s0960-9822(03) 00451-2.
[19] Hamada S, Ishiyama K, Choi S B, Wang C L, Singh S, Kawai N, Franceschi V R, Okita T W. The transport of prolamine RNAs to prolamine protein bodies in living rice endosperm cells[J]. The Plant Cell, 2003, 15(10):2253-2264.doi:10.1105/tpc.013466.
[20] Sambade A, Brandner K, Hofmann C, Seemanpillai M, Mutterer J, Heinlein M. Transport of TMV movement protein particles associated with the targeting of RNA to plasmodesmata[J]. Traffic (Copenhagen, Denmark), 2008, 9(12):2073-2088.doi:10.1111/j.1600-0854.2008.00824.x.
[21] Park H Y, Lim H, Yoon Y J, Follenzi A, Nwokafor C, Lopez-Jones M, Meng X H, Singer R H. Visualization of dynamics of single endogenous mRNA labeled in live mouse[J]. Science, 2014, 343(6169):422-424.doi:10.1126/science.1239200.
[22] K hler R H. GFP for in vivo imaging of subcellular structures in plant cells[J]. Trends in Plant Science, 1998, 3(8):317-320.doi:10.1016/S1360-1385(98) 01276-X.
[23] Wu B, Chao J A, Singer R H. Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells[J]. Biophys J, 2012, 102(12):2936-2944.doi:10.1016/j.bpj.2012.05.017.
[24] Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y, Ichinoki H, Notaguchi M, Goto K, Araki T. FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex[J]. Science, 2005, 309(5737):1052-1056.doi:10.1126/science.1115983.
[25] Wigge P A, Kim M C, Jaeger K E, Busch W, Schmid M, Lohmann J U, Weigel D. Integration of spatial and temporal information during floral induction in Arabidopsis[J]. Science, 2005, 309(5737):1056-1059.doi:10.1126/science.1114358.
[26] Luo K R, Huang N C, Yu T S. Selective targeting of mobile mRNAs to plasmodesmata for cell-to-cell movement[J]. Plant Physiology, 2018, 177(2):604-614.doi:10.1104/pp.18.00107.
[27] Tyagi S. Imaging intracellular RNA distribution and dynamics in living cells[J]. Nature Methods, 2009, 6(5):331-338.doi:10.1038/nmeth.1321.
[28] Tilsner J. Techniques for RNA in vivo imaging in plants[J]. Journal of Microscopy, 2015, 258(1):1-5.doi:10.1111/jmi.12208.
[29] Fusco D, Accornero N, Lavoie B, Shenoy S M, Blanchard J M, Singer R H, Bertrand E. Single mRNA molecules demonstrate probabilistic movement in living mammalian cells[J]. Curr Biol, 2003, 13(2):161-167.doi:10.1016/s0960-9822(02) 01436-7.
[30] Turck F, Fornara F, Coupland G. Regulation and identity of florigen:FLOWERINGLOCUS T moves center stage[J]. Annual Review of Plant Biology, 2008, 59:573-594.doi:10.1146/annurev.arplant.59.032607.092755.
[31] Li C Y, Gu M, Shi N N, Zhang H, Yang X, Osman T, Liu Y L, Wang H Z, Vatish M, Jackson S, Hong Y G. Mobile FT mRNA contributes to the systemic florigensignalling in floral induction[J]. Scientific Reports, 2011, 1:73.doi:10.1038/srep00073.

Please choose a citation manager

Content to export