Live Cell Imaging of Endogenous RNAs Using Pumilio Homology

Nov 22, 2017 - This Perspective focuses on a possible approach to the development of protein-based RNA probes using Pumilio homology domain (PUM-HD) m...
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Perspective Cite This: Biochemistry 2018, 57, 200−208

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Live Cell Imaging of Endogenous RNAs Using Pumilio Homology Domain Mutants: Principles and Applications Hideaki Yoshimura* Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ABSTRACT: Recently, dynamic changes in the location of RNA in space and time in living cells have become a target of interest in biology because of their essential roles in controlling physiological phenomena. To visualize RNA, methods for the fluorescent labeling of RNA in living cells have been developed. For RNA labeling, oligonucleotide-based RNA probes have mainly been used because of their high selectivity for target RNAs. By contrast, protein-based RNA probes have not been used widely because of their lack of design flexibility, although they have various potential advantages compared with nucleotide-based probes, such as controllability of intracellular localization, high detectability, and ease of introduction into cells and transgenic organisms in a cell type and tissue specific manner by genetic engineering techniques. This Perspective focuses on a possible approach to the development of protein-based RNA probes using Pumilio homology domain (PUM-HD) mutants. The PUM-HD is a domain of an RNA binding protein that allows custom-made modifications to recognize a given eight-base RNA sequence. PUM-HD-based RNA probes have been applied to visualize various RNAs in living cells. Here, the techniques and RNA imaging results obtained using the PUM-HD are introduced.

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amounts of the protein effectively and quickly.8 In addition, neurons must respond quickly to neural signal input to spines. To achieve this, several mRNAs related to neural signaling are localized in the spines and undergo local translation immediately upon receiving the signals, which results in brain development.9 Thus, the regulation of mRNA transportation and local translation is critical for the precise function of nervous systems. In addition to nervous systems, mRNA transportation and local translation have been observed in various physiological events, such as wound healing and early development.3 Therefore, to understand the regulation mechanisms of such physiological systems, the live cell imaging of not only related proteins but also their cognate mRNAs is important. In addition to mRNAs, RNAs that do not encode amino acid sequences or proteins, termed noncoding RNAs, are now considered to be important for the control of various physiological phenomena. The main function of noncoding RNAs is the regulation of gene expression in various physiological systems.10−12 Thus, living systems are supported by the spatiotemporal behaviors of a variety of types of RNAs. Live cell imaging techniques have developed through the use of technologies to label and visualize target proteins with higher detectability and precision.13 Generally, proteins in living cells are visualized by fusion with a fluorescent protein that is introduced through genetic manipulation. In contrast, current

RNAS AS TARGETS FOR LIVE CELL IMAGING The central dogma of molecular biology elucidates the principle common to all organisms: genetic information encoded in DNA is transcribed to mRNA, which is followed by translation to generate myriad species of proteins. Proteins, as the final products of gene expression, have been considered as the dominant substances that constitute and regulate living systems; therefore, their dynamics and localization have been intensively studied to understand the mechanisms of physiological events. In contrast, as the name indicates, mRNAs were considered simply as messengers, and the spatial and temporal behaviors of intracellular mRNAs have not been investigated as thoroughly. Recently, the intracellular dynamics and localization of mRNAs have been recognized to support the precise and effective functions of proteins, and the analysis of the intracellular behavior of RNAs is a now topic of interest in many fields of biology.1−4 A typical instance in which mRNA dynamics play an important role is neural systems.1,5,6 In response to extracellular stimuli, neurons cause morphological alterations, including axon and dendrite elongation, the expansion of spines and growth cones, and the generation of new spines and dendritic branches.7 These morphological changes are induced by the reorganization of cytoskeletal networks, which requires a large quantity of cytoskeletal proteins in the expanding or elongating sites. The transportation of large amounts of the proteins from the cell body to the site is not effective; this active transportation is performed by motor proteins, which are present in only limited numbers in cells and consume ATP. Instead of protein transportation, mRNAs that encode cytoskeletal proteins, such as β-actin, are transported to the sites for local translation to provide sufficient © 2017 American Chemical Society

Special Issue: Future of Biochemistry Received: September 30, 2017 Revised: November 21, 2017 Published: November 22, 2017 200

DOI: 10.1021/acs.biochem.7b00983 Biochemistry 2018, 57, 200−208

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Biochemistry technology is unable fuse RNAs with fluorescent proteins spontaneously in living cells. Although a fusion RNA, which consists of the target molecule and a fluorescent aptamer, can be produced in living cells, aptamers with a fluorescence intensity comparable to that of fluorescent proteins have not yet been developed. Therefore, other approaches have been adopted to monitor of RNA in living cells; the basic principle comprises the use of probes with target RNA binding regions and fluorescently detectable regions. A typical technique for RNA labeling in living cells is the MS2 method.14−16 In this method, repeats of RNA sequences, called MS2, which form a stem−loop structure, are attached to the 3′ terminus of the target RNA and then expressed in the cells of interest. An MS2 binding protein (MBP) fused with a fluorescent protein is also expressed in the cells, which binds the MBP to the MS2 repeat of the target RNA. The target RNA can then be visualized under a fluorescence microscope. The MS2 method, which is used frequently for RNA imaging in living cells and animal bodies, has provided abundant information about the spatial and temporal properties of various RNAs.2,17 In this method, the target is limited to artificially modified RNAs; however, the analysis of unmodified RNA is important and yields information that cannot be obtained through the observation of artificially modified and exogenously introduced RNA. For example, as one mRNA molecule generates a large quantity of cognate protein molecules, a difference in RNA copy number may lead to critical effects on physiological phenomena. Moreover, attachment of MBP-GFP to MS2 repeats at the 3′ terminus of target RNA blocks degradation of the RNA, resulting in generation of 3′ RNA decay fragments that potentially hampers precise analysis of intracellular localization of the target RNA.18 In addition, alternative splicing variants and sequence variations in the same RNA families are present in endogenous RNAs. Thus, endogenous RNAs are important targets that should be monitored in living samples to achieve a detailed and precise understanding of the mechanisms of biological events.

oligonucleotide part. Before the probe attaches to the target RNA, the oligonucleotide part forms a hairpin structure, in which the fluorescent dye and the quencher are brought into the proximity of each other, which quenches the dye fluorescence by the transfer of energy to the quencher. After the probe binds to the target RNA, hybridization of the oligonucleotide part resolves the hairpin structure, creating physical distance between the dye and the quencher. The target RNA is then selectively visualized under a fluorescence microscope against a low background. Another approach for nucleotide-based RNA probes uses Spinach, a fluorescent RNA−dye complex.24−26 Spinach and similar fluorescent RNA−dye complexes, Spinach227 and Broccoli,28 are RNA aptamers that bind to and switch on the fluorescence of dyes such as 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). In a recent example in which Spinach was used to monitor endogenous RNA in living cells, Spinach was fused with oligonucleotides that possess a complementary sequence to the target RNA.29 Upon hybridization of the oligonucleotide regions to the target RNA, the Spinach region folds to capture DFHBI, which produces fluorescence. This strategy allows the probe to emit fluorescence only upon binding to the target RNA, which reduces the background fluorescence. One advantage of nucleotide-based probe is the easy design of the target RNA-recognizing regions. In addition, the affinity for the target RNA can be modified on the basis of the calculated melting temperature (Tm). In contrast, existing nucleotidebased probes do not have high detectability because of their low signal-to-background ratio. In the case of molecular beacons, the limitation of the FRET efficiency results in substantial background fluorescence. The fluorescence intensities of Spinach and its derivatives are not as high as those of fluorescent proteins. For high-sensitivity detection, such as single-molecule imaging, molecular beacons and Spinach derivatives therefore require tandem arrays of the probes on a target RNA molecule.23,30,31 Comparatively, protein-based probes are not widely used for intracellular RNA imaging, predominantly because of the difficulties of the design of selective recognition for a given target RNA. Generally, an RNA binding protein has a specific target RNA sequence, and the specificity cannot be altered by rational design. However, protein-based probes have many potential advantages. One possible advantage is high detectability that results from the brightness of fluorescent proteins. The use of fluorescent proteins for live cell imaging has been proven in various studies, such as real-time single-molecule imaging in living cells.32,33 Moreover, a transfection technique can introduce expression vectors encoding the probes into a number of cultured cells at a time. The addition of functional protein domains or signal sequences can provide some functions or properties to the probes. These properties of protein-based RNA probes suggest that if it were possible to construct an RNA binding protein domain for which the specificity can be altered to recognize another specific RNA sequence in a custom manner, then RNA probes using this RNA binding protein have ideal design flexibility and functional extensibility. The Pumilio homology domain (PUM-HD), an RNA binding domain of human PUMILIO 1, is a promising candidate for an RNA binding domain that permits the custom design of RNA probes based on the target RNA sequence.34,35

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APPROACHES TO LABELING ENDOGENOUS RNA IN LIVING CELLS: NUCLEOTIDE-BASED PROBES VERSUS PROTEIN-BASED PROBES RNA probes for fluorescence live cell imaging have several requirements; the major requirements are specificity for the target RNA, detectability by fluorescence microscope observation, and the ability to be introduced into living cells effectively via a simple method. The two possible probe molecule designs that satisfy these requirements are nucleotide-based probes and protein-based probes.19−21 Nucleotide-based probes generally comprise two moieties: an oligonucleotide part, including a complementary sequence to part of the target RNA for hybridization, and a fluorescent dye part for fluorescence microscopy detection. In contrast to the fluorescence in situ hybridization method for chemically fixed and permeable cells and tissue slices, the wash-out processes to eliminate excess probe molecules cannot be applied in living cells. Therefore, nucleotide-based probes for live cell RNA imaging require a design that reduces the background fluorescence from excess probes. A representative example of oligonucleotide probes, called a molecular beacon, adopts a Förster resonance energy transfer (FRET) technique to reduce background fluorescence.16,20,22,23 In the basic design of a molecular beacon, the oligonucleotide probe has a fluorescence dye and a quencher on the respective termini of the 201

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Biochemistry

Figure 1. Specific RNA recognition by the PUM-HD. (A) Crystal structure of the PUM-HD and RNA complex. (B) Hydrogen bonding networks between amino acids in the PUM-HD and a target RNA base. (C) Amino acid combinations in the repeated motifs of the wild type PUM-HD and recognized RNA bases. (D) Amino acid combinations for recognizing each RNA base.

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RNA VISUALIZATION USING PUM-HD MUTANT-BASED PROBES The PUM-HD in human PUMILIO 1 binds to the 3′ untranslated region (UTR) of several species of mRNA; subsequently, PUMILIO 1 interacts with protein complexes such as the deadenylase complex to modulate the stability and translation of the mRNAs.36,37 The crystal structure of the PUM-HD shows the presence of eight repeated motifs in the RNA binding region (Figure 1A).38 Each motif interacts with a single RNA base, and therefore, an eight-base RNA sequence of 5′-UGUAnAUA-3′ (where n can be any base) is captured by the PUM-HD. The crystal structure also shows that the three amino acids at the third, fourth, and seventh positions in a helix of the motif form specific hydrogen bonds and van der Waals interactions with the RNA base to determine the species of the captured base (Figure 1B,C). On the basis of this RNA recognition technique, the introduction of site-directed mutagenesis on these three amino acids, especially on the third and seventh, alters the recognized RNA base (Figure 1D).35 This suggests the potential for custom-designed PUM-HD mutants that recognize a specific eight-base RNA sequence different from UGUAnAUA. Moreover, although no motifs for recognizing a cytosine base in wild type PUM-HD exist, several investigations using random amino acid substitutions succeeded in the generation of a motif to recognize a cytosine base.39,40 The dissociation constant between a mutated PUMHD (mPUM) and an eight-base RNA of the corresponding sequence is generally on the order of nanomolar when the three bases of the 5′ terminus of the recognized RNA region are UGU, and ∼100 nM when the 5′ terminus is not UGU.35 Because of the redundancy of the R4 motif, a single mPUM essentially recognizes a seven-base RNA sequence; therefore, the specificity of the target RNA sequence is limited to 4 × 107, which is inferior to the specificity of MS2 or BoxB RNA binding elements and not sufficient to label a target RNA specifically in mammalian cells by using a single mPUM species. As is the case for nucleotide-based probes, the reduction of the background signal from probe molecules that do not bind to the target RNAs is also an important issue in mPUM-based

RNA probes. One of the solutions to overcoming the potential off-target effects of mPUM is the use of fluorescent protein reconstitution technology.41,42 In this technology, the N- and C-terminal fragments of a fluorescent protein are fused to a pair of proteins of interest. When the pair of proteins forms a complex with each other, the fluorescent protein fragments are brought into the proximity of each other, which reconstitutes the full-length fluorescent protein and restores the fluorescence. In this technique, mPUM-based RNA probes are designed as follows: two mPUM molecules that recognize different but neighboring regions in a target RNA are fused to N- and Cterminal fragments of a fluorescent protein. After both mPUM molecules bind to their target sequence in the RNA molecule, the protein fragments approach each other and reconstitute the fluorescent protein (Figure 2). In this strategy, the probe

Figure 2. Schematic of mPUM-based probes adopted during the visualization of ND6 mRNA and Actb mRNA.

molecules do not emit fluorescence before attaching to the target RNA. Furthermore, the probe system recognizes 16 bases in the RNA sequence, improving the specificity. There are several issues with the design of mPUM-based RNA probes. The first is the potential formation of mPUM dimers or oligomers. Several PUMILIO family proteins, such as FBF 1/2 in Caenorhabditis elegans, are reported to interact with each other.35 If this occurs in mPUM-based probes, the reconstitution of fluorescent protein occurs in the absence of the target RNAs, which disrupts the precision of RNA detection. To avoid this artifact, negative control experiments, 202

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Figure 3. Single-molecule imaging of Actb mRNA obtained using the full-length EGFP-based probe. (A) Observed image of Actb mRNA in a living cell. (B) Time course alteration of the fluorescence intensity of a spot showing single-step photobleaching. (C) Directed motion of a fluorescent spot.

and/or dissociation.44,45 This information supports our understanding of detailed molecular mechanisms in the biological systems that include the target molecules. Considering the importance of RNA localization for various physiological phenomena, the dynamics and molecular interaction of target RNAs are critical for understanding the regulation mechanism of these phenomena. The first target for single-molecule imaging using mPUMbased RNA probes was β-actin (ACTB) mRNA.46 As the localization and dynamics of ACTB mRNA in living mammalian cells in response to extracellular stimuli have been well investigated through various approaches, ACTB mRNA represents an excellent model for the assessment of the capability of mPUM-based probes for single-molecule imaging.17,47 To label mouse Actb mRNA, two mPUMs targeting different eight-base RNA sequences in the 3′ UTR of the RNA, 5′-UGUACGUA-3′ and 5′-UGUGCUGU-3′, were prepared and named mPUM3 and mPUM4, respectively. These mPUMs were fused with the N- and C-terminal fragments of split EGFP (Figure 2) and expressed in NIH3T3 cells. When the cells were observed using a total internal fluorescence microscope equipped with an EM-CCD camera, fluorescent spots diffusing in the cytosol were detected. The analysis of the intensity of the fluorescent spots showed a single-step photobleaching of each spot, in addition to a Gaussian distribution of the spot intensities. These are typical properties seen in single-molecule fluorescent spots.48 Moreover, the positions of the spots in chemically fixed cells corresponded to those of Actb mRNAs hybridized with a complementary oligonucleotide probe conjugated with a tetramethyl rhodamine. Thus, the fluorescent spots were identified as single-molecule fluorescence from the probe molecules attached to Actb mRNAs. Yamada et al.46 then subjected the cells to serum stimulation and observed alterations in the amount and localization of Actb mRNAs; the Actb mRNAs were localized to the newly formed lamellipodium. This cellular morphological change and altered localization of the mRNA is consistent with previously reported behavior of Actb mRNA and also with the principle of lamellipodium formation, which requires the intensive construction of actin cytoskeletons.49 Moreover, the simultaneous observation of Actb mRNA and tubulin showed transportation of the Actb mRNA along a microtubule. The velocity of this transportation was consistent with those of cytoskeletal motor

such as the use of an mPUM without a target recognition sequence, should be performed. Analysis of the Mobility of Mitochondrial RNA ND6. The first trial of RNA visualization by using an mPUM-based probe in living cells was performed on NADH dehydrogenase 6 (ND6) mRNA, which is transcribed from mitochondrial DNA (mtDNA) and therefore is localized in the mitochondria.43 Ozawa et al. constructed two mPUMs that recognize two eightbase sequences in the ND6 mRNA (5′-UGAUGGUU-3′ and 5′-UGGAUAUA-3′). The dissociation constants of the target sequences and the two mPUMs were 163 and 92 nM, which are sufficient to label the target ND6 mRNA in living cells. The two mPUMs were fused with split fluorescent protein [enhanced green fluorescent protein (EGFP) or Venous] fragments, together with mitochondrial targeting signal sequences. The fluorescence microscopy observations of living cells expressing the mPUM-based ND6 mRNA probe revealed that the fluorescence from the probe overlapped intensively with that of the double-stranded DNA in the mitochondria, which indicated that the ND6 mRNA was attached to mtDNA. A fluorescence recovery after photobleaching (FRAP) experiment was then performed to estimate the dynamics of ND6 mRNA in mitochondria. The cells expressing the ND6 mRNA probe molecules showed just 20% recovery of the fluorescence intensity in the photobleaching spot 30 min after the photobleaching, whereas the cells expressing EGFP in the mitochondria as a control showed 85% fluorescence recovery. These results suggested that the diffusion motion of the probe molecules was restricted in the mitochondria. In addition, treatment with hydrogen peroxide, which induces decomposition of mtDNA by oxidation stress, increased the mobility of the ND6 mRNA. Therefore, the experiment showed that ND6 mRNA tethered on mitochondrial DNA was released upon degradation of the DNA, which demonstrated the ability of mPUM-based probes to monitor the dynamics of a target RNA in living cells. Single-Molecule Imaging of β-Actin mRNA Using an mPUM-Based Probe. An approach for further detailed analysis of molecular motion in living cells is single-molecule imaging. Single-molecule imaging is one of the most direct methods for obtaining non-averaged information about the motion of target molecules in living cells, including real-time diffusion, transportation, and molecule−molecule association 203

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Biochemistry proteins that “walk” along microtubules. This motion confirmed the previously reported transportation of Actb mRNA by motor proteins.50 Thus, the ability of the mPUMbased probe to visualize the transportation and localization of single mRNA molecules in living cells was demonstrated. The adoption of a split EGFP reconstitution technique is used to eliminate background fluorescence from excess probes. Given the high copy number of Actb mRNA, almost all the probe molecules bind to the target RNA if the expression level of the probe is maintained at a low level. Under these conditions, the background fluorescence is not severe, even without the use of the fluorescent protein reconstitution technique. Therefore, an Actb mRNA probe consisting of the two mPUM domains and full-length EGFP was examined.51 Similar to the split EGFP-containing probe, the full-length EGFP-containing probe succeeded in labeling Actb mRNA and visualized the motion of its directed transportation (Figure 3). Full-length EGFP-based probes have another advantage over the split EGFP-based probes. Contrary to the requirement for two subunits to target the RNA using split fluorescent proteinbased probes, full-length EGFP-based probes need only a single subunit for RNA labeling. This property makes it easy to introduce and control the expression level of the probe in living cells. Thus, full-length EGFP-based probes are a possible choice in cases in which the target RNA is strongly expressed. Molecular Mechanisms of a Noncoding RNA TERRA. Recently, the mPUM-based probe method was applied to investigate the molecular mechanisms of a noncoding RNA.52 The target was a telomeric repeat-containing RNA (TERRA), the transcription product from the telomere regions at the termini of chromosomal DNA.53−57 A TERRA possesses a subtelomeric region and a telomeric repeat region, derived from telomeres on chromosomal DNA, and is considered to be implicated in telomere maintenance. Two different molecular models of maintenance have been proposed: a scaffold model in which a TERRA is included in and stabilizes ribonucleoprotein (RNP) complexes formed on a telomere,58 and a decoy model in which a TERRA transports or removes protein components in the RNP complexes on telomeres.59 The direct observation of a TERRA in living cells through single-molecule imaging was strongly expected to provide sufficient information to confirm which model is correct. In contrast with Actb mRNA, a TERRA has a repeat region of (UUAGGG)n derived from a telomere. This region was addressed in the design of an mPUM-based TERRA probe.52 The probe for a TERRA consists of three domains: an Nterminal fragment of EGFP, an mPUM designed to recognize the UUAGGGUU sequence (mPUMt), and a C-terminal fragment of EGFP. Considering the nuclear localization of TERRA, three repeats of a nuclear localization signal sequence are fused to the N-terminus of the probe. Upon attachment of multiple probe molecules to the telomere repeat region of a TERRA, the N-terminal EGFP fragment in a probe and the Cterminal EGFP fragment of the next probe approach each other, reconstitute EGFP, and emit fluorescence (Figure 4). In addition, a derivative probe in which mPUMt is replaced by another mPUM that targets a sequence in Actb mRNA was prepared as a negative control. Although the TERRA probe emits fluorescence in the nucleus, the derivative did not emit fluorescence, even under the same expression condition that was used for the TERRA probe. This indicates the precise labeling of a TERRA by the probe in living cells. Because of the large number of telomeric repeats in a TERRA, many

Figure 4. Schematic of the principle of the TERRA probe.

reconstituted fluorescent proteins are expected to be generated on a TERRA molecule. However, most of the observed fluorescent spots indicated single fluorescent molecules, confirmed by single-step photobleaching. This suggested that mPUMt did not have the ability to attach to a G-quartet structure, which the telomeric repeat forms, but instead bound the flexible terminal region of a TERRA. First, a TERRA was visualized in living U2OS cells by simultaneous use of the probe with telomeres labeled with a SNAP-fused telomere marker protein, TRF1. When observed by using fluorescence microscopy, a TERRA showed diffusion motion, whereas telomeres were almost static. The detailed analysis of the obtained video revealed the presence of two modes of TERRA motion, diffusive and stationary (Figure 5A); in addition, the spatial distribution of TERRA showed a unique feature. As a TERRA performs the function of telomere maintenance, a TERRA was expected to mostly colocalize with telomeres. Although this colocalization was observed, a substantial part of TERRAs also localized around the telomeres. The distance between the TERRA and the nearest telomere was approximately 1 μm. This suggested the presence of a scaffold to capture TERRAs around the telomere, although the composition of this scaffold remains unknown. To investigate the function of the TERRAs that localize around a telomere and the implications for telomere-related proteins, triple-color simultaneous observation of TERRAs, telomeres, and telomere-related proteins was performed (Figure 5B−E). The telomere-related protein, HP1, which is included in the scaffold model,58 and hnRNPA1, which is implicated in the decoy model,59 were observed. In addition, H2A, which is considered not to interact with TERRA,60 was observed as a negative control. In this experiment, TERRA and the telomere-related proteins showed diffusion motion, whereas the telomeres were almost static. All the proteins showed colocalization with TERRA; consequently, the colocalization duration time was analyzed. For H2A, which is not reported to interact with TERRA, the colocalization of H2A and TERRA could represent accidental overlapping; the duration time was ≤0.2 s. The colocalization duration for HP1 and TERRA was almost the same as that of H2A and TERRA. However, the duration time of hnRNPA1−TERRA colocalization was significantly longer that the other two. Thus, hnRNPA1 was identified as an interacting partner of a TERRA. Importantly, the interaction between hnRNPA1 and a TERRA was concentrated in the region around the telomeres. During this colocalization event, diffusive hnRNPA1 molecules arrived and interacted with TERRAs in a stationary mode. On the basis of these single-molecule imaging results, a putative model of TERRA function was proposed (Figure 6). Initially, hnRNPA1 is included in an RNP complex on the telomere. When TERRAs appear around the telomere, hnRNPA1 interacts with the TERRAs, rather than with the telomere, which alters the state of the telomere and possibly switches the telomere 204

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Figure 5. Fluorescent images of a TERRA. (A) Image of dual-color simultaneous observation of a TERRA and a telomere: green, TERRA; red, telomere; blue, trajectory of a TERRA. The arrowhead indicates a transiently confined region of TERRA motion. (B−E) Images of triple-color simultaneous observation of a TERRA, a telomere, and hnRNPA1: (B) TERRA labeled with the present probe, (C) iRFP-TRF1 that represents telomeres, (D) hnRNPA1-SNAP-TMR, and (E) the merge image.

Figure 6. Schematic of the proposed hypothesis on a TERRA function for telomere maintenance.

another PUM family protein, FBF-2 of C. elegans, to be a platform for the recognition of endogenous RNA.64 In this study, the authors created a mutant of FBF-2 to address to a sequence in the 3′ UTR of endogenous cyclin B1 mRNA. The mutant, named Neo-PUF, was confirmed not to disturb the innate expression level of cyclin B1. Moreover, Neo-PUF fused with a segment of the poly(A) binding protein, which is known to stimulate translation, induced an increase in the cyclin B1 protein level. These results showed that FBF-2 was a potential alternative platform for binding target RNA with specificity and without the disruption of RNA properties. Pentatricopeptide repeat (PPR) proteins also provide a potential candidate for a specific RNA recognition platform. In PPR, a two-amino acid combination in each repeat motif determines the specificity for RNA base recognition; in total, an 18-base RNA is recognized by PPR. Although the complete code has not been revealed, the mechanism of the RNA binding specificity of PPR is under investigation. Thus, PPR may also be a candidate as an alternative to human mPUM. There are several potential RNA-recognizing platforms besides Pumilio family proteins. One potential candidate for providing the platform is Cas proteins. The CRISPR/Cas systems are generally targeted to double-stranded DNA, but several modifications were recently performed to enable the systems to target RNAs.65,66 Nelles et al. developed a fusion protein of dCas9 and GFP, which included a single guide RNA

maintenance function on or off. Although further analyses of the dynamics and biochemical properties of TERRA and its related molecules are required to confirm and understand the detail of this hypothesis, these findings from single-molecule imaging are unique. RNA Imaging in Non-Mammalian Cells. RNA imaging by using mPUM derived from human Pumilio 1 has been performed not only in mammalian cells but also in cells from non-mammalian organisms. A typical example of this is the visualization of genomic RNA in the tobacco mosaic virus and potato virus X.61,62 In this study, two mPUMs, which recognize adjacent eight-nucleotide sequences in the target RNA, are fused with the N- and C-terminal fragments of split mCitrine. This probe enabled the localization of the virus in the infected cells to be visualized by using fluorescence microscopy. These results showed that the ability of human mPUMs to perform RNA labeling is not limited to mammalian cells. mPUMs can be widely used in cells from various organisms.

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POTENTIAL ALTERNATIVES TO HUMAN MPUM There are some potential risks in the use of human mPUM in mammalian cells. For example, human PUF2 reportedly interacted with Argonaute and eEF1A on the 3′ UTR of an mRNA and repressed translation efficiency.63 As a result of similar potential risks in mPUM, alternatives to human mPUM are desirable. Wickens and co-workers reported the ability of 205

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Biochemistry and PAMmer that hybridizes to a target RNA. Actb mRNA localization in fixed cells was visualized using this method.

can provide powerful methodologies for exploring RNA biology.

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CONCLUSIONS AND FUTURE OUTLOOK Various RNA imaging techniques have been developed. Among them, mPUM-based probes represent an excellent system for monitoring the motion of a target RNA in living cells. There are several advantages to mPUM-based RNA probes. mPUM probes have the design flexibility to target a variety of RNAs while retaining specificity. In previous studies, mPUM-based probes have been applied to visualize ND6 mRNA,43 ACTB mRNA,46,51 TMV RNA,62 and a noncoding RNA, TERRA.52 The addition of signal peptide sequences to mPUM-based probes allows the probe to localize to a specific region in the cell samples. In the case of ND6 mRNA and TERRA visualization, the probes were localized in the mitochondria43 and nuclei,52 respectively, through the attachment of the corresponding signal sequences. As mPUM-based probes are protein-based one, fluorescent proteins such as EGFP, which has sufficient fluorescence intensity even for single-molecule live cell imaging, can be used in the probes; thus, a high sensitivity for target RNA detection in living cells can be attained. mPUM-based probes are therefore widely applied to visualize a variety of RNAs in living cells. The future evolution of mPUM-based probes can proceed in two directions. One direction is the further improvement of visualization. The development of novel fluorescent proteins with high stability, brightness, and color variation67 should enable observations with a higher time resolution, performed over a longer time, and the incorporation of multicolor RNA imaging. Other tag proteins, such as SNAP and Halo, may provide useful alternatives to fluorescent proteins. Recently reported organic fluorescent dyes that can be applied to these tag proteins have excellent properties in stability and brightness.68 In addition, improvements should focus on target specificity, which is important for precise RNA imaging. Several methods for the screening of an mPUM library in bacterial cells for obtaining mPUMs with higher affinities and specificities for target RNAs have been reported.69,70 These screening methods would be applicable not only to human mPUMs but also to PUF proteins from other organisms, which would be helpful for the development of a novel RNA-recognizing platform based on optimized mPUMs. The other possible future direction of mPUM-based probes is the development of mPUM-based molecular tools to control RNA functions.71,72 For example, Kane and co-workers have already reported a PUM-HD-based tool for controlling the translation of a reporter luciferase gene. This tool comprises two molecules: a photoreceptor protein CRY2 fused with a PUM-HD that captures the luciferase mRNA and CIB, the dimerization partner of CRY2, which is fused with translation initiator eIF4E. Upon irradiation with blue light, the CRY2− CIB interaction allows eIF4E to approach the 5′ end of the mRNA and initiate the translation of the luciferase gene. Methods that allow mPUM to control the behavior and functions of mRNA will provide useful tools for determining the mechanisms and roles of RNA in physiology. In conclusion, the properties of mPUM, such as design flexibility and high affinity for the target RNA sequence, have expanded the capability of protein-based tools for the visualization and control of RNAs in living cells. RNAs represent an important target of interest in the postgenome era, and molecular tools that make use of mPUM technologies

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +81-35841-4376. Fax: +81-3-5841-7629. ORCID

Hideaki Yoshimura: 0000-0002-7287-7712 Funding

This work was supported by the Japan Society for the Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (Grants-in-Aid for Encouragement of Young Scientists A 25708025 and Grant-in-Aid for Scientific Research (B) 16H04162). This work was also supported in part by grants from the Asahi Glass Foundation and the Sumitomo Foundation. Notes

The author declares no competing financial interest.

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