Tagging allows faithful tracing of expression and enhances biochemical detection of Ran Binding Protein 9 in vivo

The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. Opposite to RanBP9 constitutive knock-out (KO) animals, both male and female RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The presence of the tags allows unequivocal detection of RanBP9 in tissues both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. In summary, we report here the generation of a new mouse in which RanBP9 expression and interactions can be reliably studied by the use of commercially available αtag antibodies and overcome some of the existing limitations in the study of this protein in vivo.


Introduction
RANBP9 is a scaffold protein whose biological functions are not completely understood [1]. Since its initial description, it has been reported to interact with a number of proteins that are located in different cellular compartments and have different biological functions [1]. In many instances RANBP9 expression affects the half life and stability of other proteins [1]. This function is in accordance with the model in which RanBP9 is part of an E3 ligase multi-subunit structure called the CTLH (C-Terminal to LisH) complex that can induce target proteins degradation via the proteasome pathway. This E3 ligase structure is believed to be the equivalent of the GID (Glucose-Induced degradation Deficient) complex in S. cerevisiae, which is involved in responding to changes of nutrient availability in the microenvironment [2][3][4][5]. Although not well studied in mammals, the human CTLH complex has been recently reported to be a heterodecameric structure that, in addition to RANBP9, includes its paralog RANBP10 and nine other poorly studied proteins (ARMC8, GID4, GID8, MAEA, MKLN1, RMND5A, RMND5B, WDR26, and YPEL5) [5][6][7][8].
Objective difficulties impair the investigation of the cellular pathways and mechanisms in which RANBP9 takes part. Among other issues, the limited specificity and affinity of commercially available antibodies pose significant technical challenges. The existence of a paralog named RANBP10 [9,10] with high similarity in long parts of the protein limits the RANBP9 specific sequence available to raise antibodies. In addition, some of the sequences might not have biochemical features ideal to induce a robust humoral immune response. Therefore, many of the commercially available αRANBP9 antibodies fall short in their detection power, reliability, and/or specificity.
We have previously used the only existing antibody validated by the Human Protein Atlas (HPA050007; www.proteinatlas.org) for immunohistochemical (IHC) detection of human RANBP9 in formalin-fixed paraffin embedded Non-Small Cell Lung Cancer (NSCLC) specimens [11]. Although specific for human RANBP9, in our hands HPA050007 does not seem to be recognizing mouse RanBP9 with similar affinity and signal-to-noise ratio is low when assessed by Western Blot (WB). In addition, WB analysis often shows the presence of additional bands at lower molecular weight that might be due to non-specific binding.
We have used and will continue to use human cell lines for in vitro studies. However, to better recapitulate organismal physiology, a significant part of our ongoing studies on RANBP9 involvement in tumor development and response to therapy necessarily takes advantage of in vivo murine models. In this regard, we had previously generated the constitutive RanBP9 knockout (KO) animal. On a hybrid C57Bl/6-S129 genetic background, homozygous KO mice were mostly dying hours after birth. A small cohort of survivors showed small body size and severe sterility in both males and females [12].
Using reagents from the International Mouse Phenotyping Consortium (http://www.mousephenotype.org/; IKMC project nr: 44910), we have now engineered the conditional KO mouse that allows the study of RanBP9 loss of function in vivo, which recapitulates the phenotype of the constitutive KO when the gene is ubiquitously deleted [13]. However, to overcome the limitations in the detection of endogenous RanBP9, we have decided to engineer a novel RanBP9 strain in which the wild type protein is fused to commonly used tags against which there are reliable and commercially available antibodies. mouse line in which we have inserted a double epitope tag (V5-HA) at the C-terminus of the protein. We show that tagging of RanBP9 allows unequivocal detection of the protein both by IHC and biochemistry while does not affect its essential biological functions.
Because we knocked in the tags leaving intact the 3'-untranslated region (UTR) of the RanBP9 genomic locus, the expression of the protein faithfully recapitulates the wild type (WT) expression. Therefore, the RanBP9-TT strain becomes a powerful tool to dissect in vivo the biology related to RanBP9 functions allowing its unequivocal detection in murine tissues and cells.

Generation of the RanBP9-TT animals.
We used CRISPR/Cas9 to knock-in the double tag V5-HA at the C-terminus of RanBP9 (Figure 1; Figure S1). For targeting purposes, we employed the online Benchling software (https://www.benchling.com/). We selected the guide RNA (sgRNA) with the best specificity and efficiency scores closest to the insertion site before the stop codon ( Figure 1A and Figure S1A-C). C57Bl/6Tac WT fertilized eggs were used for the generation of founders (F0) mice. Two F0 animals (#1 and #2) were selected for further breeding and propagation of the RanBP9-TT colony. Both founders produced progeny (F1 mice) positive for the correct insertion of the double tag and animals from both lines were used for this work. Sanger sequencing showed that F1 animals from both founder lines contained the correct in frame insertion of the V5-HA double tag ( Figure 1C). In order to mitigate the presence of CRISPR/Cas9 potential off-target effects, we crossed F1 animals a second time to wild type C57Bl/6Tac mice and generated F2 progeny that were used for experimental purposes.
These results show that the V5-HA double tag at the C-terminus of endogenous RanBP9 was successfully inserted as designed.

Addition of tag at the C-terminus does not cause lethality or infertility.
On a mixed C57Bl/6-S129 background using gene-trapped ES cells from the Baygenomics consortium [14], homozygous inactivation of RanBP9 causes early postnatal lethality in mice [12]. The pure C57Bl/6 background seems to worsen the phenotype and homozygous KO animals immediately after birth are rarely found, if any [15].
We observed that RanBP9-TT homozygous knock-in (

Addition of V5-HA double tag at the C-terminus allows faithful immunohistochemical detection of endogenous RanBP9.
We then examined the expression of RanBP9 in mouse tissues by immunohistochemistry (IHC). We obtained tissue sections from RanBP9 WT and RanBP9-TT animals and stained them with αRanBP9 specific antibody (HPA050007), αV5, or αHA specific antibody. In Figure 2, a selected array of tissues with significant expression of RanBP9 is shown. In RanBP9-TT Cerebellum, Testis, and Lung αV5 and αHA staining are similar in pattern but sharper than the αRanBP9 specific antibody. On the other hand, αtag antibodies do not detect RanBP9 in sections of the same tissues from WT animals ( Figures S2). Finally, we also performed a more comprehensive IHC survey of several other organs/tissues using both αV5 ( Figures S3) and αHA (Figures S4).

V5-HA-tagged RanBP9 mRNA and protein expression is similar to wild type RanBP9.
Next, we sought to measure the amount of RanBP9-TT protein and messenger RNA in different tissues by qRT-PCR and WB. We extracted protein from tissues/organs with various levels of reported expression [12]. A WB analysis probing with αRanBP9 animals. Accordingly, we did not detect any significant difference between levels of RanBP9 mRNA in wild type and RanBP9-TT mice ( Figure 3D).
These results show that the expression of RanBP9-TT is similar to wild type RanBP9 and there are no perturbations of expression caused by the insertion of the V5-HA tag.

RanBP9-TT protein maintains the known RanBP9 WT interactions.
RanBP9 is part of a poorly studied multi-subunit complex called the CTLH complex [6]. Therefore, we sought to determine whether the addition of the double V5-HA tag at the C-terminus of RanBP9 interfered with its binding to the CTLH complex. To this aim, we generated homozygous RanBP9-TT mouse embryonic fibroblasts (MEFs) and performed immunoprecipitation (IP) using resin-conjugated αHA antibodies. We ran four different gels and we probed the IP fractions by WB. We were able to purchase specific antibodies These results clearly indicated that V5-HA-tagged RanBP9 interacts with known partners of WT RanBP9. However, since we were not able to probe by WB for the remaining members of the CTLH complex, we decided to perform a proof-of-principle small-scale IP using αV5 antibody followed by high-resolution mass spectrometry (MS) analysis.
Again, our experiments for IP-MS/MS analysis included lysates from RanBP9 WT MEFs as negative controls. As shown in (Table 2) (Table 2).
Taken all together these results indicate that the addition of the V5-HA to RanBP9 Cterminus does not prevent its participation into the CTLH complex.

Discussion
Specific and reliable detection of proteins in vivo is difficult and it is estimated that researchers waste millions of dollars every year on the use of non-specific antibodies [16,17]. Challenges are harder when the proteins of interest are not commonly studied and thus validated reagents are limited or missing. In some instances, the existence of paralogs with extensive homology further complicates the generation of tools that can unequivocally detect only the specific target. This is the case for the poorly studied scaffold protein RanBP9 and its paralog RanBP10 that share high homology in 4 out of the 5 annotated protein-protein interaction domains [9,10]. To date, there is only one validated antibody for detection by IHC of human RANBP9 reported in the Human Protein Atlas project (www.proteinatlas.org). This reagent is not tested and validated for detection of mouse RanBP9 (HPA050007). In our hands, this antibody did not appear to have the sensitivity at levels similar to antibodies from other sources that performed better for biochemical detection of both human and mouse RanBP9 by conventional methods such as IP and WB. While expectations are to use multiple antibodies for the detection of a target protein, this necessity generates frustration for the quality of results and cost of reagents.
To overcome these limitations and obtain a more reliable in vivo tool for the study of RanBP9 in physiological and pathological paradigms, we have decided to use CRISPR/Cas9 and generate RanBP9-TT mice by the addition of a double V5-HA tag at the C-terminus of RanBP9. In fact, biochemical tags are an excellent solution for detection problems and are widely employed for research purposes [18].
In order to minimize steric hindrance, we have chosen to use relatively small tags of 9 amino acids. In addition, we have also chosen to use two of them (V5 and HA) in sequence to increase the detection power and have at least two different options.
The engineering of RANBP9-TT mice has been possible thanks to the use of CRISPR/Cas9. In fact, compared to the classical use of ES cells [19], in addition to being faster and efficient in different genetic backgrounds, this new technology offers the critical advantage of not leaving any "scar" in the edited allele other than the desired engineered mutation [20,21]. Therefore, in RanBP9-TT mice RanBP9 C-terminus as well as the 3'-untranslated region remained intact preserving not only the protein structure, but also the endogenous regulation of expression (Figure 1 and Figure S1). Figure 3A-C clearly shows that RanBP9 protein levels in all analyzed organs/tissues are comparable between RanBP9 WT and TT mice. Interestingly, detection of RanBP9 by the use of αV5 ( Figure 3B) appears to have superior sensitivity and specificity (lower background) compared not only to the αHA (Figure 3C), but also the αRanBP9 specific antibody ( Figure 3A). qRT-PCR measurements confirmed that there are no differences in RanBP9 mRNA levels in different organs/tissues from RanBP9-TT compared to RanBP9 WT mice ( Figure 3D). However, it is interesting to note that levels of mRNA not always correlate with protein levels. While testes yield the highest amount of protein and mRNA, lungs showing the second highest level of protein display a level of messenger lower than cerebellum where RanBP9 protein is significantly less. Similarly, liver contains the lowest amount of RanBP9 protein, but a level of mRNA higher than both spleen and lungs. These results might be explained as technical artifacts or suggest the existence of post-transcriptional regulatory mechanisms of gene expression. In this regard, we previously observed this poor correlation between levels of RanBP9 mRNA and protein in human NSCLC samples [11]. This phenomenon leads us to believe that it is always necessary in cancer cells and tissues to measure protein levels to evaluate the expression of RanBP9 rather than relying on the amount of RNA. Therefore, a tool like the RanBP9-TT mice allowing the unequivocal detection of RanBP9 protein becomes invaluable for studies in vivo. Indeed, the IHC analysis in If crossed to in vivo models of different malignancies, RANBP9-TT will be of value in studying the role of this protein during tumorigenesis. One priority for our group is to show how relevant this protein is in lung tumorigenesis and how it is specifically expressed in tumor cells versus populations of the microenvironment. Further, we have shown that DNA damage elicits post-translational modifications and alters RANBP9 subcellular localization [22]. Since in vitro studies completely miss the complexity of organismal responses and adaptive mechanisms that ensue in vivo, we are planning to use our new mouse model to study how the protein is modified and changes localization following genotoxic stress, for example.

The WB analysis in
We have deliberately decided the placement of the TT tag based on in vitro evidence from our lab and other groups indicating that C-terminus tagging of RanBP9 was not impairing RANBP9 movement inside cells upon specific stimulation [22][23][24]. However, such a strategy still presents the intrinsic and real risk that the added tag might block RanBP9 interactions with other proteins and interfere with its biological functions. Here, we show that vital functions and known interactions of RanBP9 are preserved in RanBP9-TT mice. In fact, RanBP9 constitutive KO mice show a severe perinatal lethality and the occasional survivors, both males and females, are runt and sterile displaying gonads completely devoid of germ cells [12,15]. On the contrary, RanBP9-TT homozygous animals do not show any obvious phenotype. A basic observational and anatomical examination shows that TT-knock-in animals develop and grow similarly to WT littermates. Gross anatomic parameters and reproduction abilities are similar in RanBP9-TT mice compared to WT littermates ( Table 1). Specifically, RanBP9-TT mice display normal histological architecture of both testes and ovaries. Sperm and oocytes are present in RanBP9-TT animals without any appreciable difference compared to WT controls (Figure 2, Figure S2, Figure S3, Figure S4). Since sperm and oocytes are the cells in which RanBP9 is expressed at its highest levels, these results clearly show that the TT tag does not have undesired deleterious effects and it is not interfering with fundamental biological functions of RanBP9 necessary for germ cell development and maturation [12,25].
Obviously, it is impossible for us to completely exclude the possibility that the V5-HA tag is interfering at the molecular level with yet unknown biological functions of RanBP9. However, this protein is part of an evolutionary conserved and ubiquitously expressed multi-subunit E3-ligase called the CTLH (C-Terminal to LisH) complex [4,[6][7][8]. Therefore, a valid, if not the best, strategy for us to test whether the TT tag interfered  Figure 4D). Identification of 6 of the 10 known CTLH members (not including RanBP9) strongly indicates that the tagged protein is incorporated into the complex and retains its activity and functionality. As reliable antibodies for the remaining CTLH members (Gid4, RanBP10, Rmnd5B, and Ypel5) are not commercially available, to further validate our findings, we performed αV5 IPs from RanBP9-TT and WT MEFs followed by tandem mass spectrometry analysis ( Table 2) In summary, we show here that we have engineered a new mouse strain bearing a tagged RanBP9 protein that retains all the known features and abilities of the endogenous wild type molecule. The TT tag allows reliable immunohistochemical detection and enrichment from cell lysates of RanBP9 and its known main binding partners and members of the CTLH macromolecular complex. This new tool will be invaluable in studying RanBP9 functions in the context of the entire organism in physiological and pathological conditions.

CRISPR/Cas9 RanBP9-TT mouse generation
The Genetically Engineered Mouse Modeling Core of the Ohio State University Comprehensive Cancer Center generated RanBP9-TT mice by using CRISPR/Cas9 technology. Briefly, CRISPR targeting strategy was designed using the online algorithm

Adult mouse tissues were sectioned and stained at the Comparative Pathology & Mouse
Phenotyping Shared Resource of the Ohio State University Comprehensive Cancer Center as previously described [26]. Briefly, tissues were fixed in 10% (v/v) formalin, routinely processed, and embedded in paraffin for immunohistochemical characterization.
Formalin-fixed, paraffin-embedded tissues were cut into 4-µm-thick sections, were dewaxed in xylene and rehydrated through graded ethanol solutions to water. Antigens were retrieved by heating the tissue sections at 96-98°C for 25 minutes in citrate solution (10 mmol/L, pH 6.0) using a vegetable steamer. Sections were cooled at room temperature for 15 minutes, and immersed in methanol in the presence of 0.3% hydrogen peroxide for 15 minutes to block the endogenous peroxidase activity, rinsed in tap water, and then subsequently rinsed in PBS for 5 minutes. The sections were then stained with the indicated antibodies using a Dako Autostainer. Rabbit anti-RanBP9 antibody (Sigma-Aldrich; cat. nr. HPA050007) at room temperature for 1hr. As negative controls, slides were incubated with rabbit IgG, and antibody diluent instead of the primary antibody.
The sections were rinsed with buffer, and then incubated with horseradish peroxidaselabeled goat against rabbit secondary antibody for 30 minutes. The sections were rinsed and then incubated with VECTASTAIN® Elite® ABC HRP Reagent, R.T.U for 30 minutes. DAB was used as the chromogen, and hematoxylin as the nuclear counterstain.
Sections were then dehydrated, cleared, and mounted permanently with glass coverslip.
Slides were photographed with an Olympus BX45 light microscope with attached DP27 digital camera and corresponding CellSen software (B&B Microscopes Limited, Pittsburgh, PA).

WB analysis of RANBP9 expression.
Mouse tissues were homogenized on ice in NP-40 buffer supplemented with Halt Protease and Phosphatase Inhibitor cocktail (Thermo Fisher Scientific cat. nr. 78442).
Protein concentration was determined using the Bio-Rad protein assay dye (Bio-Rad cat. nr. 5000006). Western blot analysis was performed using 30 to 50 µg proteins run on Mini-PROTEAN TGX precast gels (Bio-Rad cat. nr. 456-1094). αRANBP9 primary antibody was purchased from Abcam (cat. number ab140627) and used at a dilution of 1:2,000 in 5% non-fat milk in TBS-T. Signals were detected with HRP-conjugated secondary antibodies (GE Healthcare) and the chemiluminescence substrate Supersignal wet pico PLUS (Thermo Fisher Scientific). Equivalent loading among samples was confirmed with αGAPDH (Cell Signaling cat. nr. 3683S).

RT-PCR analysis of RanBP9 expression.
Murine tissues were homogenized in ice using 1 ml TRIzol Reagent (Life technologies cat. nr. 15596018) following the manufacturer's protocol. The extracted RNA was

Immunoprecipitation
RanBP9 was immunoprecipitated from total protein extracts using monoclonal αHA-Agarose antibody (Sigma cat. nr. A2095-1ML). Briefly, 1mg of total protein extracts were pre-cleared by incubating with Pierce TM Protein A/G Plus Agarose (Thermo Scientific cat. nr. 20423) for 1 hr at 4°C. Lysates were centrifuged at 4°C, the supernatant was collected and incubated with 5 µg of the primary antibody overnight at 4°C. Proteins interacting with RanBP9 were detected in the immunoprecipitates of RanBP9 using wettransfer WB. Interactors of CTLH complex were detected using the following antibodies,

Immunoprecipitation Tandem Mass Spectrometry (IP-MS/MS)
Cells were separated into cytoplasmic and nuclear fractions prior to immunoprecipitation as described in Mohammed et al [27]. Briefly, αV5 antibody (5 µg, Invitrogen cat. nr.

R96025) was incubated with Pierce TM Protein A/G Magnetic Beads (Thermo Scientific
cat. nr. 88803) at 4°C with rotation overnight. Following washes to remove unbound antibody, 1 mg of cytoplasmic or nuclear protein extract was incubated with the beadantibody mixture at 4°C with rotation overnight. After stringent washes to remove nonspecific proteins, beads were equilibrated in 100 mM ammonium bicarbonate with 3 washes prior to on-bead digestion with trypsin (800 ng; Promega cat. nr. V5280) overnight at 37°C 800 rpm. Digestion continued the next day for 4 hours after samples were supplemented with an additional bolus of trypsin (800 ng). Beads were placed in magnetic stand to collect supernatants and dried down in Speedvac concentrator.
Prior to mass spectrometry analysis, peptides were resuspended in loading buffer (2% ACN, 0.003% TFA) and quantified via Nanodrop. Chromatography separation and mass spectrometry methods were performed as described in Scheltema et al for data dependent acquisition on a Q-Exactive HF (Thermo Fisher Scientific) [28]. Tryptic peptides (1000 ng) were isocratically loaded onto a PepMap C18 trap column (300µm x 5mm, 100Å, 5µm) at 5µL min -1 and analytical reversed phase separations were performed on a Dionex UltiMate 3000 RSLCnano HPLC system coupled to an EASYSpray PepMap C18 column         Prism®) did not result in any significant difference between the expected and the observed number of animals of a specific genotype or gender.