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. 2022 Dec 16;3(4):101823.
doi: 10.1016/j.xpro.2022.101823. Epub 2022 Nov 16.

Deconvolution of in vivo protein-RNA contacts using fractionated eCLIP-seq

Giulia Biancon  1 , Emma Busarello  2 , Poorval Joshi  3 , Bluma J Lesch  4 , Stephanie Halene  5 , Toma Tebaldi  6
Affiliations

Deconvolution of in vivo protein-RNA contacts using fractionated eCLIP-seq

Giulia Biancon et al. STAR Protoc. .

Abstract

Thousands of RNA-binding proteins orchestrate RNA processing and altered protein-RNA interactions frequently lead to disease. Here, we present experimental and computational analysis pipelines of fractionated eCLIP-seq (freCLIP-seq), a modification of enhanced UV-crosslinking and RNA immunoprecipitation followed by sequencing. FreCLIP-seq allows transcriptome-wide analysis of protein-RNA interactions at single-nucleotide level and provides an additional level of resolution by isolating binding signals of individual RNA-binding proteins within a multicomponent complex. Binding occupancy can be inferred from read counts and crosslinking events. For complete details on the use and execution of this protocol, please refer to Biancon et al. (2022).

Keywords: Bioinformatics; Cell Biology; Molecular Biology; Protein Biochemistry; Protein expression and purification; RNAseq; Sequencing.

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Conflict of interest statement

Declaration of interests S.H., consultancy, Forma Therapeutics.

Figures

None
Graphical abstract
Figure 1
Figure 1
Autoradiographic visualization and membrane size-selection Top: representative U2AF1 freCLIP-seq membrane cutting to obtain U2AF1 light and heavy fractions in U2AF1 wildtype and mutant (S34F or Q157R) conditions. Figure adapted and reprinted with permission from (Biancon et al., 2022). Bottom: representative AGO2 eCLIP-seq membrane cutting to obtain AGO2-RNA complexes. See also Figure S1.
Figure 2
Figure 2
Quality control of representative U2AF1 freCLIP-seq libraries before sequencing (A) Visualization by TBE-Urea gel. Yellow lines mark the expected library size. (B) Visualization by Agilent 2100 Bioanalyzer. Peak size is reported. Lower and upper marker peaks are indicated, respectively, in green and purple. These libraries are ready for sequencing: 175–350 bp range (yellow box), good concentration, no adapter dimers (<10%), primer excess can be ignored.
Figure 3
Figure 3
Characterization of transcriptome-wide protein-RNA interactions (A) Distribution of AGO2 eCLIP-seq peaks in genome regions. (B) Distribution of crosslinked nucleotides in AGO2 eCLIP-seq peaks. (C) Sequence logo of AGO2 eCLIP-seq peak regions (10 nucleotide on each side from the crosslinked nucleotide).
Figure 4
Figure 4
Comparison of freCLIP-seq occupancies between different conditions in specific transcript regions (A) Metaprofile (mean±SEM) of U2AF1 freCLIP-seq occupancies in all intron-exon junctions, comparing wildtype with the S34F mutant samples. (B) Profile (mean±SEM) of U2AF1 freCLIP-seq occupancies in a selected intron-exon junction, comparing wildtype with the S34F mutant samples.
Figure 5
Figure 5
Issues during autoradiographic visualization Representative U2AF1 freCLIP-seq samples obtained from not-infected HEL cells.
Figure 6
Figure 6
Presence and removal of adapter dimers (A) Visualization by TBE-Urea gel of representative U2AF1 freCLIP-seq libraries. Yellow lines mark the expected library size. Purple stars indicate adapter dimers around 150 bp in post-AMP libraries. Gray-labeled samples are the purple-labeled samples after re-amplification changing PCR cycles or adapters. (B) Visualization by Agilent 2100 Bioanalyzer of representative U2AF1 freCLIP-seq libraries obtained from not-infected HEL cells. Yellow boxes mark the expected library size. Peak size is reported. Purple stars indicate adapter dimers’ peak around 150 bp in post-AMP libraries. Black-labeled samples are the respective purple-labeled samples after gel purification.
None
See this image and copyright information in PMC

References

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