PAR-CLIP and streamlined small RNA cDNA library preparation protocol for the identification of RNA binding protein target sites

Benhalevy, Daniel; McFarland, Hannah L.; Sarshad, Aishe A.; Hafner, Markus

doi:10.1016/j.ymeth.2016.11.009

Cited by 35 publications

(31 citation statements)

References 19 publications

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“…HuR (ELAVL1) has been shown to interact with mRNA and pre-mRNA in several CLIP studies and has a well-documented binding motif (5'-UUUUUU -3') (26). After in vivo labeling of cellular RNA using 4-thiouridine (4SU) and UV irradiation at 365 nm, we performed i) classical PAR-CLIP analysis (PAR-CLIPclassic) (11,27) of HuR, ii) a PAR-CLIP variant using onbead ligation of adapters (PAR-CLIP-on-beads) (28,29), and iii) a version in which we use phenol extraction (termed pCLIP) for removal of unbound RNA instead of PAGE/membrane excision (Fig. 4a).…”

Section: Purification Of Rbps From Animal Tissuementioning

confidence: 99%

“…As an alternative, the ligation of the 3´/ 5´adapters can be achieved directly on the beads used in the affinity capture of the selected clRNP (28,29). Onbeads adapters ligation was done by incubating the FLAG-clHuR-RNA beads with the 3´adapter in the presence of Rnl2(1-249)K227Q ligase and PEG-8000 overnight at 4°C.…”

Section: Par-clip On-beadsmentioning

confidence: 99%

“…Onbeads adapters ligation was done by incubating the FLAG-clHuR-RNA beads with the 3´adapter in the presence of Rnl2(1-249)K227Q ligase and PEG-8000 overnight at 4°C. After washes, the 5´adapter was ligated using the Rnl1 enzyme, 2 h at 37°C as described in (29). Followed by protein electrophoresis (4-12% Nu-PAGE MOPS, Invitrogen), blotting to nitrocellulose membranes and band excision at the defined size-range (50 to 60 kDa).…”

Section: Par-clip On-beadsmentioning

confidence: 99%

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Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Urdaneta

Vieira-Vieira

Hick

et al. 2018

Preprint

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Recent methodological advances allowed the identification of an increasing number of RNAbinding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to nonadenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). To overcome these limitations, we have developed a novel protocol, Phenol Toluol extraction (PTex), that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a first global RNA-bound proteome of human HEK293 cells and Salmonella Typhimurium as a bacterial species.

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Section: Purification Of Rbps From Animal Tissuementioning

confidence: 99%

Section: Par-clip On-beadsmentioning

confidence: 99%

Section: Par-clip On-beadsmentioning

confidence: 99%

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Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Urdaneta

Vieira-Vieira

Hick

et al. 2018

Preprint

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“…To generate a simpler and faster way of producing CLIP-seq datasets, a method was developed using on-bead ligations 5–7 of 3′ adapters (termed L3) and 5′ adapters (termed L5), each with a different fluorescent dye 8 (Figure 1A, B). After running an SDS-PAGE gel and transferring to a nitrocellulose membrane, single- and dual-ligated RNA were clearly visible (Figure 1C).…”

Section: Resultsmentioning

confidence: 99%

easyCLIP Quantifies RNA-Protein Interactions and Characterizes Recurrent PCBP1 Mutations in Cancer

Porter

Khavari

2019

Preprint

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35RNA-protein interactions mediate a host of cellular processes, underscoring the need 36 for methods to quantify their occurrence in living cells. RNA interaction frequencies 37 for the average cellular protein are undefined, however, and there is no quantitative 38 threshold to define a protein as an RNA-binding protein (RBP). Ultraviolet (UV) cross-39 linking immunoprecipitation (CLIP)-sequencing, an effective and widely used 40 means of characterizing RNA-protein interactions, would particularly benefit from 41 the capacity to quantitate the number of RNA cross-links per protein per cell. In 42 addition, CLIP-seq methods are difficult, have high experimental failure rates and 43 many ambiguous analytical decisions. To address these issues, the easyCLIP 44 method was developed and used to quantify RNA-protein interactions for a panel 45of known RBPs as well as a spectrum of random non-RBP proteins. easyCLIP 46 provides the advantages of good efficiency compared to current standards, a 47 simple protocol with a very low failure rate, troubleshooting information that 48 includes direct visualization of prepared libraries without amplification, and a new 49 form of analysis. easyCLIP, which uses sequential on-bead ligation of 5' and 3' 50 adapters tagged with different infrared dyes, classified non-RBPs as those with a 51 per protein RNA cross-link rate of <0.1%, with most RBPs substantially above this 52 threshold, including Rbfox1 (18%), hnRNPC (22%), CELF1 (11%), FBL (2%), and 53 STAU1 (1%). easyCLIP with the PCBP1 L100 RBP mutant recurrently seen in cancer 54 quantified increased RNA binding compared to wild-type PCBP1 and suggested a 55 potential mechanism for this RBP mutant in cancer. easyCLIP provides a simple, 56 efficient and robust method to both obtain both traditional CLIP-seq information 57and to define actual RNA interaction frequencies for a given protein, enabling 58 quantitative cross-RBP comparisons as well as insight into RBP mechanisms. 59 60 61 62 65challenges of integrating such data between RNA-binding proteins (RBPs) was recently 66 highlighted 1 . The physical reality of RNA-protein interactions is their individual occurrence in 67individual cells, which may be abstracted to an average complex number per-cell in a 68population. The RNA-protein complex count per-cell may be normalized to derive the number 69 of complexes per-interaction partner. It is these frequencies, per-cell and per-interaction 70 partner, that are the most basic characterizations of RNA-protein interaction networks. 71Determining the targets of an RBP by enrichment over negative control immunopurifications, 72or by clustering of cross-links, or many such other approaches, are all ultimately inferring that 73the absolute count of an RNA-protein complex in the cell is abnormally high. The estimation 74of per-cell and per-protein absolute quantities provide the ultimate framework for describing 75 a global and widely reproducible view of RNA-protein interactions. 76 77There is currently no general method to estimate abs...

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“…PAR-CLIP was performed as described previously (40,68). Briefly, for each PAR-CLIP experiment, 500-600 x 10 6 HAP1 RBM4 KO 2 cells transduced with FLAG-RBM4 grown in 15 cm plates were used for each biological replicate.…”

Section: Par-clipmentioning

confidence: 99%

Post-transcriptional regulation of human endogenous retroviruses by RNA-Binding Motif Protein 4, RBM4

Foroushani

Chim

Wong

et al. 2020

Preprint

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The human genome encodes for over 1,500 RNA-binding proteins (RBPs), which coordinate regulatory events on RNA transcripts (Gerstberger et al., 2014). Most studies of RBPs concentrate on their action on mRNAs that encode protein, which constitute a minority of the transcriptome. A widely neglected subset of our transcriptome derives from integrated retroviral elements termed endogenous retroviruses (ERVs) that comprise ~8% of the human genome. Some ERVs have been shown to be transcribed under physiological and pathological conditions suggesting that sophisticated regulatory mechanisms to coordinate and prevent their ectopic expression exist. However, it is unknown whether RBPs and ERV transcripts directly interact to provide a post-transcriptional layer of regulation. Here, we implemented a computational pipeline to determine the correlation of expression between individual RBPs and ERVs from single-cell or bulk RNA sequencing data. One of our top candidates for an RBP negatively regulating ERV expression was RNA-Binding Motif Protein 4 (RBM4). We used PAR-CLIP to demonstrate that RBM4 indeed bound ERV transcripts at CGG consensus elements. Loss of RBM4 resulted in elevated transcript level of bound ERVs of the HERV-K and -H families, as well as increased expression of HERV-K envelope protein. We pinpointed RBM4 regulation of HERV-K to a CGG-containing element that is conserved in the long terminal repeats (LTRs) of HERV-K-10 and -K-11, and validated the functionality of this site using reporter assays. In summary, we identified RBPs as potential regulators of ERV function and demonstrate a new role for RBM4 in controlling ERV expression.Significance StatementThe expression of endogenous retroviruses (ERVs) appears to have broad impact on human biology. Nevertheless, only a handful of transcriptional regulators of ERV expression are known and to our knowledge no RNA-binding proteins (RBPs) were yet implicated in positive or negative post-transcriptional regulation of ERVs. We implemented a computational pipeline that allowed us to identify RBPs that modulate ERV expression levels. Experimental validation of one of the prime candidates we identified, RBM4, showed that it indeed bound RNAs made from ERVs and negatively regulated the levels of those RNAs. We hereby identify a new layer of ERV regulation by RBPs.

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PAR-CLIP and streamlined small RNA cDNA library preparation protocol for the identification of RNA binding protein target sites

Cited by 35 publications

References 19 publications

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

easyCLIP Quantifies RNA-Protein Interactions and Characterizes Recurrent PCBP1 Mutations in Cancer

Post-transcriptional regulation of human endogenous retroviruses by RNA-Binding Motif Protein 4, RBM4

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