A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol

Abstract

Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which can efficiently crosslink to interacting proteins using UVA and UVB light. Crosslinking of 4SU or 6SG to interacting amino acids changes their base-pairing properties and results in characteristic mutations in cDNA libraries prepared for high-throughput sequencing, which can be computationally exploited to remove abundant background from non-crosslinked sequences and help pinpoint RNA binding protein binding sites at nucleotide resolution on a transcriptome-wide scale. Here we present a streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. It is based on direct ligation of a fluorescently labeled adapter to the 3′end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the adapter-ligated RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation by half to 2 days and increases sensitivity by 10–100-fold.

Matrin3 regulates cell proliferation and spindle dynamics by regulating CDC14B alternative splicing

“…Matrin3 is a well-characterized RNA-binding protein (RBP) and we hypothesized that its effect on cell proliferation may be mediated by a set of specific RNA targets. Therefore, we mapped Matrin3 binding sites on RNAs on a transcriptome-wide scale and at nucleotide resolution in HCT116 cells using 4-thiouridine (4SU) PAR-CLIP 26,27 . For PAR-CLIP we immunoprecipitated (IP) endogenous, UV-crosslinked Matrin3 RNP (ribonucleoprotein) with a specific antibody.…”

“…We found that Matrin3 is overexpressed in CRC and provides growth advantage in CRC cell lines. To understand the molecular mechanism(s) by which Matrin3 mediates its effects, we performed PAR-CLIP (Photoactivatable Ribonucleoside-enhanced CrossLinking and Immunoprecipitation), a UV-crosslinking based technique that identifies RNAs directly interacting with RNA-binding proteins (RBPs) at nucleotide resolution on a transcriptome-wide scale 26,27 . We found that in HCT116 cells (CRC), Matrin3 binds to thousands of pre-mRNAs at pyrimidine-rich sequence elements repressing inclusion of nearby exons.…”

Cellular abundance shapes function in piRNA-guided genome defense

“…We used unique molecular identifiers (UMIs) to accurately represent the original number of small RNA molecules and varying adapter-terminal nucleotides to minimize ligation bias ( Supplemental Figs. S2, S3 ; Hafner et al 2011 ; Kivioja et al 2012 ; Fu et al 2018 ; Anastasakis et al 2021 ). Combining calculations from eight biological replicates, we estimated that the total number of piRNAs in a single cell ranges from ∼500,000 to ∼800,000 and does not exceed one million ( Fig.…”

“…To quantify the average number of piRNAs in a single cell, we used an established reference approach ( Farazi et al 2011 ; Gainetdinov et al 2018 ). Small RNA samples were prepared according to Hafner et al (2012) and Anastasakis et al (2021) . In brief, total small RNAs from one million OSCs were extracted using a PureLink miRNA Isolation kit (Thermo Fisher Scientific K157001) and were spiked with a calibrator mix (see below for preparation, calibrators 1–4) ( Supplemental Table S5 ; Supplemental Fig.…”

RNA Binding Proteins as Pioneer Determinants of Infection: Protective, Proviral, or Both?

“…To mitigate the possibility of RNP assembly post-cell lysis, crosslinking procedures were developed in which RNA and proteins would be chemically linked in living cells or tissue [60]. There are a wide variety of UV-based and chemical-based cross-linking and immunoprecipitation (CLIP) approaches, all of which rely on the use of antibodies to isolate an RBP of interest to enrich for corresponding target transcripts [59][60][61][62][63][64][65][66][67][68][69][70][71][72][73]. While the discovery and characterization of regulated transcripts of a single RBP remains an essential strategy towards building a global understanding of PTGR, an analogous rationale can be made for investigations that aim to discover and characterize the constellation of RBPs that act upon groups of related RNAs, or even a singular transcript.…”