easyCLIP analysis of RNA-protein interactions incorporating absolute quantification

Porter, Douglas F.; Miao, Weili; Yang, Xue; Goda, Grant A.; Ji, Andrew L.; Donohue, Laura K.; Aleman, Maria M.; Domínguez, Daniel; Khavari, Paul A.

doi:10.1038/s41467-021-21623-4

Cited by 37 publications

(28 citation statements)

References 43 publications

(67 reference statements)

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“…The PAR-CLIP protocol was adapted from [ 103 , 104 ]. 3×10 6 HeLa cells were plated in 15 cm dishes.…”

Section: Methodsmentioning

confidence: 99%

“…RNA-protein complexes were visualized on a Typhoon imaging device (GE Healthcare), cut out of the membrane, and extracted by digestion with 10 U proteinase K (Thermo Fisher Scientific) in SDS-proteinase K buffer (10 mM TrisCl pH 7.5, 5 mM NaCl, 0.1 mM EDTA, 0.02% [w/v] SDS) at 55 °C for 45 min. Ligated RNA was purified via oligo-dT magnetic beads (Thermo Fisher Scientific) and reverse transcribed as previously described [ 104 ]. NEBNext small RNA primers were used to prepare the sequencing library from cDNA.…”

Section: Methodsmentioning

confidence: 99%

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Control of immediate early gene expression by CPEB4-repressor complex-mediated mRNA degradation

et al. 2022

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Background Cytoplasmic polyadenylation element-binding protein 4 (CPEB4) is known to associate with cytoplasmic polyadenylation elements (CPEs) located in the 3′ untranslated region (UTR) of specific mRNAs and assemble an activator complex promoting the translation of target mRNAs through cytoplasmic polyadenylation. Results Here, we find that CPEB4 is part of an alternative repressor complex that mediates mRNA degradation by associating with the evolutionarily conserved CCR4-NOT deadenylase complex. We identify human CPEB4 as an RNA-binding protein (RBP) with enhanced association to poly(A) RNA upon inhibition of class I histone deacetylases (HDACs), a condition known to cause widespread degradation of poly(A)-containing mRNA. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis using endogenously tagged CPEB4 in HeLa cells reveals that CPEB4 preferentially binds to the 3′UTR of immediate early gene mRNAs, at G-containing variants of the canonical U- and A-rich CPE located in close proximity to poly(A) sites. By transcriptome-wide mRNA decay measurements, we find that the strength of CPEB4 binding correlates with short mRNA half-lives and that loss of CPEB4 expression leads to the stabilization of immediate early gene mRNAs. Akin to CPEB4, we demonstrate that CPEB1 and CPEB2 also confer mRNA instability by recruitment of the CCR4-NOT complex. Conclusions While CPEB4 was previously known for its ability to stimulate cytoplasmic polyadenylation, our findings establish an additional function for CPEB4 as the RNA adaptor of a repressor complex that enhances the degradation of short-lived immediate early gene mRNAs.

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“…The PAR-CLIP protocol was adapted from [ 103 , 104 ]. 3×10 6 HeLa cells were plated in 15 cm dishes.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Control of immediate early gene expression by CPEB4-repressor complex-mediated mRNA degradation

et al. 2022

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“…8a,b). Restricting the comparison to genes with similar expression levels between 293T and HepG2 cells, or comparing TLC-CLIP and easyCLIP 18 , which were both performed in 293T cells, increases the overlap to up to 72%, which is similar to technical variation observed between replicates, given the stochastic nature of RNA binding (Fig. 2b and Supplementary Fig.…”

mentioning

confidence: 88%

Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP

Ernst

Duc

Trono

2022

Preprint

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RNA-binding proteins are instrumental for post-transcriptional gene regulation, yet transcriptomewide methods to profile RNA-protein interactions remain technically challenging. We present an improved library preparation strategy for crosslinking and immunoprecipitation (CLIP) that involves tailing and ligation of cDNA molecules (TLC) for increased sensitivity and efficiency. TLC-CLIP eliminates time-consumingpurifications, reduces sample loss and minimises experimental steps, allowing precise profiling of RNA-protein interactions from limited starting material at nucleotide resolution.

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“…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.…”

Section: Surveying Vrna-rbp Interactions In Cells Enables the Discovery And Characterization Of Rbps; New And Oldmentioning

confidence: 99%

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

Lisy

Rothamel

Ascano

2021

Viruses

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As the first intracellular host factors that directly interact with the genomes of RNA viruses, RNA binding proteins (RBPs) have a profound impact on the outcome of an infection. Recent discoveries brought about by new methodologies have led to an unprecedented ability to peer into the earliest events between viral RNA and the RBPs that act upon them. These discoveries have sparked a re-evaluation of current paradigms surrounding RBPs and post-transcriptional gene regulation. Here, we highlight questions that have bloomed from the implementation of these novel approaches. Canonical RBPs can impact the fates of both cellular and viral RNA during infection, sometimes in conflicting ways. Noncanonical RBPs, some of which were first characterized via interactions with viral RNA, may encompass physiological roles beyond viral pathogenesis. We discuss how these RBPs might discriminate between an RNA of either cellular or viral origin and thus exert either pro- or antiviral effects—which is a particular challenge as viruses contain mechanisms to mimic molecular features of cellular RNA.

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easyCLIP analysis of RNA-protein interactions incorporating absolute quantification

Cited by 37 publications

References 43 publications

Control of immediate early gene expression by CPEB4-repressor complex-mediated mRNA degradation

Control of immediate early gene expression by CPEB4-repressor complex-mediated mRNA degradation

Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP

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

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