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• W2921310588 abstract "Article12 March 2019free access Source DataTransparent process ALYREF links 3′-end processing to nuclear export of non-polyadenylated mRNAs Jing Fan State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Ke Wang State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Xian Du Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China Search for more papers by this author Jianshu Wang State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Suli Chen State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yimin Wang State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Min Shi State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Li Zhang State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Xudong Wu Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Search for more papers by this author Dinghai Zheng Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Changshou Wang State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Lantian Wang State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Bin Tian Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Guohui Li orcid.org/0000-0001-8223-705X Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Search for more papers by this author Yu Zhou Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China Search for more papers by this author Hong Cheng Corresponding Author [email protected] orcid.org/0000-0002-1965-2776 State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Jing Fan State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Ke Wang State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Xian Du Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China Search for more papers by this author Jianshu Wang State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Suli Chen State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yimin Wang State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Min Shi State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Li Zhang State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Xudong Wu Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Search for more papers by this author Dinghai Zheng Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Changshou Wang State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Lantian Wang State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Bin Tian Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Guohui Li orcid.org/0000-0001-8223-705X Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Search for more papers by this author Yu Zhou Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China Search for more papers by this author Hong Cheng Corresponding Author [email protected] orcid.org/0000-0002-1965-2776 State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Author Information Jing Fan1, Ke Wang1, Xian Du2, Jianshu Wang1, Suli Chen1, Yimin Wang1, Min Shi1, Li Zhang1, Xudong Wu3, Dinghai Zheng4, Changshou Wang1, Lantian Wang1, Bin Tian4, Guohui Li3, Yu Zhou2 and Hong Cheng *,1 1State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China 2Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China 3Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China 4Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School, Newark, NJ, USA *Corresponding author. Tel: +86 21 54921160; E-mail: [email protected] EMBO J (2019)38:e99910https://doi.org/10.15252/embj.201899910 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 The RNA-binding protein ALYREF plays key roles in nuclear export and also 3′-end processing of polyadenylated mRNAs, but whether such regulation also extends to non-polyadenylated RNAs is unknown. Replication-dependent (RD)-histone mRNAs are not polyadenylated, but instead end in a stem-loop (SL) structure. Here, we demonstrate that ALYREF prevalently binds a region next to the SL on RD-histone mRNAs. SL-binding protein (SLBP) directly interacts with ALYREF and promotes its recruitment. ALYREF promotes histone pre-mRNA 3′-end processing by facilitating U7-snRNP recruitment through physical interaction with the U7-snRNP-specific component Lsm11. Furthermore, ALYREF, together with other components of the TREX complex, enhances histone mRNA export. Moreover, we show that 3′-end processing promotes ALYREF recruitment and histone mRNA export. Together, our results point to an important role of ALYREF in coordinating 3′-end processing and nuclear export of non-polyadenylated mRNAs. Synopsis The RNA-binding protein ALYREF functions in coupling nuclear export and 3′-end processing of polyadenylated mRNAs. New data identify a parallel mechanism for ALYREF also linking 3′-end processing to nuclear export of non-polyadenylated, replication-dependent histone mRNAs. ALYREF prevalently binds to a 3′ region of histone mRNAs in an SLBP-dependent manner. ALYREF promotes histone pre-mRNA processing by ensuring U7-snRNP recruitment. The TREX complex facilitates histone mRNA export by recruiting NXF1. 3′-end processing promotes ALYREF recruitment and histone mRNA export. Introduction In eukaryotes, most pre-mRNAs undergo 3′ processing in a coupled cleavage/polyadenylation (CPA) reaction, which consists of an endonucleolytic cleavage of the nascent RNAs followed by synthesis of the polyA tail (reviewed in Shi & Manley, 2015). In contrast, replication-dependent (RD)-histone pre-mRNAs, which are synthesized during S-phase of the cell cycle, undergo one-step U7-snRNP-dependent 3′-end cleavage and end in a stem-loop (SL) structure (Mowry & Steitz, 1987; Cotten et al, 1988; Marzluff, 2005; Dominski & Marzluff, 2007; Marzluff et al, 2008). The SL structure is recognized by SL-binding protein (SLBP), which stabilizes the interaction of U7-snRNP with the histone downstream element (HDE) of the pre-mRNA (Schaufele et al, 1986; Williams & Marzluff, 1995; Dominski et al, 1999; Battle & Doudna, 2001; Skrajna et al, 2017). Together, SLBP and U7-snRNP direct RNA cleavage 4–5 nt downstream from the SL. It remains unclear how SLBP facilitates U7-snRNP recruitment, as no physical interaction was identified between these critical processing factors. mRNAs are transported from the nucleus to the cytoplasm through a pathway that utilizes NXF1 as an export receptor. NXF1 is recruited to polyadenylated mRNAs mostly via the highly conserved TREX complex (TREX; Strasser & Hurt, 2000; Hautbergue et al, 2008; Katahira et al, 2009; Hung et al, 2010; Viphakone et al, 2012). The core of TREX mainly comprises three parts: the RNA-binding protein ALYREF, the RNA helicase UAP56/URH49, and the multi-subunit THO sub-complex (THO1/2/3/5/6/7; Strasser et al, 2002; Masuda et al, 2005; Chi et al, 2013). Among these, ALYREF and THO mediate the interaction between the mRNA and NXF1 (Strasser & Hurt, 2000; Hautbergue et al, 2008; Katahira et al, 2009; Hung et al, 2010; Viphakone et al, 2012). Different from polyadenylated mRNAs, RD-histone mRNAs are known to recruit NXF1 via SR proteins 9G8 and SRp20, which bind to specific cis-elements in the coding regions (Huang & Steitz, 2001). In addition to SR proteins, SLBP also plays a critical role in RD-histone mRNA export, but the underlying mechanism remains unknown (Sullivan et al, 2009). The physical and functional coupling between 3′-end processing and nuclear export has been well established for polyadenylated mRNAs. In yeast, the ALYREF homologue, Yra1, is recruited via the 3′-end processing factor Pcf11 and regulates alternative polyadenylation (APA; Johnson et al, 2009, 2011). In mammalian cells, the THO subunits associate with the 3′-end processing machinery and influence APA (Katahira et al, 2013; Tran et al, 2014). Further, ALYREF was recently found to bind to a 3′ region of polyadenylated mRNAs in a polyA-binding protein (PABPN1)-dependent manner (Shi et al, 2017). Thus, the coupling between 3′-end processing and mRNA export seems to ensure efficient mRNA export and modulate APA. Currently, the roles of TREX in regulating the metabolism of non-polyadenylated mRNAs remain unknown, and whether nuclear export is linked to 3′-end processing for these mRNAs is still controversial. Initially, 3′-end processing was shown to promote histone mRNA export (Eckner et al, 1991). However, later studies reported that the RNA length, but not 3′-end processing, is a major determinant of histone mRNA export efficiency (Erkmann et al, 2005). In this study, a deeper analysis of ALYREF iCLIP-seq (individual-nucleotide-resolution UV crosslinking and immunoprecipitation and sequencing) data led us to the finding that ALYREF universally binds to a region next to the SL on RD-histone mRNAs (for simplicity, histone mRNAs in this study). This binding is ensured by its direct interaction with SLBP and has two functional consequences: ALYREF stimulates proper histone mRNA 3′-end formation by facilitating efficient U7-snRNP recruitment and promotes histone mRNA export by enhancing NXF1 recruitment. Importantly, we demonstrate that 3′-end processing promotes ALYREF recruitment and histone mRNA export. Thus, ALYREF is shared by polyadenylated and non-polyadenylated mRNA metabolism pathways, coordinating processing and nuclear export of both types of mRNAs. Results ALYREF binds to a region 5′ of the SL on histone mRNAs Our recent iCLIP study revealed that ALYREF binds to a region near the 3′ end of polyadenylated mRNAs in a nuclear polyA-binding protein (PABPN1)-dependent manner (Shi et al, 2017). To investigate whether ALYREF also binds to non-polyadenylated RNAs, we further analyzed ALYREF iCLIP data. To increase resolution of the data, we identified clustered binding sites (Konig et al, 2010; Rossbach et al, 2014). Significantly, of the total 55 histone mRNAs that were easily detected in HeLa nuclei (Fan et al, 2017), clustered ALYREF binding sites were apparently and reproducibly detected on 52 (Fig 1A). This binding was not due to the high abundance of histone mRNAs, as compared to those in nuclear total RNA-seq, histone reads were significantly more enriched in ALYREF iCLIP-seq (Fig 1B). Note that such enrichment was not observed with short or long ncRNAs (Fig EV1A). ALYREF binding on histone mRNAs was confirmed by RNA immunoprecipitation (RIP) and RT–qPCRs (Fig 1C). In addition to ALYREF, TREX components UAP56 and THO were also easily detected on histone mRNAs (Fig EV1B). Note that the THOC2 antibody co-precipitates other THO subunits (Masuda et al, 2005; Chi et al, 2013). Figure 1. ALYREF binds to a region next to the SL on histone mRNAs The Venn diagram depicts the overlapping of histone mRNAs detected in a previously performed rRNA-depleted nuclear RNA-seq (RPM > 1) and in ALYREF iCLIP-seq (with clustered binding sites in at least two out of three biological replicates). The ratio of unique histone iCLIP-seq read population to unique histone RNA-seq read population was calculated and is shown, with the unique histone RNA-seq read population set as “1”. Error bars represent standard deviations from three replicates of ALYREF iCLIP data. Statistical analysis was performed using Student's t-test. ***P < 0.001. ALYREF RIP–qPCRs to examine ALYREF binding on histone mRNAs. Relative RIP efficiencies are shown. A tRNA was used as a negative control for ALYREF binding. Error bars represent standard deviations from biological repeats (n = 3). Statistical analysis was performed using Student's t-test. **P < 0.01, ***P < 0.001, n.s.: not significant. The distribution of histone reads in 5′ UTR, CDS, and 3′ UTR in rRNA-depleted nuclear RNA-seq and ALYREF iCLIP-seq. Metagene analysis of normalized ALYREF signals on histone mRNA based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot. Screenshots of two histone mRNAs showing clustered ALYREF binding sites. The 3′ UTR regions are highlighted. Distribution profile of clustered ALYREF binding sites on histone mRNA 3′ UTRs relative to the cleavage site (CS). The CS is set as “0” in the x-axis, and the relative position to the CS is marked. The y-axis shows the total occurrence of binding sites in three repeats at each nucleotide. Graphic displays that ALYREF binds next to SLBP on histone mRNAs. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. TREX associates with histone mRNAs The ratios of unique histone iCLIP-seq read population to unique short or long ncRNA RNA-seq read population were calculated and are shown, with the unique histone RNA-seq read population set as “1”. Statistical analysis was performed based on three replicates of ALYREF iCLIP data using Student's t-test. ***P < 0.001. ALYREF, UAP56, and THO RIP–qPCRs to examine their binding on multiple histone mRNAs. Relative RIP efficiencies are shown. Screenshots of several histone mRNAs with ALYREF iCLIP reads. Data information: In (A and B), error bars represent standard deviations from biological repeats (n = 3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Relative to histone read distribution in RNA-seq, ALYREF iCLIP reads are preferentially enriched in the 3′ UTR (Fig 1D). Interestingly, metagene analysis of iCLIP reads showed an apparent enrichment of ALYREF binding at the 3′ region of histone mRNAs (Fig 1E). Clustered ALYREF binding sites on two exemplified histone mRNAs, HIST1H2AG and HIST1H1E, are shown in Fig 1F, and ALYREF iCLIP read distribution on more histone mRNAs is shown in Fig EV1C. To understand the ALYREF binding principle at the 3′ region of histone mRNAs, we aligned clustered binding sites relative to the cleavage site (CS). Strikingly, ALYREF binds at a region spanning ~100 nt upstream of the CS, with the peak at −50 nt (Fig 1G). Considering that SLBP binds at the SL that is located at the −6~−21 region, our data demonstrate that ALYREF and SLBP bind adjacent to each other at the SL region of histone mRNAs (Fig 1H). In support of this view, a previous study identified an SLBP-independent protected region 5′ of the SL (Brooks et al, 2015). ALYREF interacts with SLBP in vivo and in vitro Since histone mRNA 3′ UTR sequences are not conserved and ALYREF is a nonspecific RNA-binding protein, it is possible that SLBP is a primary determinant of ALYREF binding neighboring to the SL. To test this possibility, we first determined whether ALYREF associates with SLBP by carrying out immunoprecipitations (IPs) from RNase A-treated S-phase HeLa cell lysate. Significantly, ALYREF was co-precipitated by the SLBP antibody, but not the control IgG (Fig 2A). Conversely, when the ALYREF antibody or IgG was used for IPs, SLBP was specifically detected in the ALYREF immunoprecipitate (Fig 2B). These results indicate that ALYREF and SLBP associate with each other through protein–protein interaction. To confirm this association and examine whether UAP56 and THO associate with SLBP, we next carried out Flag IPs from Flag-SLBP or Flag-Cntl expression cells in the presence of RNase A. ALYREF and UAP56 and THOC2 were all co-precipitated with Flag-SLBP, but not Flag-Cntl (Fig EV2A). These results indicate that ALYREF interacts with SLBP in the context of TREX. Figure 2. SLBP interacts with ALYREF and facilitates its binding on histone mRNAs A, B. IPs from RNase A-treated S-phase HeLa cell lysate using the SLBP antibody or IgG (A), or the ALYREF antibody or IgG (B). Western blotting was performed with the indicated antibodies. 2% of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blots. C. Pull-downs of in vitro translated 35S-labeled SLBP and luciferase (Cntl) using MBP or MBP-ALYREF in the presence of RNase A. The proteins pulled down were visualized by Coomassie staining (left) or PhosphorImager (right). 3% of input was loaded. D. GST-SLBP, GST-UAP56, and GST were used for pull-down of purified MBP-ALYREF or MBP in the presence of RNase A. Proteins pulled down were separated by SDS–PAGE, followed by Coomassie staining. 37.5% of input proteins were loaded. E. (Top) Domain schematic representation of SLBP. (Bottom) Flag IPs from RNase A-treated HeLa cell lysate individually expressing the indicated Flag-tagged proteins, followed by Western blotting using Flag and ALYREF antibodies. 3% of input was loaded. * indicates a band that probably resulted from degradation of Flag-SLBP. The white line delineates the boundary where irrelevant lanes have been removed from the same blot. F. Same as (E), except that Flag IPs were carried out from HA-SLBP stable expression cells transfected with plasmids expressing ALYREF fragments. G. Western blotting to examine the KD efficiency of SLBP. GAPDH was used as a loading control. The white line delineates the boundary where irrelevant lanes have been removed from the same blot. H, I. Cntl- or SLBP siRNA-treated HeLa cells were used for IPs with IgG or the ALYREF antibody. The immunoprecipitates were subjected to Western blot analysis (H) and RT–qPCRs (I). Error bars represent standard deviations from biological repeats (n = 3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, n.s.: not significant. J. Metagene analysis of normalized ALYREF signals on histone mRNAs in Cntl and SLBP KD cells based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot. K. Graphic displays that SLBP functions in recruiting ALYREF to histone mRNAs. Source data are available online for this figure. Source Data for Figure 2 [embj201899910-sup-0005-SDataFig2.jpg] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. TREX associates with SLBP Flag IPs from RNase A-treated HeLa cell lysate expressing Flag-Cntl (DDX3) or Flag-SLBP, followed by Western blotting using the indicated antibodies. 3% of input was loaded. GST-SLBP, GST-ALYREF, and GST were used for pull-down of purified His-UAP56 or His-Cntl (PGK1) in the presence of RNase A. Proteins pulled down were separated by SDS–PAGE, followed by Coomassie staining and Western blotting. 18.75% of input was loaded. Metagene analysis of normalized ALYREF signals on polyadenylated mRNAs in Cntl and SLBP KD cells based on two replicates of iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot. Download figure Download PowerPoint To further examine the ALYREF-SLBP interaction, we in vitro translated SLBP and luciferase (Cntl) and carried out pull-downs using MBP-ALYREF or MBP. Significantly, SLBP, but not Cntl, was pulled down by MBP-ALYREF, whereas neither of these in vitro translated proteins interacted with MBP (Fig 2C). This result provides additional evidence for the ALYREF-SLBP interaction and suggests that this interaction might be direct. Indeed, GST-SLBP, but not GST, pulled down purified MBP-ALYREF (Fig 2D). In contrast, His-UAP56 did not interact with GST-SLBP, although it was efficiently pulled down by GST-ALYREF (Fig EV2B). Together, these data demonstrate that ALYREF interacts with SLBP in vivo and in vitro. SLBP interacts with ALYREF via its C-terminal region To decipher how SLBP interacts with ALYREF, we constructed two Flag-SLBP mutants, with either the N- or C-terminal region truncated (ΔN or ΔC; Fig 2E). These constructs, as well as Flag-SLBPFL (full-length SLBP) and Flag-Cntl, were separately expressed in HeLa cells, followed by Flag IPs. Western blot analysis showed that ALYREF was efficiently co-precipitated with Flag-SLBPFL and Flag-SLBPΔN, but not Flag-SLBPΔC or Flag-Cntl, indicating that the C-terminal region (SLBPC) is required for interacting with ALYREF (Fig 2E, left lower panel). Further, using Flag-GST-SLBPC, we found that SLBPC is sufficient for this interaction (Fig 2E, right lower panel). Thus, SLBP interacts with ALYREF via its C-terminal region. Notably, this region is important for facilitating U7-snRNP recruitment (Skrajna et al, 2017). We also mapped the interaction of ALYREF with SLBP. Neither N- nor C-terminal region of ALYREF is required for the SLBP interaction (Fig 2F, left lower panel), raising the possibility that the RRM domain might be important. However, when ALYREF was separated into N-terminal, RRM, and C-terminal fragments, none of them was associated with SLBP (Fig 2F, right lower panel). It is possible that both the RRM and the surrounding residues are important for ALYREF interacting with SLBP. SLBP is required for ALYREF binding at the SL region on histone mRNAs To examine whether SLBP is required for ALYREF binding on histone mRNAs, we carried out ALYREF RIP in Cntl- and SLBP-knockdown (KD) cells. SLBP was efficiently knocked down, and ALYREF was equally immunoprecipitated from these cells (Fig 2G and H). Significantly, RT–qPCR data showed that ALYREF association with histone mRNAs was reproducibly reduced in SLBP KD cells, as compared to that in Cntl cells (Fig 2I). In contrast, its binding with the three randomly picked polyadenylated mRNAs, including CBP80, DNAJC30, and KLLN, was not apparently affected (Fig 2I). To examine how SLBP KD impacts ALYREF distribution along the histone mRNA, we next carried out ALYREF iCLIP in Cntl and SLBP KD cells. Low RNase I digestion efficiency could possibly result in biased iCLIP read enrichment at the SL region. We have thus optimized the experiment and ensured the digestion efficiency of the 3′ region containing the SL is not generally lower than that of a 5′ region of histone mRNAs (Appendix Fig S1; See Materials and Methods). Under this condition, in Cntl cells, ALYREF binding was still mostly enriched at the 3′ region, while it was also partially detected at other regions (Fig 2J). Significantly, in both biological replicates, SLBP KD led to a preferential reduction in ALYREF binding at the 3′ region (Fig 2J). In contrast, ALYREF distribution along the polyadenylated mRNA was not apparently affected (Fig EV2C). Taken together, the data suggest that SLBP plays a determinant role in ALYREF binding at the SL region of histone mRNAs (Fig 2K). ALYREF facilitates 3′-end processing of histone pre-mRNAs Considering the critical roles of SLBP in histone mRNA 3′-end formation, we reasoned that ALYREF might also be involved in this process. RD-histone genes have a canonical polyadenylation signal downstream of the CS. Defects in histone mRNA 3′-end processing usually cause the usage of this signal, resulting in upregulation in polyA+ forms and concomitant downregulati" @default.
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• W2921310588 title "<scp>ALYREF</scp> links 3′‐end processing to nuclear export of non‐polyadenylated <scp>mRNA</scp> s" @default.
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