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Chemical Crosslinking Enhances RNA Immunoprecipitation for Efficient Identification of Binding Sites of Proteins That Photo-Crosslink Poorly with RNA

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Chemical Crosslinking Enhances RNA Immunoprecipitation for Efficient Identification of Binding Sites of Proteins That Photo-Crosslink Poorly with RNA Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA ROBERT D. PATTON,1,2 MANU SANJEEV,2,3 LAUREN A. WOODWARD,2,3 JUSTIN W. MABIN,2,3 RALF BUNDSCHUH,1,2,4,5 and GURAMRIT SINGH2,3 1Department of Physics, The Ohio State University, Columbus, Ohio 43210, USA 2Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA 3Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210, USA 4Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA 5Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio 43210, USA ABSTRACT In eukaryotic cells, proteins that associate with RNA regulate its activity to control cellular function. To fully illuminate the basis of RNA function, it is essential to identify such RNA-associated proteins, their mode of action on RNA, and their preferred RNA targets and binding sites. By analyzing catalogs of human RNA-associated proteins defined by ultraviolet light (UV)-dependent and -independent approaches, we classify these proteins into two major groups: (i) the widely recognized RNA binding proteins (RBPs), which bind RNA directly and UV-crosslink efficiently to RNA, and (ii) a new group of RBP-associated factors (RAFs), which bind RNA indirectly via RBPs and UV-crosslink poorly to RNA. As the UV crosslink- ing and immunoprecipitation followed by sequencing (CLIP-seq) approach will be unsuitable to identify binding sites of RAFs, we show that formaldehyde crosslinking stabilizes RAFs within ribonucleoproteins to allow for their immunoprecip- itation under stringent conditions. Using an RBP (CASC3) and an RAF (RNPS1) within the exon junction complex (EJC) as examples, we show that formaldehyde crosslinking combined with RNA immunoprecipitation in tandem followed by se- quencing (xRIPiT-seq) far exceeds CLIP-seq to identify binding sites of RNPS1. xRIPiT-seq reveals that RNPS1 occupancy is increased on exons immediately upstream of strong recursively spliced exons, which depend on the EJC for their inclusion. Keywords: CLIP-seq; RNA binding proteins; exon junction complex; UV crosslinking; formaldehyde crosslinking; pre- mRNA splicing INTRODUCTION teins, and its eventual degradation (Müller-McNicoll and Neugebauer 2013; Singh et al. 2015). Therefore, to fully As cells grow, divide, and respond to their environment, comprehend the intricate workings of cellular processes, they critically depend on RNA-associated proteins to reg- it is important to identify RNA-associated proteins and elu- ulate RNA biogenesis and function. RNA-associated pro- cidate their functions. teins participate in all aspects of RNA biology—they The post-genomic era has witnessed a revolution in assemble RNA into ribonucleoprotein (RNP) machines our ability to catalog RNA-associated proteins encoded (e.g., spliceosome, ribosome), membraneless RNP organ- in the human and other genomes. Early efforts to build cat- elles (e.g., nuclear speckles, stress granules), and gene/ alogs of RNA-associated proteins mainly relied on se- chromatin regulatory complexes (e.g., Polycomb repres- quence similarity to well-known RNA binding domains sive complex 2). In the case of messenger RNA (mRNA), (Anantharaman et al. 2002). More recent efforts have taken RNA-associated proteins control its processing, subcellu- advantage of the ability of RNA and proteins in direct phys- lar location, intracellular transport, translation into pro- ical contact to form “zero-length” covalent bonds when Corresponding author: [email protected] Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna. © 2020 Patton et al. This article, published in RNA, is available under a 074856.120. Freely available online through the RNA Open Access Creative Commons License (Attribution-NonCommercial 4.0 Interna- option. tional), as described at http://creativecommons.org/licenses/by-nc/4.0/. 1216 RNA (2020) 26:1216–1233; Published by Cold Spring Harbor Laboratory Press for the RNA Society Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Formaldehyde versus UV crosslinking of RNPs exposed to shortwave ultraviolet (UV) light (Hockensmith manner (Mabin et al. 2018). To identify RNAs bound to EJC et al. 1993). Such covalent crosslinking “freezes” dynamic inside human cells, we have devised a UV crosslinking-in- intermolecular RNA:protein interactions as they occur in dependent tandem purification approach termed RNA IP situ to enable their biochemical analysis. This property of in tandem (RIPiT) (Singh et al. 2012, 2014). To investigate direct RNA:protein contacts has been exploited to identify binding sites of more transient and/or labile complexes, the protein interactome of polyadenylated RNA (Baltz et al. such as the EJCs containing alternate factor RNPS1, we 2012; Castello et al. 2012; Hentze et al. 2018), and more re- have also combined RIPiT with formaldehyde-based chem- cently to unveil the proteins that come in contact with all ical-crosslinking (Singh et al. 2014). We and others have cellular RNA (Queiroz et al. 2019; Trendel et al. 2019; Urda- also successfully applied RIPiT-seq with and without form- neta et al. 2019). These studies conservatively estimate that aldehyde crosslinking to investigate binding profiles of the human genome encodes more than 1200 proteins that RNA-associated proteins beyond the EJC, that is, directly contact RNA, and hence function as RNA binding Staufen1 (Ricci et al. 2014) and WDR5 (Yang et al. 2014). proteins (RBPs). The UV reactivity of the RNA:protein Formaldehyde is a small, cell permeable, rapid, and re- contacts has also transformed our understanding of versible crosslinker that can covalently link proteins to nu- global-scale RNA cargoes of individual RBPs. UV crosslink- cleic acids and other proteins when they exist in close ing-immunoprecipitation (CLIP)-based methods allow puri- proximity (Hoffman et al. 2015). Thus, formaldehyde is an fication of an RBP of interest and its crosslinked RNAs, attractive alternative to UV to crosslink both RBPs and which can then be identified via high-throughput sequenc- RAFs within cellular RNPs. The utility of formaldehyde ing (Lee and Ule 2018). Advantageously, the protein ad- crosslinking prior to RIP to enrich RBP-bound RNAs was first ducts on crosslinked RNA can be leveraged to map RNA shown nearly two decades ago (Niranjanakumari et al. positions directly in contact with RBPs to obtain a single-nu- 2002). Ever since, formaldehyde crosslinking has been uti- cleotide resolution view of in vivo RNA:protein interactions. lized to capture RNA cargoes of RNA-associated proteins While UV crosslinking-based approaches have illuminat- from diverse eukaryotic systems (e.g., Huang and Hopper ed many areas of RNA biology, like any other method, they 2015; Hendrickson et al. 2016; Chatterjee et al. 2017). too come with limitations. For example, the UV crosslink- More recently, formaldehyde crosslinking has been com- ing ability of an RBP is likely to be influenced by sequence bined with a CLIP-seq workflow to identify binding sites composition of RNA and protein at binding interfaces of DROSHA, a double-stranded RBP that poorly UV-cross- (Smith 1969; Hockensmith et al. 1986), and also by the links with RNA (Kim and Kim 2019). Despite such general strength and duration of interactions. Importantly, UV use, several fundamental issues regarding formaldehyde crosslinking is limited in applicability to proteins that are crosslinking remain to be tested: its degree of influence in direct contact with RNA (i.e., RBPs) and will be ill-suited on specificity of RIP signal, its performance in comparison to study proteins that interact with RNA indirectly via RBPs to UV crosslinking, and its applicability to RBPs versus (RBP-associated factors, RAFs). Although we currently lack RAFs, to name a few. full understanding of the prevalence of RAFs, a closer ex- Here we describe a comparative analysis of published amination of cellular RNPs reveals many proteins that catalogs of human RNA-associated proteins that were de- play a critical role in RNA biology without directly contact- fined based on UV crosslinking ability of proteins to RNA or ing RNA (e.g., nuclear export factors GLE1 and NXT1; protein:protein interaction networks of annotated RBPs (a EIF4E binding proteins). RAFs can function along with UV crosslinking-independent approach). This analysis en- RBPs either as their regulators or as subunits of multipro- ables us to categorize human RNA-associated proteins tein complexes that act on RNA, or both. It is therefore im- into RBPs and RAFs. We find that RAFs are prevalent in portant to gain insights into the prevalence, properties, all steps of RNA metabolism and display a wide array of and functions of RAFs. Thus, UV crosslinking-independent molecular functions and biochemical activities. This analy- methods are critical to investigate interactions of RAFs with sis also confirms the classification of EJC factors into RBPs RNA inside the cells. (EIF4A3, CASC3) and RAFs (MAGOH, RNPS1). To investi- Our perspective on RBPs and RAFs is shaped by our in- gate binding sites of EJC proteins, we have previously vestigation of the exon junction complex (EJC), a multipro- used RIPiT-seq either from uncrosslinked human cells tein complex that mainly assembles ∼24 nt upstream of (nRIPiT-seq) (Singh et al. 2012; Mabin et al. 2018) or from exon–exon junctions during pre-mRNA splicing (Boehm formaldehyde-crosslinked cells (xRIPiT-seq) (Mabin et al. and Gehring 2016; Le Hir et al. 2016; Woodward et al. 2018). Other groups have instead used CLIP-seq to map 2017). The EJC contains both RBPs (EIF4A3) and RAFs binding sites of RBPs and RAFs within EJCs (Saulière (MAGOH, RBM8A) within its core. The EJC core also inter- et al. 2012; Hauer et al. 2016). Here we use these existing acts with several peripheral proteins that link it to various nRIPiT-seq, xRIPiT-seq, and CLIP-seq data sets for CASC3 steps in post-transcriptional mRNA regulation.
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