CPSF30 and Wdr33 Directly Bind to AAUAAA in Mammalian Mrna 39 Processing
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Downloaded from genesdev.cshlp.org on November 3, 2014 - Published by Cold Spring Harbor Laboratory Press CPSF30 and Wdr33 directly bind to AAUAAA in mammalian mRNA 39 processing Serena L. Chan,1 Ina Huppertz,2,3 Chengguo Yao,1 Lingjie Weng,1,4,5 James J. Moresco,6 John R. Yates III,6 Jernej Ule,2,3 James L. Manley,7 and Yongsheng Shi1 1Department of Microbiology and Molecular Genetics, School of Medicine, University of California at Irvine, Irvine, California 92697, USA; 2Department of Molecular Neuroscience, University College London Institute of Neurology, London WC1N 3BG, United Kingdom; 3Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom; 4Institute for Genomics and Bioinformatics, 5Department of Computer Science, University of California at Irvine Irvine, California 92697, USA; 6Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA; 7Department of Biological Sciences, Columbia University, New York, New York 10027, USA AAUAAA is the most highly conserved motif in eukaryotic mRNA polyadenylation sites and, in mammals, is specifically recognized by the multisubunit CPSF (cleavage and polyadenylation specificity factor) complex. Despite its critical functions in mRNA 39 end formation, the molecular basis for CPSF–AAUAAA interaction remains poorly defined. The CPSF subunit CPSF160 has been implicated in AAUAAA recognition, but direct evidence has been lacking. Using in vitro and in vivo assays, we unexpectedly found that CPSF subunits CPSF30 and Wdr33 directly contact AAUAAA. Importantly, the CPSF30–RNA interaction is essential for mRNA 39 processing and is primarily mediated by its zinc fingers 2 and 3, which are specifically targeted by the influenza protein NS1A to suppress host mRNA 39 processing. Our data suggest that AAUAAA recognition in mammalian mRNA 39 processing is more complex than previously thought and involves multiple protein–RNA interactions. [Keywords: mRNA 39 processing; cleavage and polyadenylation; polyadenylation signal] Supplemental material is available for this article. Received August 13, 2014; revised version accepted September 23, 2014. mRNA 39 processing, typically involving an endonucleo- these sequences, the AAUAAA hexamer (or its close vari- lytic cleavage and subsequent polyadenylation, is not only ant, AUUAAA) is the most highly conserved, and almost all an essential step in eukaryotic gene expression but also a single-nucleotide substitutions in this hexamer reduce or critical mechanism for gene regulation (Colgan and Manley abolish mRNA 39 processing in vitro (Sheets et al. 1990; 1997; Zhao et al. 1999; Millevoi and Vagner 2009; Chan et al. Proudfoot 2011). Genetic mutations in this hexamer can 2011). The majority of eukaryotic genes produce multiple significantly impact the efficiency of mRNA 39 processing mRNA isoforms by using alternative polyadenylation sites in vivo and cause human diseases, including thalassemia (PASs), and PAS selection is dynamically regulated during and thrombophilia (Danckwardt et al. 2008). development (Shi 2012; Tian and Manley 2013). Aberrant The AAUAAA hexamer is specifically recognized by CPSF mRNA alternative polyadenylation has been associated with (cleavage and polyadenylation specificity factor) (Takagaki a number of diseases, including cancer (Di Giammartino et al. 1988; Keller et al. 1991), a protein complex that con- et al. 2011). As such, a central question in the mRNA 39 tains six subunits: CPSF160, Wdr33, CPSF100, CPSF73, processing field has been to understand how PASs are Fip1, and CPSF30 (Mandel et al. 2008; Shi et al. 2009). It recognized and how PAS selection can be regulated. In has been suggested that CPSF160 is responsible for recog- metazoans, most PASs are defined by several key cis- nizing AAUAAA based on several lines of evidence. First, elements, including the AAUAAA hexamer located 10–30 two proteins of ;160 kDa and ;30 kDa in the purified CPSF nucleotides (nt) upstream of the cleavage site, a U/GU-rich complex that were thought to correspond to CPSF160 and downstream element located ;30 nt downstream from the cleavage site, and other auxiliary sequences (Colgan and Ó Manley 1997; Zhao et al. 1999; Chan et al. 2011). Among 2014 Chan et al. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). Corresponding author: [email protected] After six months, it is available under a Creative Commons License Article published online ahead of print. Article and publication date are (Attribution-NonCommercial 4.0 International), as described at http:// online at http://www.genesdev.org/cgi/doi/10.1101/gad.250993.114. creativecommons.org/licenses/by-nc/4.0/. 2370 GENES & DEVELOPMENT 28:2370–2380 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/14; www.genesdev.org Downloaded from genesdev.cshlp.org on November 3, 2014 - Published by Cold Spring Harbor Laboratory Press CPSF30 and Wdr33 directly bind to AAUAAA CPSF30, respectively, can be specifically cross-linked to complexes tended to vary, making it difficult to carry out AAUAAA-containing RNA substrates by UV irradiation detailed functional studies. To overcome these technical (Keller et al. 1991). However, the identities of these proteins limitations, we established HEK293 cell lines that stably were not experimentally determined, and, more impor- express Flag-tagged CPSF73 near the endogenous levels tantly, it was unclear whether either of these two proteins (Shi et al. 2009). To purify the CPSF complex, we prepared directly binds to AAUAAA. Second, the yeast homolog nuclear extract from these cells, carried out immunopre- of CPSF160, Yhh1/Cft1p, has been shown to bind RNA cipitation under stringent conditions using anti-Flag anti- (Dichtl et al. 2002). Third, recombinant human CPSF160 bodies, and eluted immunoprecipitated proteins under binds to RNAs in a pull-down assay and shows a preference native conditions using Flag peptides. Analyses of the eluted for AAUAAA-containing RNAs (Murthy and Manley 1995). proteins by mass spectrometry and Western blotting sug- However, CPSF160 has been shown to bind to an enhancer gested that the purified CPSF complex was intact, contain- element located 76 nt upstream of the AAUAAA in HIV ing all known CPSF subunits plus an additional associated mRNA PASs (Gilmartin et al. 1995). Additionally, a recent protein, symplekin (Table 1; Shi et al. 2009). It was noted study that aimed to map protein–RNA interactions in vivo that the purified CPSF complex contained two CPSF30 for mRNA 39 processing factors failed to detect specific isoforms that are encoded by alternatively spliced mRNAs CPSF160 binding at the AAUAAA hexamer (Martin et al. (Fig. 1A). The gel staining profile (Fig. 1A) and mass spec- 2012). Therefore, it remains unclear how CPSF recognizes trometry analyses results (Table 1) suggest that all subunits AAUAAA in mammalian mRNA 39 processing. are present in the purified CPSF complex at comparable The smallest subunit of the CPSF complex, CPSF30, levels. Furthermore, mass spectrometry analyses of biologi- contains five zinc finger (ZF) domains and, in metazoans, a cal replicates revealed that the molecular composition and zinc knuckle (ZK) domain (Barabino et al. 1997). Recombi- apparent stoichiometry of the purified CPSF complex were nant CPSF30 has RNA-binding activity, and its ZK domain highly consistent (Table 1). Therefore, these cell lines enhances this activity (Barabino et al. 1997). Interestingly, provide a reliable and efficient system for purifying CPSF. the influenza virus protein NS1A specifically interacts with Next we tested whether the immunopurified CPSF spe- CPSF30 via its ZF2 and ZF3 domains, and this interaction cifically recognizes the AAUAAA hexamer and functions is responsible for suppressing host mRNA 39 processing in mRNA 39 processing using multiple in vitro assays. As (Nemeroff et al. 1998; Twu et al. 2006). However, the role shown in Figure 1B, gel mobility shift assays demonstrated of CPSF30–RNA interaction in mRNA 39 processing re- that a specific RNP complex was formed when CPSF was mains poorly characterized, and it is unclear why CPSF30 incubated with an AAUAAA-containing RNA substrate is specifically targeted by the viral protein. (WT: wild type) derived from the commonly used SV40 In an effort to define the molecular basis for CPSF– late (SVL) transcript PAS. A single-nucleotide substitution AAUAAA interaction, we unexpectedly found that CPSF30 in the hexamer (AAGAAA) completely abolished this inter- and Wdr33 directly bind to the AAUAAA hexamer. We action (Fig. 1B, MT: mutant). In addition, we also carried demonstrated that CPSF30–RNA interaction is essential for out in vitro polyadenylation assays with purified CPSF mRNA 39 processing and is primarily mediated by its ZF2 and and recombinant poly(A) polymerase (PAP), which have ZF3 domains. By combining these new results with previous been shown to be sufficient for reconstituting AAUAAA- findings, we propose a new model for AAUAAA recognition dependent polyadenylation (Christofori and Keller 1988; by CPSF that requires multiple protein–RNA interactions. Takagaki et al. 1988). Consistent with the gel shift results (Fig. 1B), our in vitro polyadenylation assays showed that the AAUAAA-containing, but not the AAGAAA-containing, Results RNA substrate was specifically polyadenylated when in- cubated with CPSF and PAP (Fig. 1C).