The tRNA pseudouridine synthase TruB1 regulates the maturation of let‐7 miRNA
2020; Springer Nature; Volume: 39; Issue: 20 Linguagem: Inglês
10.15252/embj.2020104708
ISSN1460-2075
AutoresRyota Kurimoto, Tomoki Chiba, Yoshiaki Ito, Takahide Matsushima, Yuki Yano, Kohei Miyata, Yuka Yashiro, Tsutomu Suzuki, Kozo Tomita, Hiroshi Asahara,
Tópico(s)MicroRNA in disease regulation
ResumoArticle14 September 2020free access Source DataTransparent process The tRNA pseudouridine synthase TruB1 regulates the maturation of let-7 miRNA Ryota Kurimoto Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Search for more papers by this author Tomoki Chiba Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Search for more papers by this author Yoshiaki Ito Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Research Core, Research Facility Cluster, Institute of Research, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Search for more papers by this author Takahide Matsushima Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Search for more papers by this author Yuki Yano Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Search for more papers by this author Kohei Miyata Department Obstetrics and Gynecology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan Search for more papers by this author Yuka Yashiro Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan Search for more papers by this author Tsutomu Suzuki orcid.org/0000-0002-9731-1731 Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan Search for more papers by this author Kozo Tomita Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan Search for more papers by this author Hiroshi Asahara Corresponding Author [email protected] [email protected] orcid.org/0000-0002-5215-8745 Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Department of Molecular and Experimental Medicine, The Scripps Research Institute, San Diego, CA, USA Search for more papers by this author Ryota Kurimoto Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Search for more papers by this author Tomoki Chiba Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Search for more papers by this author Yoshiaki Ito Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Research Core, Research Facility Cluster, Institute of Research, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Search for more papers by this author Takahide Matsushima Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Search for more papers by this author Yuki Yano Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Search for more papers by this author Kohei Miyata Department Obstetrics and Gynecology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan Search for more papers by this author Yuka Yashiro Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan Search for more papers by this author Tsutomu Suzuki orcid.org/0000-0002-9731-1731 Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan Search for more papers by this author Kozo Tomita Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan Search for more papers by this author Hiroshi Asahara Corresponding Author [email protected] [email protected] orcid.org/0000-0002-5215-8745 Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Department of Molecular and Experimental Medicine, The Scripps Research Institute, San Diego, CA, USA Search for more papers by this author Author Information Ryota Kurimoto1, Tomoki Chiba1, Yoshiaki Ito1,2, Takahide Matsushima1, Yuki Yano1, Kohei Miyata3, Yuka Yashiro4, Tsutomu Suzuki5, Kozo Tomita4 and Hiroshi Asahara *,*,1,6 1Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan 2Research Core, Research Facility Cluster, Institute of Research, Tokyo Medical and Dental University (TMDU), Tokyo, Japan 3Department Obstetrics and Gynecology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan 4Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan 5Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan 6Department of Molecular and Experimental Medicine, The Scripps Research Institute, San Diego, CA, USA *Corresponding author (lead contact). Tel: +81 03 5803 5015; Fax: +81 03 5803 5810; E-mails: [email protected]; [email protected] EMBO J (2020)39:e104708https://doi.org/10.15252/embj.2020104708 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Let-7 is an evolutionary conserved microRNA that mediates post-transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let-7 biogenesis is tightly regulated by several RNA-binding proteins, including Lin28A/B, which represses let-7 maturation. To identify new regulators of let-7, we devised a cell-based functional screen of RNA-binding proteins using a let-7 sensor luciferase reporter and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let-7 family members. Rather than inducing pseudouridylation of the miRNAs, high-throughput sequencing crosslinking immunoprecipitation (HITS-CLIP) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem-loop structure of pri-let-7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri-let-7 and the microprocessor DGCR8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let-7. Altogether, we reveal an unexpected function for TruB1 in promoting let-7 maturation. Synopsis The tRNA pseudouridine synthase TruB1 positively and directly regulates the maturation step of miRNA let-7 in an enzyme activity-independent manner. This uncovered TruB1—let-7 axis suppresses cell proliferation. A devised systematic cell-based gain of function screen revealed that the tRNA pseudouridine synthase TruB1 promotes let-7 expression. TruB1 selectively enhanced let-7 family member maturation independent of its enzymatic activity. Endogenous TruB1 directly binds to the stem-loop structure of pri-let-7 and promotes the maturation steps of let-7. Cell proliferation can be regulated by the TruB1 and let-7 molecular cascade. Introduction MicroRNAs (miRNA) are short non-coding RNAs of about 22 bases that regulate gene expression by inhibiting either the stability of target mRNAs or protein synthesis in association with Argonaute (AGO) family proteins (Lee et al, 1993; Wightman et al, 1993). Lin-4 and let-7 were the first reported miRNAs in Caenorhabditis elegans (Lee et al, 1993; Reinhart et al, 2000). Since then, more than 1,500 conserved miRNAs have been identified across many species including mammals (Ambros, 2004). The expression and maturation of miRNAs are tightly regulated by multi-step mechanisms involving protein-RNA associations with enzymatic activities and molecular shuttling, specifically: (i) Pol-II-mediated primary miRNA (pri-miRNA) transcription (Cai et al, 2004; Lee et al, 2004), (ii) Drosha/DiGeorge syndrome critical region 8 (DGCR8)-mediated processing producing a precursor-miRNA (pre-miRNA) in the nucleus (Lee et al, 2003; Denli et al, 2004; Gregory et al, 2004; Han et al, 2004; Landthaler et al, 2004; Zeng et al, 2005), (iii) nuclear export of pre-miRNA by exportin-5 (Yi et al, 2003; Bohnsack et al, 2004; Lund et al, 2004), (iv) additional cleavage by the RNase III enzyme Dicer in the cytoplasm (Grishok et al, 2001; Hutvágner et al, 2001; Ketting et al, 2001; Knight & Bass, 2001), and (v) incorporation into the RNA-induced silencing complex (RISC) with AGO family proteins to generate the final miRNA (Mourelatos et al, 2002). Although the first transcription step for pri-miRNA expression can be driven by transcription factors and RNA Pol-II as well as mRNA, the following maturation steps, (ii–v), are also critical and unique to miRNA biogenesis and dynamics (Krol et al, 2010; Ha & Kim, 2014; Bartel, 2018). The comparison of gene expression in tumor tissues showed discrepancies in the levels of miRNAs and their pri-miRNAs (Thomson et al, 2006). In particular, mutually exclusive and reciprocal regulation between the let-7 miRNA and the RNA-binding protein (RBP) Lin-28 at these maturation process have been well demonstrated in ES cells, C. elegans, and cancer pathology (Heo et al, 2008; Newman et al, 2008; Piskounova et al, 2008; Viswanathan et al, 2008; Chang et al, 2009). Lin28A/B specifically binds to the preE loop sequence of pri-/pre-let-7 and suppresses the processing of let-7 by causing oligo-uridylation of the let-7 precursor through TUT4/7 activity (Heo et al, 2008; Piskounova et al, 2011). Based on these fundamental findings, the factors mediating let-7 multi-step regulation, which may be critical for various biological and pathological events, have been extensively explored by several approaches (Newman et al, 2008; Trabucchi et al, 2009; Treiber et al, 2017). Recently, it has been reported that METTL1 promotes processing of microRNA let-7 by m7G methylation (Pandolfini et al, 2019). However, to date, only a few factors have been shown to regulate let-7 family-specific maturation (Newman et al, 2008; Trabucchi et al, 2009; Michlewski & Caceres, 2010; Choudhury et al, 2014). Taking advantage of the completion of genome-wide comprehensive full-length cDNA libraries (Chanda et al, 2003; Conkright et al, 2003; Iourgenko et al, 2003; Huang et al, 2004; Liu et al, 2005), we and others successfully developed cell-based screening systems to quantify miRNA targets and functions, including destabilization of target mRNA and suppression of protein synthesis (Wolter et al, 2014, 2015; Ito et al, 2017). Here we applied the above strategy and introduced a new cell-based screening to identify genes that regulate let-7 miRNA by using a luciferase reporter assay with a set of expression plasmid libraries mainly encoding proteins with RNA-binding properties. We identified a new regulatory mechanism: TruB1, an RNA-modifying enzyme, selectively regulates let-7 levels in an enzymatic activity-independent manner. Results Functional screening to detect RNA-binding proteins (RBPs) that promote let-7 expression We developed a cell-based, functional screen using an expression plasmid library and a let-7 sensor reporter to identify proteins that regulate let-7 biogenesis. This type of screen enables the identification of proteins that regulate endogenous let-7 levels, as opposed to other aspects of let-7 biology, such as binding, which is assayed using affinity-based (pull-down) screens. The gain-of-function format also enables the identification of regulatory factors that may cause lethality under loss of function conditions, such as with Crispr-Cas9 or RNAi-KD screens. The let-7 sensor reporter was designed to monitor let-7 expression levels by inserting a let-7a target site into the luciferase 3′UTR region driven by the SV-40 promoter in pLuc2 plasmids (Fig 1A). Thus, luciferase activity is repressed in the presence of let7 miRNA (Fig EV1A). In this screen, we focused on identifying molecules directly regulating the miRNA maturation process; thus, we prepared a sib-selection library covering molecules annotated with RNA-binding protein (RBP) features from the Center for Cancer systems Biology (CCSB)-Broad Lentiviral overexpression library (Yang et al, 2011). As many zinc-finger-proteins have not been directly tested for their RNA or DNA binding preference, the genes with zinc-finger domains were also included in the sib-selection library. Ultimately, 1,469 genes were selected and prepared for the screening. This library was transfected into HEK293FT cells, along with the let-7 sensor reporter. If let-7 maturation is inhibited by coexpression of one of the cDNAs in the RBP library, expression of the luciferase reporter would be induced. As a positive control for the assay, we overexpressed Lin28A from the same backbone plasmid transfection along with the let-7 sensor reporter with or without overexpression of pri-let-7a in HEK293FT cells. This significantly promoted luciferase activity reflecting reduction of endogenous let-7 expression (Fig 1B). Next, we screened the 1,469 genes in our library utilizing the luciferase assay with the let-7 sensor reporter in HEK293FT cells seeded into 384-well plates (Fig 1C). pRL-SV40 Renilla luciferase activity was used as a transfection efficiency control. We only included genes that did not change the luciferase activity of the let-7 sequence minus the reporter (0.5 2.0) (results are shown in Table EV1). Of these, we found that overexpression of 259 genes significantly reduced relative luciferase activity (< 0.50)compared with the GFP expression plasmid control (Fig 1D), indicating that they potentially promote let7 miRNA maturation. From the top five hits, we performed a larger scale (96-well plate) luciferase reporter assay for validation, whereby four of the top five genes reproducibly repressed the luciferase activity of the let-7 sensor (Fig 1E). To confirm the effect on endogenous let-7a expression, we examined the expression levels of a panel of ten miRNAs including let-7a by qPCR. Consistent with the screening results, overexpression of all of the top five candidate genes significantly promoted endogenous let-7a expression. Only TruB1 selectively induced let-7 expression but did not increase expression of the other miRNAs, whereas the other four hits, SF3A3, LARP7, GLTSCR2, and EF1E1, also increased the other miRNAs (Fig 1F). This suggests the specific promotion of let-7 biogenesis by TruB1. TruB1 is among the RNA-modifying enzymes that mediate pseudouridylation of tRNA. TruB1 has also recently been shown to regulate mRNA via pseudouridylation (Schwartz et al, 2014; Safra et al, 2017), but, to date, there has been no reported function of TruB1 in miRNA biogenesis. Figure 1. Functional screen using a luciferase reporter assay to identify RBPs inducing let-7-expression Design of the let-7 sensor reporter and sequence of let-7a. Sensor (+) vector has the antisense sequence of let-7a inserted into the 3′UTR of the luciferase gene. Relative luciferase activity of the let-7 sensor reporter (sensor (+)) or the negative control sensor (−) reporter contransfected with pLX-Lin28A in HEK293FT cells with or without pcDNA-pri-let-7a transfection. Error bars show SD; n = 3. Significance was assessed using 2-tailed Student's t-test, < 0.05*. The screening model. A screening in 384-well plates with a library of 1,469 genes was performed. Screening results: relative luciferase activity of each gene in the library. Only genes that did not affect the luciferase activity of the sensor (−) reporter (0.5 2.0) were analyzed. The full results of the screen are also in Table EV1. Relative luciferase activity of the sensor (+) reporter or sensor (−) reporter induced by expression vectors of the top 5 genes identified in the screen or the GFP vector in HEK293-FT cells. Error bars show SD; n = 3. Significance was assessed using 2-tailed Student's t-test, 0.05*. Heat map of relative miRNA expression of various miRNAs in HEK-293FT cells transfected with expression vectors of the top 5 genes from the screen, or the ctrl (GFP) vector. Red color represents suppressed expression compared with ctrl (GFP). n = 3. Source data are available online for this figure. Source Data for Figure 1 [embj2020104708-sup-0008-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Schematic model of cell-based functional screen and knockdown of TruB1 repressed expression of relative miRNAs in various human cells Repressors of maturation such as Lin28 lead to high luciferase activity, and activators lead to low luciferase activity. This cell-based screen is assaying for a function directly on endogenous let-7 maturation, rather than affinity/binding. The gain-of-function screen also enables to identify regulatory factors that may cause lethality under loss of function conditions, such as with Crispr-Cas9 or RNAi-KD screens. qRT–PCR analysis for TruB1 mRNA expression normalized to GAPDH with TruB1 KD or ctrl (siRNA). TruB1 KD by siRNA significantly repressed the expression of TruB1 mRNA in HEK-293FT cells. Error bars show SD; n = 3. Significance was assessed using 2-tailed Student's t-test, < 0.05*. qRT–PCR analysis for let-7 families levels in A549 cells with TruB1 KD or ctrl (siRNA). TruB1 KD by siRNA significantly repressed the expression of the let-7 family. Error bars show SD; n = 3. Significance was assessed using 2-tailed Student's t-test, < 0.05*. Source data are available online for this figure. Download figure Download PowerPoint TruB1 selectively promotes the expression of the let-7 family Although a few proteins, such as Lin28A/B, KSRP, hnRNPA1, have been shown to be involved in the biogenesis of a specific set of miRNAs, most other miRNA regulators act without specificity, or their miRNA selectivity remains unclear. To test whether TruB1-dependent let-7 promotion is specific for the let-7 family, as our preliminary results suggested, we comprehensively analyzed the effect of TruB1 knockdown by siRNA on miRNA biogenesis in HEK293FT cells by TaqMan array. TruB1 knockdown downregulated the expression of four let-7 family members (let-7a, b, c and g) (Figs 2A and EV1B, Table EV2). These data were further confirmed by large scale TaqMan PCR for miRNA and Northern blotting (NB) in HeLa cells, revealing that almost all endogenous let-7 family genes were significantly downregulated upon TruB1 knockdown (Fig 2B–D). We confirmed these findings in A549 cells (Fig EV1C). Although there was some variation in the effects observed, the large scale qPCR experiments revealed a similar trend for each member of the let-7 family upon TruB1 knockdown in three cell types (HEK293FT cells, HeLa cells, and A549 cells). Even the members that did not show a statistically significant difference still showed a tendency to decline. TruB1 knockdown also decreased the level of pre-let-7a1 (Fig 2B). In contrast, TruB1 knockdown increased the levels of endogenous, immature, primary let-7 (Fig 2E). When we measured the processing rate, TruB1 knockdown significantly decreased the processing rate both of pri-to-pre and pre-to-mature processing. These results indicate that TruB1 promotes let-7 family miRNA biogenesis specifically at their maturation step, rather than at the transcriptional level. Figure 2. TruB1 selectively promotes the expression of let-7 family miRNAs Volcano plot of TaqMan array showing the mean average expression and P-value of miRNA expression profiles between TruB1 KD and ctrl (scramble). N = 3. Significance was assessed using 2-tailed Student's t-test. MiRNAs suppressed (Relative expression TruB1 KD/ctrl < 0.8 and P-value < 0.1) are enclosed by the red square. Northern blotting for let-7a, miR-34a, and RNU6B in HeLa cells with TruB1 KD or ctrl (siRNA). Processing rates were quantified and normalized to ctrl based on hybridization intensities of (B). Significance was assessed using 2-tailed Student's t-test, < 0.05*. Relative expression of let-7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl (siRNA) determined by qRT–PCR. Significance was assessed using 2-tailed Student's t-test, < 0.05*. Relative expression of primary let-7 family miRNAs as in (D) determined by qRT–PCR. Significance was assessed using 2-tailed Student's t-test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD. Source data are available online for this figure. Source Data for Figure 2 [embj2020104708-sup-0009-SDataFig2.zip] Download figure Download PowerPoint TruB1 promotes miRNA processing independently of its enzymatic activity We analyzed whether let-7 regulation by TruB1 is dependent on the pseudouridine enzyme activity of TruB1. Critical residues involved in enzymatic activity (D48, D90) and RNA-binding ability (K64) have been identified in Escherichia coli TruB (Wright et al, 2011; Friedt et al, 2014; Keffer-Wilkes et al, 2016). Given the TruB amino acid sequences for these enzyme activity and RNA-binding ability are highly conserved (Zucchini et al, 2003), we were able to insert mutations to modify the function of TruB1 by amino acid substitution. We generated two TruB1 mutants: Mt1, with inactivated enzyme activity, and mt2, with suppressed RNA-binding ability (Fig 3A). An in vitro enzymatic activity assay with a tRNAphe substrate using the wt, mt1, and mt2 recombinant proteins showed that the pseudouridylation enzyme activities of both mt1 and mt2 were completely attenuated (Fig 3B). An EMSA study using recombinant proteins revealed a physical interaction between tRNAphe and wt TruB1 and mt1, but not with mt2 (Fig EV2A). Next, we tested the function of these mutants in cells. Western blotting (WB) revealed that all proteins were overexpressed largely to the same levels (Fig EV2B). Overexpression of Wt and mt1 TruB1 significantly increased let-7a maturation, whereas mt2 did not (qPCR, NB). Although no change in pre-let-7 was observed, the processing rate of pre-to-mature was elevated by overexpression of Wt and mt1 (Fig 3C–E). These results suggest that promotion of let-7 maturation by TruB1 is independent of its enzyme activity, but dependent on RNA binding. Figure 3. TruB1 promotes let-7 processing independently of its enzymatic activity Amino acid sequences encoding for the enzyme activity and RNA-binding ability in Escherichia coli TruB and human TruB1 (Top). Design of mutant 1 (mt1) and mutant 2 (mt2) (bottom). In vitro enzyme activity assay. 32p-UTP-labeled tRNAphe were treated with recombinant TruB1, mt1, or mt2. The strong upper bands represent uridine (U), and the lower weaker bands represent pseudouridine (Ψ) on autoradiographs of the TLC plate. Relative miRNA expression of let-7a in HEK-293 cells infected with tetracycline-inducible expressing lentiviruses for TruB1, mt1, mt2, or GFP 5 days after doxycycline treatment, as determined by qRT–PCR. Significance was assessed using 2-tailed Student's t-test, < 0.05*. Northern blotting for let-7a and RNU6B in HeLa cells infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP, 5 days after doxycycline treatment. Hybridization intensities of (D) were quantified and normalized to ctrl (GFP). Significance was assessed using 2-tailed Student's t-test, < 0.05*. Pseudouridylation activity of TruB1 for tRNA and pri-miRNAs. 32p-UTP-labeled tRNAphe, pri-let-7a1, or pri-miR-10a were treated with recombinant TruB1. Upper bands represent uridine (U), lower bands represent pseudouridine (Ψ) in autoradiographs of the TLC plate. Location of pseudouridine sites detected by the CMC primer extension method. Total RNA purified from HEK-293FT cells were treated with CMC. CMC-treated RNA were reverse-transcribed with RI-labeled specific primers for tRNAphe or pri-let-7b. ddATP was used for sequence control. Pseudouridines are indicated by black arrows. Data information: All experiments were performed in triplicate. Error bars show SD. Source data are available online for this figure. Source Data for Figure 3 [embj2020104708-sup-0010-SDataFig3.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Impact of pseudouridylation enzyme activity on microprocessing of let-7 EMSA of 32p-ATP-labeled tRNAPhe mixed with recombinant TruB1, mt1, or mt2 at several doses. RNP: Ribonucleoprotein complexes. Protein expression of Flag-tagged TruB1 and its mutants. Protein expression was evaluated by Western blotting. Protein was isolated from HEK-293FT cells infected with tetracycline-inducible lentiviruses expressing TruB1, mt1, or, mt2, 5 days after doxycycline treatment. Location of pseudouridine sites were detected by CMC primer extension. Total RNA purified from HEK-293FT cells was treated with CMC. CMC-treated RNA were reverse-transcribed with RI-labeled specific primers for pri-let-7a1. ddATP was used as a sequence control. In vitro processing analysis for pri-let-7 with pseudouridine. 32p-ATP-labeled pri-let-7a1 was synthesized using UTP: pseudouridine at a ratio of 1:1 or 1:0. This labeled RNA was treated with whole cell lysate from TruB1 expressing HEK293FT cells transfected with pcDNA3.1-TruB1. Autoradiographed image (D) and the relative processing rate (E) are shown. Error bars show SD; n = 3. Significance was assessed using 2-tailed Student's t-test. Source data are available online for this figure. Download figure Download PowerPoint Next, we performed an in vitro enzyme assay to verify that let-7 could not be pseudouridylated by recombinant TruB1. Pseudouridine of tRNAphe was clearly increased in the assay, whereas no pseudouridylation of pri-let-7 and another primary miRNA was observed (Fig 3F). We also examined the presence of pseudouridine directly by treatment with CMC (N-cyclohexyl-N0-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate) followed by a primer extension assay. We monitored the position of the UTP using ddATP during a primer extension assay. If a band is found in CMC-treated RNA that does not match the height of this UTP, it can be determined to be non-specific binding. Furthermore, the bands observed in non-CMC-treated RNA cannot indicate the presence of pseudouridine. Based on these conditions, the multiple thin bands found around 35 nt of CMC-treated RNA in let-7 are likely to be non-specific bands. The bands at the height of the UTP were also only of the same intensity as the other non-specific bands in let-7. In contrast, in tRNAs, a dense band of CMC-treated RNA consistent with the height of UTP was observed, indicating the presence of pseudouridine. These results indicate that pseudouridine is not present in endogenous let-7 (Figs 3G and EV2C). We also synthesized RI-labeled pri-let-7a1 in which pseudouridine was randomly introduced using UTP: pseudouridine at a ratio of 1:1. An in vitro processing assay using this labeled pri-let-7a1 showed that the presence of pseudouridine in pri-let-7a1 did not affect its processing (Fig EV2D and E). These results indicate that the regulation of let-7 by TruB1 does not depend on its enzyme activity. TruB1 binds directly to primary let-7 To determine how TruB1 regulates let-7 processing, we first tested whether TruB1 binds to pri-let-7. RNA immunoprecipitation (RIP) assays revealed that overexpression of wt and mt1 TruB1 immunoprecipitated pri-let-7a1, whereas mt2 did not (Fig 4A). An EMSA study using recombinant proteins revealed a physical interaction between pri-let7a1 and wt and mt1 TruB1, but not with mt2 (Fig 4B). Furthermore, EMSA was performed using mutant RNA in which the loop structure of pri-let-7a1 was modified (loop mt). As a result, no binding to TruB1 was observed in the loop mt (Figs 4B and EV3A). To comprehensively survey the RNA-protein binding property under physiological conditions, we performed high-throughput crosslinking immunoprecipitation (HITS-CLIP) using a cell line in which the 3xFLAG tag was knocked into the TruB1 N-terminal end by using the CRISPR/Cas9 system (Figs 4C and EV3B–F). This revealed the expected, but not previously shown, direct interaction between TruB1 and its known substrate tRNA (positive control). We also observed a physical interaction between TruB1 and pri-let-7a1 (Fig 4D, Table EV3). Direct binding sites were also found in other miRNA sequences, including miR-29b, miR-139, and miR-107 whose expression levels of their mature forms were also specifically decreased upon TruB1 KD in the TaqMan array (Fig 4E, Table EV3). Looking at the entire mapped reads, mRNA, lncRNA, and miRNA were detected in addition to tRNA (Fig 4F). It has been reported that mRNA is modified by TruB1, which is consistent with this result (Schwartz et al, 2014; Safra et al, 2017). When a read mapped to tRNA was extracted and motif analysis was performed, the sequence was similar to the pseudouridylation site reported for Saccharomyces cerevisiae PUS4 (Fig 4G), which is homologous with TruB1 (Fig 4H; Becker et al
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