View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Kosmopolis University of Pennsylvania ScholarlyCommons Departmental Papers (Biology) Department of Biology 1-2016 Cytoplasmic Poly(A) Binding Protein-1 Binds to Genomically Encoded Sequences Within Mammalian mRNAs Hemant K. Kini University of Pennsylvania Ian M. Silverman University of Pennsylvania Xinjun Ji University of Pennsylvania, [email protected] Brian D. Gregory University of Pennsylvania, [email protected] Stephen A. Liebhaber University of Pennsylvania, [email protected] Follow this and additional works at: http://repository.upenn.edu/biology_papers Part of the Biology Commons Recommended Citation Kini, H. K., Silverman, I. M., Ji, X., Gregory, B. D., & Liebhaber, S. A. (2016). Cytoplasmic Poly(A) Binding Protein-1 Binds to Genomically Encoded Sequences Within Mammalian mRNAs. RNA, 22 (1), 61-74. http://dx.doi.org/10.1261/rna.053447.115 This paper is posted at ScholarlyCommons. http://repository.upenn.edu/biology_papers/44 For more information, please contact [email protected]. Cytoplasmic Poly(A) Binding Protein-1 Binds to Genomically Encoded Sequences Within Mammalian mRNAs Abstract The functions of the major mammalian cytoplasmic poly(A) binding protein, PABPC1, have been characterized predominantly in the context of its binding to the 3′ poly(A) tails of mRNAs. These interactions play important roles in post-transcriptional gene regulation by enhancing translation and mRNA stability. Here, we performed transcriptome-wide CLIP-seq analysis to identify additional PABPC1 binding sites within genomically encoded mRNA sequences that may impact on gene regulation. From this analysis, we found that PABPC1 binds directly to the canonical polyadenylation signal in thousands of mRNAs in the mouse transcriptome. PABPC1 binding also maps to translation initiation and termination sites bracketing open reading frames, exemplified most dramatically in replication-dependent histone mRNAs. Additionally, a more restricted subset of PABPC1 interaction sites comprised A-rich sequences within the 5′ UTRs of mRNAs, including Pabpc1 mRNA itself. Functional analyses revealed that these PABPC1 interactions in the 5′ UTR mediate both auto- and trans-regulatory translational control. In total, these findings reveal a repertoire of PABPC1 binding that is substantially broader than previously recognized with a corresponding potential to impact and coordinate post-transcriptional controls critical to a broad array of cellular functions. Keywords CLIP-seq, poly(A) binding protein, polyadenylation signal, and translational control Disciplines Biology This technical report is available at ScholarlyCommons: http://repository.upenn.edu/biology_papers/44 Downloaded from rnajournal.cshlp.org on July 10, 2017 - Published by Cold Spring Harbor Laboratory Press Cytoplasmic poly(A) binding protein-1 binds to genomically encoded sequences within mammalian mRNAs HEMANT K. KINI,1,4 IAN M. SILVERMAN,2,3,4 XINJUN JI,1 BRIAN D. GREGORY,2 and STEPHEN A. LIEBHABER1 1Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 19104, USA 2Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA 3Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA ABSTRACT The functions of the major mammalian cytoplasmic poly(A) binding protein, PABPC1, have been characterized predominantly in the context of its binding to the 3′ poly(A) tails of mRNAs. These interactions play important roles in post-transcriptional gene regulation by enhancing translation and mRNA stability. Here, we performed transcriptome-wide CLIP-seq analysis to identify additional PABPC1 binding sites within genomically encoded mRNA sequences that may impact on gene regulation. From this analysis, we found that PABPC1 binds directly to the canonical polyadenylation signal in thousands of mRNAs in the mouse transcriptome. PABPC1 binding also maps to translation initiation and termination sites bracketing open reading frames, exemplified most dramatically in replication-dependent histone mRNAs. Additionally, a more restricted subset of PABPC1 interaction sites comprised A-rich sequences within the 5′ UTRs of mRNAs, including Pabpc1 mRNA itself. Functional analyses revealed that these PABPC1 interactions in the 5′ UTR mediate both auto- and trans-regulatory translational control. In total, these findings reveal a repertoire of PABPC1 binding that is substantially broader than previously recognized with a corresponding potential to impact and coordinate post-transcriptional controls critical to a broad array of cellular functions. Keywords: CLIP-seq; poly(A) binding protein; polyadenylation signal; translational control INTRODUCTION (Kd = 1.8 nM), while RRMs 3 and 4 can bind to nonhomopo- lymeric AU sequences (K = 2.9 nM) (Sladic et al. 2004). The biogenesis of eukaryotic messenger RNAs (mRNAs) is d With the exception of non-redundant impacts of PABPC4 tightly linked to the post-transcriptional addition of poly- ′ in erythroid maturation (Kini et al. 2014) and vertebrate de- adenylate [poly(A)] tails to their 3 ends. These poly(A) tails velopment (Gorgoni et al. 2011), the levels of functional spe- contribute to regulation of mRNA transcription, transport, cificity and/or redundancy of the mammalian cytoplasmic stability, and translation (Mangus et al. 2003; Goss and PABPs remain unexplored. Kleiman 2013). Poly(A) tail-dependent functions are mediat- PABPC1 is the major cytoplasmic PABP isoform in adult ed in large part via the association of one or more poly(A) mouse somatic cells and is abundantly expressed in all tissues binding proteins (PABPs). In mammals, there are six defined (Kleene et al. 1994). The interaction of PABPC1 with poly(A) PABP isoforms; a single nuclear isoform, PABPN1, that im- tails is well documented and defined in multiple contexts pacts on the addition of poly(A) tails in the nucleus and five (Blobel 1973; Mangus et al. 2003). The corresponding func- cytoplasmic PABPs, ePAB, PABPC1, PABPC2, PABPC4, and tions of the PABPC1/poly(A) tail complex are primarily PABPC5, that are thought to play roles in regulating mRNA mediated in pathways of mRNA stabilization and translation stability and translation in the cytoplasm (Wahle 1991; enhancement (Wang and Kiledjian 2000; Wilusz et al. 2001; Mangus et al. 2003; Good et al. 2004). The overall structures Kahvejian et al. 2005). These functions may be linked to the and RNA-binding specificities of the five cytoplasmic ′ interactions of PABPC1 with the 5 cap-binding complex via PABPs are highly conserved (Adam et al. 1986; Burd and heterodimerization with eIF4G (Tarun and Sachs 1995; Wells Dreyfuss 1994). They each contain four RNA recognition et al. 1998; Peixeiro et al. 2012) Limited evidence points to motifs (RRMs). RRMs 1 and 2 are primarily responsible for additional binding sites and functions for PABPC1 within the high-affinity binding to homopolymeric adenosines © 2015 Kini et al. This article is distributed exclusively by the RNA Society 4These authors contributed equally to this work. for the first 12 months after the full-issue publication date (see http:// Corresponding author: [email protected] rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available Article published online ahead of print. Article and publication date are at under a Creative Commons License (Attribution-NonCommercial 4.0 Inter- http://www.rnajournal.org/cgi/doi/10.1261/rna.053447.115. national), as described at http://creativecommons.org/licenses/by-nc/4.0/. RNA 22:61–74; Published by Cold Spring Harbor Laboratory Press for the RNA Society 61 Downloaded from rnajournal.cshlp.org on July 10, 2017 - Published by Cold Spring Harbor Laboratory Press Kini et al. the eukaryotic mRNA transcriptome. For example, PABPC1 PABPC1 band (blue line, Fig. 1B) were excised for analysis. has been shown to bind to an A-rich element in the 5′ un- The slower running complexes were excluded from library translated region (UTR) of its own mRNA (mouse and hu- preparation as they were assumed to represent PABPC1 mul- man), establishing an autoregulatory translation control timers bound to poly(A) (red line, Fig. 1B). RNA fragments circuit (de Melo Neto et al. 1995; Bag and Wu 1996; were isolated from the PABPC1 RNP complexes and used as Hornstein et al. 1999). A recent study in Saccharomyces templates for the construction of high-throughput sequenc- cerevisiae using a photoactivatable-ribonucleoside-enhanced ing libraries (see Materials and Methods). crosslinking immunoprecipitation approach (PAR-CLIP) Sequencing of the libraries generated from the PABPC1- demonstrated in vivo binding of yeast poly(A) binding bound RNA fragments yielded 14.8 million unique sequences protein Pab1 to AU-rich elements in mRNAs (Baejen et across three biological replicates (Table 1). The mean size of al. 2014), including binding to the efficiency element the unique sequences protected from the RNase I treatment (UAUAUA) of the yeast polyadenylation signal (Guo and was 24 nt. These unique sequences were mapped to the Sherman 1995; Tuck and Tollervey 2013). Importantly, the mouse genome (mm10) with Novoalign, resulting in 7.5 mil- PAR-CLIP approach used in this yeast study only identifies lion uniquely mapping CLIP tags that were used for down- crosslinking events at uridines, which may bias the overall stream analysis