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Transcriptome wide high throughput mapping of protein-RNA interactions across human cell lines using POP-seq

ABSTRACT: Protein-RNA interactions are critical for post-transcriptional regulatory processes that govern RNA metabolism. Several existing high throughput methods based to detect these interactions are reported for the biases associated to the inherent crosslinking approaches. In this study, we used Protein Occupancy Profile-Sequencing (POP-seq), a phase separation based method that does not require crosslinking, to examine the regulatory protein RNA interactions in cancer cell lines: K562, HepG2, A549, MCF7, Jurkat, and HEK293. POP-seq provides unbiased protein occupancy profiles in a transcriptome wide manner due to its crosslinking free approach. Our study demonstrates that POP-seq can enrich the protein occupied sites with over 60,000 peaks identified across multiple cancer cell lines. In our initial analysis, we compared POP-seq identified interactions with two protocols in presence and absence of UV crosslinking in K562 and HepG2 cells and results showed >70% overlap between the two approaches at the gene level, indicating that POP-seq can efficiently map the unbiased protein occupied sites in transcriptome wide manner. Using downstream bioinformatics analysis we found majority of POP-seq peaks on the exonic region of the transcript followed by introns and 3’ UTRs, while less than 10% abundance was recorded for repetitive elements like SINEs and LINEs. Among the various gene types, we found abundance of POP-seq peaks on protein coding genes followed by non-coding RNAs, that are typically highly expressed in cancer cell lines. We used CRISPR Cas 9 system to evaluate the functionality of POP-seq sites and observed that identified interactions could potentially impact the expression of nearby exons. Overall, this study provides the first comprehensive resource of transcriptome wide protein-RNA interaction maps in multiple cell line using a crosslinking free approach that further opens the opportunity to implement the method in primary tissues for detecting the regulatory interactions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE198655 | GEO | 2024/12/23

REPOSITORIES: GEO

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Project description:Protein-RNA interactions are integral components of nearly every aspect of biology including regulation of gene expression, assembly of cellular architectures, and pathogenesis of human diseases. However, studies in the past few decades have only uncovered a small fraction of the vast landscape of the protein-RNA interactome in any organism, and even less is known about the dynamics of protein-RNA interactions under changing developmental and environmental conditions. Here, we describe the gPAR-CLIP (global photoactivatable-ribonucleoside-enhanced crosslinking and immunopurification) approach for capturing regions of the transcriptome bound by RNA-binding proteins (RBPs) in budding yeast. We report over 13,000 RBP crosslinking sites in untranslated regions (UTR) covering 72% of protein-coding transcripts encoded in the genome, confirming 3M-bM-^@M-^Y UTRs as major sites for RBP interaction. Comparative genomic analyses reveal that RBP crosslinking sites are highly conserved, and RNA folding predictions indicate that secondary structural elements are constrained by protein binding and may serve as generalizable modes of RNA recognition. Finally, 38% of 3M-bM-^@M-^Y UTR crosslinking sites show changes in RBP occupancy upon glucose or nitrogen deprivation, with major impacts on metabolic pathways as well as mitochondrial and ribosomal gene expression. Our study offers an unprecedented view of the pervasiveness and dynamics of protein-RNA interactions in vivo. Duplicate gPAR-CLIP and mRNA-seq libraries were sequenced from yeast strains for each of three conditions: log-phase growth, growth after 2 hour glucose starvation, and growth after 2 hour nitrogen starvation. Additional duplicate mRNA-seq libraries were sequenced from yeast strains grown in the absence of 4-thiouracil. gPAR-CLIP libraries were used to determine regions of mRNA bound by proteins. mRNA-seq libraries served as controls for mRNA abundance. A Puf3p PAR-CLIP library was sequenced to determine how well gPAR-CLIP captured the binding signatures of a single RNA-binding protein.

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Transcriptome wide high throughput mapping of protein-RNA interactions across human cell lines using POP-seq

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