RNA–protein analysis using a conditional CRISPR nuclease

Abstract

RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA-protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5′ ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.non-coding RNA | RNA processing | miRNA | mass spectrometry R NA molecules function together with specific binding proteins to regulate cellular pathways at the levels of transcription, posttranscriptional modification, and translation (1-4). For example, microRNAs (miRNA), siRNAs, and piwi-interacting RNAs (piRNAs) regulate more than 30% of mammalian gene expression (5). Small nucleolar RNAs (snoRNAs) govern the sites and efficiencies of RNA chemical modifications in cells (6), whereas long noncoding RNAs (lncRNAs), such as HOTAIR and MALAT1, have been implicated in chromatin remodeling, transcriptional activation, and tumorigenesis (7,8). UTRs of mRNA transcripts are also known to interact with regulatory proteins to control their expression level as well as their stability. Understanding how these RNAs function in cells and how they may be manipulated for therapeutic purposes is an important goal that spans many areas of biology.Although new ncRNAs are being discovered at a rapid pace, determination of their biochemical activities is often slow. A major barrier to such functional analysis is the current difficulty of identifying RNA-binding partners that associate with specific transcripts and participate in their biological behavior. Despite the development of various RNA affinity purification methods, the expense and/or technical challenges associated with each approach has precluded its use by nonspecialists or in high-throughput discovery experiments. Current strategies for identifying RNA-binding proteins that associate with specific transcripts involve the use of affinity tags, including biotin, aptamers, and particular proteinbinding sequences (9-13). In each case, however, the modest affinity or specificity of tag recognition and the difficulty of selective elution complicate sample analysis. A strategy that will allow simple and rapid identification of proteins that associate selectively with particu...

ARiBo pull-down for riboproteomic studies based on label-free quantitative mass spectrometry

“…RNA-binding proteins (RBPs; black dot) are inferred based on recent genome-wide AP-MS studies of mRNA-binding proteins (Baltz et al 2012;Castello et al 2012;Kwon et al 2013;Beckmann et al 2015). Results from previous AP-MS studies are provided for let-7a-1 stem-loops (7a-1; 1: WCE from Huh7 cells [Heo et al 2008]; 2: with nuclear extract from HeLa cells [Michlewski et al 2008;Michlewski and Caceres 2010]; 3: WCE from Lin28A-transfected HEK203T cells [Heo et al 2009]; 4: WCE from P9 cells [Chang et al 2013]; and 5: WCE from HeLa and NT2 cells [Lee et al 2013]) and a let-7g stem-loop (7g; 6: with P19 cells ). a The SAINTexpress scores for the identification of the YBX1 protein from the SL-let-7a-1 and SL-let-7g pull-down versus the mock control are just below the cutoff (0.65 > score ≥ 0.60).…”

“…The ARiBo tag itself contributes significantly to the high sensitivity and specificity of the RNA pulldown by affording high-affinity immobilization via the λboxB/N peptide complex (K d of 10 pM) (Austin et al 2002;Di Tomasso et al 2011) as well as highly efficient elution of the RNA-protein complex by activatable cleavage of the glmS ribozyme. Similarly, activatable cleavage of an RNA tag has previously been exploited for AP-MS studies using the activatable Csy4 enzyme of a cognate RNA substrate tag, where it was shown to dramatically reduce the amount of background protein contaminants (Lee et al 2013). In comparison to a noncleavable system, elution by cleavage of the target RNA from the immobilized tag prevents the elution of proteins that are bound to the tag and thus likely reduces the amount of eluted proteins that are not specifically associated with the target RNA.…”

“…We performed quantitative LC-MS/MS of RNA pull-downs using biological triplicates and two experimental controls to identify proteins that specifically bind to the stem-loops of let-7a-1 and let-7g. Several proteins were identified that were previously shown to bind immature forms of let-7 miRNAs (Heo et al 2008(Heo et al , 2009Michlewski et al 2008;Viswanathan et al 2008;Michlewski and Caceres 2010;Chang et al 2013;Lee et al 2013). In addition, we identified an extensive group of novel protein factors not previously found to bind these RNAs.…”

“…Thus, there is considerable interest in developing quick single-step affinity purification protocols that maximize the capture of true interaction partners and minimize nonspecific interactions (Oeffinger 2012;Faoro and Ataide 2014;McHugh et al 2014). Moreover, given that only a few studies have combined RNA pull-down with quantitative MS (Butter et al 2009;Lee et al 2013;Adachi et al 2014;Leppek and Stoecklin 2014), there is a need to develop robust RNA affinity purification procedures that can complement recent developments in label-free quantitative proteomics (Oeffinger 2012;Faoro and Ataide 2014;McHugh et al 2014).…”

“…The RNA pulldown procedure was developed using in vitro transcribed ARiBo-tagged stem-loops present in the immature forms of miRNAs (miRNA stem-loops) to capture RNA-associating proteins from whole cell extracts (WCEs). Stem-loops derived from the precursors of let-7a-1 and let-7g were used (Bussing et al 2008;Roush and Slack 2008;Viswanathan and Daley 2010;Thornton and Gregory 2012;Nguyen and Zhu 2015;Rehfeld et al 2015), since several proteomic studies have been reported for these RNAs (Heo et al 2008(Heo et al , 2009Michlewski et al 2008;Viswanathan et al 2008;Michlewski and Caceres 2010;Chang et al 2013;Lee et al 2013). In addition, let-7a-1 and let-7g are two of the 12 human let-7 miRNAs that play important roles in mammalian development, metabolism, and cancer (Bussing et al 2008;Roush and Slack 2008;Viswanathan and Daley 2010;Thornton and Gregory 2012;Nguyen and Zhu 2015;Rehfeld et al 2015), and there is still significant interest in identifying proteins that control biogenesis of these miRNAs though interactions with the stem-loop structures present in their immature forms.…”