Development of a Split Fluorescent Protein-Based RNA Live-Cell Imaging System to Visualize mRNA Distribution in Plants

doi:10.21203/rs.3.rs-994002/v1

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Development of a Split Fluorescent Protein-Based RNA Live-Cell Imaging System to Visualize mRNA Distribution in Plants

https://doi.org/10.21203/rs.3.rs-994002/v1

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published 08 Feb, 2022

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Background: RNA live-cell imaging systems have been used to visualize subcellular mRNA distribution in living cells. The MS2-based RNA imaging system exploits the coat protein (MCP) of MS2 bacteriophage with RNA-binding activity and the RNA hairpin structure MS2 binding sequences (MBS) recognized by MCP to indirectly label mRNAs. Co-expression of fluorescent protein (FP)-fused MCP and target mRNA conjugated with multiple copies of MBS allows for visualizing of mRNA localization by confocal microscopy. To minimize the background fluorescence in cytosol, the nucleus localization sequence was used to sequester the MCP-FP not bound to mRNA in the nucleus. However, strong fluorescence in the nucleus may limit the application of RNA visualization in the nucleus and sometimes may interfere in detecting fluorescence signals in cytosol, especially in cells with low signal-to-noise ratio.

Results: By using the split FP-based approach, we eliminated the background fluorescence derived from MCP-FP not bound to mRNAs. MCP and PCP, a coat protein of PP7 bacteriophages with RNA-binding activity, were fused with the N- or C-terminus of split FPs (FP_N or FP_C). On co-expression of MCP-FP_N and PCP-FP_C with the target mRNA conjugated with multiple copies of the hairpin structure recognized by MCP and PCP, the interaction of RNA and proteins can bring split FPs together to reconstitute functional FPs that allow for visualizing target mRNAs in living cells. We optimized the combinations of MCP- and PCP-FPs with minimal background fluorescence and applied the imaging system to visualize mRNAs in living plant cells.

Conclusions: We established a background-free RNA live-cell imaging system that provides a platform to visualize subcellular mRNA distribution in living plant cells.

Plant Molecular Biology and Genetics

RNA live-cell imaging

MCP

PCP

split fluorescent proteins

puncta distribution of mobile mRNAs

agro-infiltration

Most cells transport mRNAs to specific subcellular compartments for localized RNA translation. Instead of transporting a quantity of proteins, the transport of mRNA provides an efficient strategy to distribute proteins to specific subcellular compartments. In eukaryotic cells, asymmetric mRNA localization is a conserved mechanism to create translation hotspots for regulating spatial gene expression in response to environmental cues [1]. The intracellular mRNA asymmetric distribution in plants can be extended to the intercellular level in which specific plant mobile mRNAs are transcribed in local tissues and undergo long-distance movement through phloem to exert non-cell autonomous functions, including regulation of leaf development [2], floral transition [3, 4], and potato tuber formation [5]. Thus, understanding the mRNA subcellular distribution may provide information to unravel RNA logistics and post-transcriptional gene regulation.

In living plant cells, directly visualizing mRNAs by microscopy is a challenge task. Many approaches have been developed to allow for visualizing mRNAs in living cells. Directly labeling mRNAs can be achieved by incorporating in vitro-synthesized mRNA with fluorescein-labeled nucleotides or introduce molecular beacons into cells to allow the detection of fluorescein-labeled mRNAs or mRNAs specifically hybridized with molecular beacons [6, 7]. However, these in vitro RNA labeling systems require invasive injection of labeled RNA into plant cells such as by microinjection or particle bombardment. In addition, the in vitro-synthesized RNAs may behave differently from the in vivo RNAs because the RNA synthesized in vitro may bypass the RNA modification occurring in nucleus, which may change the properties of RNA.

Alternatively, RNA can be indirectly visualized by an RNA aptamer-based approach. In this system, the target mRNA is genetically modified by tagging with an RNA aptamer recognized by RNA-binding proteins (RBPs). Co-expression of fluorescent proteins (FPs) conjugated with RBPs with aptamer-tagged mRNA allows for indirectly labelling mRNA in living cells. MCP, a coat protein derived from MS2 bacteriophage with RNA-binding activity, and the stem loop structures recognized by MCP have been widely used to monitor mRNA localization in yeast, fruit fly, mammals and plants [8-13]. To reduce the background GFP fluorescence detected in the cytosol, the nucleus localization sequence (NLS) has been used to sequester the MCP-GFP not bound to RNA in the nucleus [8, 13]. In addition, the signal-to-noise ratio of RNA detected in the cytosol can be greatly improved by using the plant-specific NLS tightly restricts MCP in the nucleus [13]. Although the MCP-based approach has been successfully used to visualize the intracellular distribution of mobile and non-mobile mRNA in plants [13], the background GFP fluorescence derived from nucleus-localized MCP-GFP may interfere in detecting mRNA with low expression level. In addition, cells that undergo active cell division are smaller and the nucleus is relatively large [14], so the high nucleus-to-cytoplasmic ratio may disturb the detection of RNA localization due to strong background GFP fluorescence in the nucleus.

Here, we improved the RNA live-cell imaging system to reduce the background fluorescence in the nucleus by using a split FP-based approach. We conjugated split FPs with bacterial phage MS2 or PP7 coat protein (MCP or PCP, respectively). The target mRNA was flanked with the MS2 binding sites (MBS) and PP7 binding sites (PBS) in tandem repeats [15]. The fluorescence only shines when split MCP-FP and PCP-FP binds to the mRNA-MBS-PBS that bring two split proteins together to reconstitute a mature FP.

Development of split FP-based RNA live-cell imaging system

To develop the mRNA live-cell imaging system without background GFP fluorescence in the nucleus, we exploited the bimolecular fluorescence complementation (BiFC) system to reduce background GFP fluorescence derived from MCP-GFP not bound to mRNA. In this system, two different RNA-binding proteins (RBPs) were fused with N- or C-terminus of split FPs, respectively, to reduce the fluorescence produced from RBP-FPs not bound to mRNA. The interaction between RBPs and RNA motifs recognized by RBPs may bring two different split FPs together to allow the detection of fluorescence (Figure 1A). We selected two coat proteins of bacteriophages, MCP and PCP, which specifically bind to a RNA hairpin structure of MBS or PBS, respectively [15]. MCP and PCP were conjugated with the N-terminus (FP_N, amino acids 1-173 from Venus) or C-terminus (FP_C, amino acids 156-239 from SCFP3A) fragment of the split FP protein [16]. To identify the optimized fusion proteins for the system, we generated different MCP/PCP-FP fusion proteins by flanking FP_N or FP_C with MCP or PCP (Figure 1B). In addition, the NLS derived from Arabidopsis FD [13] was used to locate MCP or PCP to the nucleus (Figure 1B). To generate the target mRNAs that can be recognized by MCP and PCP, the fragments containing 12 copies of MBS-PBS tandem repeats were conjugated with FT or RFP cDNA to create FT_HSL12(FT-hybrid stem loops, 12 copies) or RFP_HSL12, respectively (Figure 1A).

Background GFP fluorescence was under the detection limit in cells expressing MCP-FP_Nand PCP-FP_C

To identify the combinations of MCP and PCP with a minimal background GFP fluorescence in plant cells, we transiently expressed different MCP- and PCP-split FPs in leaves of 3-weak-old Nicotiana benthamiana by agro-infiltration and examined GFP fluorescence under a confocal laser scanning microscope at 3 days after infiltration. When NLS-containing M_NLS-FP_N was co-expressed with P-FP_C or FP_C-P, weak GFP fluorescence was detected in both cytosol and nucleus (Figure 2A-D), which suggests that the separation of MCP and PCP into different subcellular compartments may not significantly reduce the background GFP fluorescence. Consistently, when NLS-containing P_NLS-FP_C was co-expressed with FP_N-M, the background GFP fluorescence was again detected in the cytosol and nucleus (Figure 3A, 3D, 3G). However, when P_NLS-FP_C was co-expressed with M-FP_N or NLS-containing M_NLS-FP_N, the background GFP fluorescence that formed a speckle-like spot was detected only in the nucleus (Figure 3B-C, 3E-F, 3H-I), which suggests that the topology of fusion proteins rather than subcellular localization may contribute to the background GFP fluorescence.

We next examined background GFP fluorescence in cells expressing MCP-FP and PCP-FP without NLS. When M-FP_N was co-expressed with P-FP_C, we consistently obtained the images without detectable background GFP fluorescence (Figure 4A and 4E). However, the other 3 combinations of MCP-FP and PCP-FP conferred weak (Figure 4B and 4F) or strong GFP fluorescence (Figure 4C-D, and 4G-H). We summarize these results in Table 1. Taken together, our results revealed that the combination of MCP-FP_N and PCP-FP_C produced undetectable background GFP fluorescence. In addition, the combination of P_NLS-FP_C with M-FP_N or M_NLS-FP_N produced a speckle-like background GFP fluorescence in the nucleus, which may also provide a useful positive control for presence of the infiltrated constructs.

Detection of mRNA localization in background-free split FP-based imaging system

To verify the application of the split-FP system for localization of mRNAs, we co-infiltrated M-FP_N and P-FP_C with FT_HSL12or RFP_HSL12, two mRNAs with a differential subcellular distribution pattern [13] in tobacco leaves. At 3 days after infiltration, no fluorescence was detected in control cells co-expressing M-FP_N and P-FP_C without mRNA (Figure 5A, 5E). However, GFP fluorescence was detected in the cytosol of cells co-expressing M-FP_N and P-FP_C with RFP_HSL12 or FT_HSL12mRNA. Of note, the fluorescence in the cytosol showed distinct distribution patterns in cells expressing RFP_HSL12 or FT_HSL₁₂ mRNA: in cells expressing RFP_HSL12, the fluorescence was evenly distributed at the cell periphery (Figure 5B, 5F), whereas with FT_HSL₁₂, the fluorescence showed a punctate distribution (Figure 5C, 5G). These subcellular distribution patterns were reminiscent of the previous MS2-based mRNA live-cell imaging [13]. However, in the split FP-based imaging system, the GFP fluorescence spots were detected inside or in the boundary of the nucleus (Figure 5D-H), which were not detected in the previous MS2-based system [13]. These results suggest the broader use of a split FP-based imaging system for visualizing mRNA distribution.

We further verify the application of the split FP-based imaging system with speckle-like background GFP fluorescence for mRNA visualization. In control cells co-expressing M_NLS-FP_Nand P_NLS-FP_C, speckle-like GFP fluorescence was detected in the nucleus (Figure 6A and 6C), whereas in cells co-expressing M_NLS-FP_Nand P_NLS-FP_C with FT_HSL₁₂ mRNA, the GFP fluorescence was detected in the cytosol with a puncta distribution pattern (Figure 6B and 6D). Of note, the GFP fluorescence spots were detected in the nucleus (Figure 6D), suggesting that the speckle-like background GFP may not greatly disturb mRNA visualization in the nucleus (Figure 5B, 5D).

Plant materials and growth conditions

Nicotiana benthamiana plants were grown in a growth chamber under 27^oC, 16-h/8-h day/night cycles and light intensity of 100 mmol m^-2 s^-1. The 3-week-old plants were used for agro-infiltration assay.

Plasmid construction and Arabidopsis transformation

The DNA fragments of MCP and PCP were PCR-amplified with the following primers: MCP-For (5’-GGATCCATGGCTTCTAACTTTACTCAGTTCG-3’), MCP-Rev (5’-CTCGAGGTAGATGCCGGAGTTTGCTGCGATT-3’), PCP-For (5’-GGATCCATGTCCAAAACCATCGTTCTTTCGG-3’), PCP-Rev (5’-CTCGAGACGGCCCAGCGGCACAAGGTTGACG-3’). The DNA fragments of MCP and PCP were confirmed by sequencing and cloned into split FP-containing binary vectors pVYNE, pVYNE(R), pSCYCE, or pSCYCE(R) [16] to generate M-FP_N, FP_N-M, P-FP_C and FP_C-P, respectively. The C-terminal fragment (211-240 a.a.) of Arabidopsis FD, which contains the nucleus localization sequence (NLS) [13], was inserted in M-FP_N or P-FP_C to generate M_NLS-FP_N and P_NLS-FP_C, respectively.

For the 12 copies of the MBS-PBS hybrid stem loop sequence (HSL12), the Pcr4-12X-MBS-PBS [15] DNA was digested with BamHI and SpeI, followed by the Klenow fill-in reaction. The 1402-bp HSL12 fragment was gel-eluted and ligated with the binary vector pCAMBIA1390 that contains the 35S-FT or 35S-RFP to form the p1390-35S-FT_HSL12 or p1390-35S-RFP_HSL12 construct, respectively.

Agro-infiltration of N. benthamiana

Agrobacterium tumefaciens strain AGL1 carrying individual constructs was cultured in 20 mL LB broth (10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl) containing 50 mg mL^-1 kanamycin, 10 mM MES, pH 5.7 and 20 mM acetosyringone at 28^oC with 220 rpm shaking for 2 days. Bacteria were pelleted by centrifugation and resuspended in infiltration medium (10 mM MgCl2, 10 mM MES, pH 5.7, and 200 mM acetosyringone) to OD600 = 1.0. The bacterial solution was placed at room temperature for at least 1 h. For agro-infiltration, bacteria solution was infiltrated into the abaxial side of the 4^th or 5^th leaves of 3-week-old N. benthamiana by using a needle-removed 5-mL syringe. For co-infiltration with 2 or 3 different constructs, different bacterial solution or infiltration medium was mixed at a 1:1 volume ratio and infiltrated into leaves. The infiltrated plants were cultured in growth chambers for 3 days, then infiltrated leaves were excised for confocal laser scanning microscopy.

Confocal laser scanning microscopy

GFP fluorescence was observed by confocal laser scanning microscopy (LSM880, Carl Zeiss) with the argon laser was set to 488/500-530 for excitation/emission.

BiFC: bimolecular fluorescence complementation

MCP: MS2 bacteriophage coat protein

PCP: PP7 bacteriophage coat protein

MBS: MCP binding sequences

PBS: PCP binding sequences

FP: fluorescent protein

NLS: nucleus localization sequence

Acknowledgements

We would like to thank Mrs. Mei-Jane Fang for assistance in troubleshooting the confocal microscopy.

Authors’ contributions

NCH and TSY planed the research. NCH carried out experiments and drafted the manuscript. KRL carried out experiments of RFP localization. TSY contributed to writing the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by grants (MOST 108-2311-B-001-025-MY3) from the Ministry of Science and Technology, Taiwan and (AS-TP-108-L01) from Academia Sinica, Taiwan.

Availability of data and materials

All data and materials are available upon request to TSY ([email protected]).

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors read and agreed to publish the final manuscript.

Competing interests

The authors declare that they have no competing interests.

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Table 1. Summary of background GFP fluorescence detection with different combinations of split fluorescent proteins

MCP varieties	PCP varieties	Background	Fluorescence
M_NLS-FP_N	P-FP_C	Yes	nucleus, cytosol
M_NLS-FP_N	FP_C-P	Yes	nucleus, cytosol
FP_N-M	P_NLS-FP_C	Yes	nucleus, cytosol
M-FP_N	P_NLS-FP_C	Yes	speckle in nucleus
M_NLS-FP_N	P_NLS-FP_C	Yes	speckle in nucleus
M-FP_N	P-FP_C	No	Non-detectable
FP_N-M	P-FP_C	Yes	cytosol
M-FP_N	FP_C-P	Yes	nucleus, cytosol
FP_N-M	FP_C-P	Yes	cytosol

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Journal Publication

published 08 Feb, 2022

Read the published version in Plant Methods →

Editorial decision: Minor revision
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You are reading this latest preprint version

Development of a Split Fluorescent Protein-Based RNA Live-Cell Imaging System to Visualize mRNA Distribution in Plants

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