Visualizing nuclear‐localized RNA using transient expression system in plants
Abstract
By modifying the existing cytosolic RNA visualization tool pioneered by Schönberger, Hammes, and Dresselhaus (2012), we developed a method to visualize nuclear‐localized RNA. Our method uses (i) an RNA component that consists of an RNA of interest that is fused to a bacteriophage‐derived MS2 sequence; and (ii) GFP fused to MS2 coat protein (MSCP), which binds specifically to MS2 as is also the case in the method for cytosolic RNA visualization. The nuclear localization sequence (NLS) at the C‐terminal of MSCP‐GFP tethers the probe to the nucleus. To reduce background signals in the nucleus, we replaced the NLS with a nuclear export sequence (NES) that anchors the MSCP‐GFP probe in the cytosol. Our nuclear RNA visualization method differs from previous methods in two aspects: (i) We used an NES to reduce nuclear background signal so that the MSCP‐GFP probe localizes in the cytosol by default; (ii) We added mCherry as a visual marker in the RNA component to increase its efficient usage in a transient system.
1 INTRODUCTION
Comprehensive transcriptome analyses suggest that intergenic and noncoding regions are transcribed at a low level genome‐wide (Liu et al., 2012; Matsui et al., 2010; Okamoto et al., 2010).
RNA‐seq results using next‐generation sequence technology strengthened these findings with better coverage and precision, especially regarding 5′ and 3′ of the transcript. These results confirm and clarify the near‐ubiquitous presence of lncRNAs in plants (Liu et al., 2012).
lncRNA genes fall into two classes depending on their relative location to the nearest protein‐coding genes: (i) those overlapping with or at the vicinity of protein‐coding genes—this type includes antisense, sense and intronic RNAs; and (ii) long intergenic noncoding RNA (lincRNA). These are at least 500 bp away from protein‐coding genes and do not encode peptides longer than 100 amino acids (Liu et al., 2012).
Some overlapping types of lncRNAs are found to regulate gene expression in cis‐acting manner. For example, antisense RNA and COOLAIR, as well as an intronic RNA COLDAIR, mediate the epigenetic silencing of FLC, a repressor of flowering, during vernalization (Heo & Sung, 2011; Swiezewski, Liu, Magusin, & Dean, 2009). APOLO regulates the expression of the key auxin efflux facilitator, PIN, in a cis‐acting manner (Ariel et al., 2014). However, Guttman et al. (2011) found that a large number of lincRNA act in trans and regulate gene expressions throughout the genome. The regulatory potential of lincRNA remains greater in trans, as this mechanism does not limit the physical locations of the lincRNA genes with respect to its target genes. In Arabidopsis, Seo et al. (2017) have recently shown that the lincRNA, ELENA1, activate PR1 expression during innate immunity response by binding to Med19a.
Aside from protein‐coding potential and average expression levels, there are many common characteristics between protein‐coding and lncRNA genes. These characteristics include capped 5′ ends, introns and polyadenylation in many lncRNAs (Guttman et al., 2009; Liu et al., 2012). Additionally, transcript levels for many lncRNAs are regulated in a tissue‐/organ‐specific and/or stress‐dependent manner (Jin, Liu, Wang, Wong, & Chua, 2013; Matsui et al., 2010; Okamoto et al., 2010).
Given that the molecular functions of many lincRNAs are entirely unknown, clarifying subcellular localization provides information to better understand their molecular mechanisms.
One way to observe in vivo subcellular localization for RNA is to introduce fluorescence‐labeled RNA to the cell. Although Christensen et al. (2009) visualized tobacco mosaic virus RNA with this technique, their protocol is not amenable to high‐throughput analyses in intact plant tissues due to the difficulties posed by turgor pressure and limited cytoplasmic volume.
A more straightforward solution is to use a highly specific RNA‐binding protein that is fused to a fluorescence protein as an indirect way to track subcellular localization of RNA (Schönberger et al., 2012).
This method uses two constructs: (i) an RNA component—the RNA of interest is fused to a highly structured RNA, which serves as a specific binding site of RNA‐binding protein (RBP); and (ii) a fluorescence protein (FP) component. FP is fused to a RBP. An example of such a RBP and its binding site is the RNA bacteriophage MS2 coat protein (MSCP) and its binding site MS2. MS2 is a 19‐nucleotide RNA stem‐loop structure (Fouts, True, & Celander, 1997; Valegârd et al., 1997).
By itself, RBP‐FP localizes in both cytosol and the nucleus resulting in inherent background noise. Tethering RBP‐FP to the nucleus using NLS (nuclear localization sequence) successfully reduced background noise in the cytosol while achieving a higher sensitivity.
We introduce a new approach by modifying the method developed by Schönberger et al. (2012). The key features of this method are as follows: (i) reducing the background signal in the nucleus; and (ii) modifying the vector system to better suit transient expression protocol.
2 RESULTS AND DISCUSSION
2.1 Reducing the nuclear background signal by introducing NES
We modified the cytosolic RNA visualization method to be used for nuclear localization and focused on bacteriophage‐derived MS2 stem‐loop RNA and MSCP (MS2 coat protein). MS2 binds to MSCP in a highly specific manner (Figure 1a). This technique enables the visualization of cytoplasmic and nuclear body localized RNAs (Campalans, Kondorosi, & Crespi, 2004; Fujioka, Utsumi, Ohba, & Watanabe, 2007; Schönberger et al., 2012).
To visualize cytosolic localized RNA, RBP‐fluorescent protein (FP) is usually tethered to the nucleus by tagging the FP with an NLS.
However, there are two exceptions: (i) RNA binding leads to a significant redistribution including shifting general nucleocytoplasmic signals to either nuclear or cytosolic‐specific signals; and (ii) the formation of distinct particles or granules within these subcellular locations (Campalans et al., 2004; Fujioka et al., 2007).
The challenge of applying this method to visualizing RNA nuclear localization is that the FP is already in the nucleus. To observe nuclear‐localized RNAs, we used eGFP because of the intensity of its fluorescence signal and its photo‐stability (Shaner, Steinbach, & Tsien, 2005). We also replace NLS with NES (nuclear export sequence). The latter is derived from the heat‐stable inhibitor (PKI) of cAPK (cAMP‐dependent protein kinase). NES from PKI triggers a rapid, active exclusion of the catalytic subunit of the PKI complex from the nucleus (Lefkimmiatis, Moyer, Curci, & Hofer, 2009; Schönberger et al., 2012; Wen, Meinkoth, Tsien, & Taylor, 1995; Figure 1).
This ensures that MSCP‐GFP‐NES is localized in the cytosol, thus lowering the background noise in the nucleus (Figure 1b). By itself (without the RNA component) the GFP signal is specifically found in the cytosol (Figures 1b, 3b top panel).
It is likely that the NLS version of Schönberger et al. (2012) functions by targeting cytosolically synthesized MSCP‐GFP‐NLS into the nucleus. By contrast, our NES version allows export of nuclear diffused MSCP‐GFP‐NES back into the cytosol (Figure 1b left). We suspect that there is a constant shuttling of MSCP‐GFP‐NES proteins into the nucleus as well as active translocation of the protein from the nucleus into the cytosol.
MSCP is 14 kDa, GFP is 27 kDa, and NES (14 amino acids) is 1.5 kDa. Therefore, the size of the fusion protein MSCP‐GFP‐NES is 42.5 kDa (Hill, Stonehouse, Fonseca, & Stockley, 1997; Gasteiger et al.,2005; Sarkar, Koushik, Vogel, Gryczynski, & Gryczynski, 2009). This estimate is consistent with Western blot data from Schönberger et al. (2012). They demonstrated that size of MSCP‐VENUS‐NLS was approximately 43 kDa. They made this calculation by adding VENUS, which is a single amino acid mutation of GFP, and NLS, which is 1.5 kDa (12 amino acids) to MSCP.
The historical view for the maximal size for protein diffusion through the nuclear pore was estimated to be 60 kDa (Görlich, 1998). More recently, Wang and Brattain (2007) demonstrated that proteins with sizes from 90 to 110 kDa diffuse through the nuclear pore (Figure 1; Wang & Brattain, 2007; Schönberger et al., 2012).
In sum, the new fluorescent fusion protein (MSCP‐GFP‐NES) is small enough to function as a probe for nuclear‐localized RNAs.
2.2 Alteration of RNA component vector
We modified Schonberger's RNA component in the transient expression system because of its fast turnaround time and higher sensitivity due to an increased probe protein expression levels.
To facilitate the visual selection of the transformed cells, we inserted mCherry in place of the herbicide‐resistant gene BAR (bialaphos resistance) as the mCherry signal does not overlap with GFP signal. In addition, we attached the NLS to mCherry to concentrate the fluorescence signal in the nucleus. The promoter and terminator regions from the mannopine synthase gene controlled the expression of mCherry as in the stable transgenic construct.
The mCherry–NLS fusion gene and the RNA component (MS2‐RNA fusion gene) were co‐located within the same T‐DNA even though they were expressed from independent transcription units (Figure 1a). The use of mCherry facilitated the identification of transformed cells with the visual marker, and it also served as a nuclear marker (Figures 1 and 3b middle panel).
2.3 Subcellular localization of lncRNA in the nucleus
To test the modified versions of above‐mentioned vectors, we investigated whether NPC60 RNA was localized in the nucleus (nonprotein coding 60; Hirsch et al., 2006; Ben Amor et al., 2009). Our work confirmed that NPC60 RNA expression was pronounced in floral tissues with an expected size of approximately 500 nt (nucleotides; Figure 2).
We fused the NPC60 sequence downstream of the MS2 sequence because NPC60 contains putative polyA addition signals at the 3′ end.
After infiltrating Nicotiana benthamiana with Agrobacterium tumefaciens carrying MSCP‐GFP‐NLS, we observed a nuclear‐localized GFP signal (Figure 3a). This is consistent with the previous study (Schönberger et al., 2012). Upon MSCP‐GFP‐NES infiltration, we observed that the GFP signal was depleted from the nucleus (Figure 3b top panel). When infected only with Agrobacterium tumefaciens carrying MS2‐NPC60 (with mCherry–NLS), the mCherry fluorescence signal displayed a punctuated nuclear pattern (Figure 3b middle panel).
When the two plasmids were co‐expressed, the GFP signal translocated to the nucleus (Figure 3b bottom panel). These nuclear‐localized GFP patterns were never observed when MSCP‐GFP‐NES was expressed alone (Figure 3b top panel). Our results suggest that NPC60 is a nuclear‐localized RNA.
To reduce background noise from the diffused RBP‐FP signals, either NLS or NES was attached to the reporter fluorescence protein. In the NES method, localization of the RNA‐fluorescence protein complex was dependent upon a combination of (i) RNA affinity to the target destination of the RNA of interest, and (ii) the affinity of the fluorescence probe to remain in the cytosol. We anticipated that nuclear localization of the fluorescent probe occurred only when the affinity of the RNA of interest to localize in the nucleus was greater than the effect of NES.
Among the cells expressing both the NPC60 RNA and the MSCP‐GFP probe, 59% exhibited nuclear RNA localization in the transient expression system. At the same time, the MSCP‐GFP probe by itself never displayed nuclear localization (Figure S1).
There are at least two probable explanations for the nonuniform nuclear localization: (i) the nuclear localization of NPC60 may require multiple components. Not all N. benthamiana epidermal cells are equipped with all the components to translocate cytosolically localized probe to the nucleus; (ii) this technique entails ectopic expression and the addition of a foreign, structured stem loop. It is possible that this experimental setup may interfere with the endogenous RNA localization machinery.
Our subcellular localization data suggest that the NPC60 RNA is partially localized in the nucleus. It is probable that the NPC60 RNA also resides in the cytosol itself. To clarify the relative subcellular distribution of NPC60 RNA, parallel analyses using NLS and NES versions may provide additional insight into these intracellular localization processes.
3 CONCLUSION
We developed a high‐throughput‐compatible method to visualize nuclear‐localized RNA, based on the Schönberger et al. (2012) protocol for cytosolic mRNAs. This was accomplished by placing a visual marker onto the RNA component, thus permitting the identification of cells with both components for localization experiments using the transient expression system. The turnaround time to analyze localization in the transient expression system is much shorter compared to using stable transgenic lines. Expression levels of the transgenes are also higher than in stable transgenic lines.
To test our hypothesis that NPC60 localizes in the nucleus, we inserted NES in place of NLS at the C‐terminal of MSCP‐GFP to reduce the background signal in the nucleus.
The primary advantage of the system we have developed is that it provides an RNA component with a visible marker. Together with fluorescence protein of the visualization probe, these visible markers enable us to identify cells that are transformed with both plasmids.
Our new transient expression system allows for higher expression levels on average, thus making the method more sensitive. Moreover, the turnaround time for localization analysis is shorter than that of the stable transgenic method.
Given that the functions of most plant lncRNAs remain largely unknown, the availability of tools for identifying subcellular localization, especially nuclear localization, has the potential to aid progress toward a better understanding their molecular mechanisms.
4 EXPERIMENTAL PROCEDURES
4.1 Plant material and growth conditions
All experiments concerning Arabidopsis thaliana were carried out with the Columbia (Col.0) ecotype. For tobacco infiltration experiment, 1‐month‐old pot‐grown Nicotiana benthamiana plants with occasional fertilizer (Miracle‐Gro) were used.
4.2 Cloning and vectors
pENTR‐D‐TOPO with full‐length NPC60 was generated by amplifying full‐length NPC60 DNA fragments using PCR with KOD enzyme (Novagen) and cloned into pENTR‐D‐TOPO vector, followed by LR reaction with the binary vector pSCJ216 (35S‐6xMS2‐GW).
4.3 RNA extraction, RT‐qPCR and Northern blot analyses
Total RNA was extracted from plant tissues using Trizol reagent. Reverse transcription was carried out with 1 μg of total RNA using SuperScript III reverse transcriptase (Invitrogen) and oligo (dT) or specific primer.
cDNA was analyzed by qPCR using SYBR Premix Ex Taq (Takara) and was carried out using a BioRad CFX96 real‐time PCR analysis with the primer listed in Table S1. All quantitative RT (qRT)‐PCR reactions were carried out with at least three technical triplicates. Northern blot analysis was carried out according to Kinoshita et al. (2012).
4.4 Nuclear localization
Full‐length entry clones encoding NPC60 were recombined with pSCJ216 (35S‐6xMS2‐GW) to acquire 35S‐6xMS2‐NPC60 (Schönberger et al., 2012). Subsequently, the selectable marker bialaphos resistance (BAR) gene was replaced by mCherry using in‐fusion technology (Clontech).
35S‐MSCP‐GFP‐NES was obtained by mutating NLS in the original construct to NES (Wen et al., 1995). Agrobacterium‐mediated transient expression of MS2‐NPC60 RNA and MSCP‐GFP in Nicotiana benthamiana was carried out as previously described (Guo, Fei, Xie, & Chua, 2003). Briefly, A. tumefaciens cultures carrying 35S‐MS2‐NPC60 (P531) or 35S‐MSCP‐GFP (P481, pSCJ380 derivative), respectively, were harvested by centrifugation at 4,000 g for 10 min at 4°C and the pellets were resuspended in the infiltration medium (10 mM MgCl2 10 mM MES, and 100 μM acetosyringone).
After standing for 3 hr at room temperature, the cell suspensions were mixed and infiltrated into mature N. benthamiana mesophyll cells via syringe. Two days after inoculation, fluorescence signals were observed using LSM 780 laser scanning confocal microscope (Zeiss) at the Bio‐Imaging Resource Center at Rockefeller University with excitation–emission wavelength of 488–525/35 nm for GFP, and 561–617/30 for mCherry.
ACKNOWLEDGMENTS
We thank Dr. Ulrich Z. Hammes for the in vivo RNA localization plasmid tools and the Bio‐Imaging Resource Center, Rockefeller University for technical assistance. MS. Coral Martínez Martínez and Lic. Xochitl Alvarado Affantranger provided technical support. We also thank John Harris and Barry Lustig for their critical comments and suggestions on the manuscript and Takuhiko Yokoyama for help with the illustration. N.K. was supported by the Uehara Memorial Foundation Postdoctoral Fellowship, Swiss National Science Foundation Fellowship for Prospective Researchers, and Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. C.A. was supported by a Consejo Nacional de Ciencia y Tecnologia Postdoctoral Fellowship. This research was, in part, supported by Tomizawa Jun‐ichi & Keiko Fund of Molecular Biology Society of Japan for Young Scientists, KAKENHI Grant‐in‐Aid for Young Scientists (B) 15K18550, Tobe Maki Foundation, Grant‐in‐Aid for JSPS Fellows from Science and Technology Agency of Japan, Urakami Foundation for Food and Food Culture Promotion, and Canon Foundation Grant “Pursuit of Ideal.” Members of the Laboratory of Plant Molecular Biology, Rockefeller University, and colleagues at the Faculty of Life and Environmental Sciences, University of Tsukuba, provided helpful comments on the manuscript.
