The human proteome with direct physical access to DNA

Trendel J, Trendel S, Sha S, Greulich F, Goll S, Wudy SI, Kleingrewe K, Kubicek S, Uhlenhaut NH, Kuster B. Cell 188, 4424–4440 (2025).

• Metabolic labeling with the photo-reactive nucleotide 4-thiothymidine (4ST) and rapid irradiation in a high-intensity 365 nm irradiation device crosslink proteins to DNA at zero distance in living cells.
• Unlike formaldehyde fixation, which also links proteins indirectly through other proteins or RNA in chromatin, XDNAX captures only direct protein-DNA contacts.
• We drafted the first human DNA-interacting proteome, with almost 700 peptide-DNA crosslinks that pinpointed contact sites at single-amino-acid resolution, some overlapping disease-associated variants.
• Quantifying about 1,800 DNA-interacting proteins in breast cancer cells treated with estrogen, genotoxic drugs or a BAF-complex inhibitor followed transcription factors, DNA repair proteins and chromatin remodelers within minutes.
• XDNAX also works in primary cells, as we showed in mouse bone marrow-derived macrophages during LPS stimulation.

XRNAX and cytosolic XRNAX, with label-free protein-level quantification

Trendel J, Prokofeva P, Chen ZA, Horn L, Trendel S, Mema M, Stuiver M, Rappsilber J, Kuster B. Nucleic Acids Research 54, gkag339 (2026).

This is the current version of XRNAX, without trypsin pre-digestion and with label-free quantification at protein level. We use a high-intensity 365 nm irradiation device that enables crosslinking in seconds and add a simplified XRNAX version for the quantification of protein-RNA interactions in the cytosol.

• Streamlined and improved XRNAX protocols isolate RNA-crosslinked proteins for label-free quantification at protein level, with no trypsin pre-digestion or peptide-level quantification required.
• 22-minute LC-MS runs quantify near-complete RNA-interacting proteomes and resolved responses to 12 RNA-binding drugs after 5 minutes.
• Cytosolic XRNAX showed the ASCC complex, ZNF598 and ubiquitin arriving on cytosolic RNA 2 minutes after UV damage.

The human RNA-binding proteome and its dynamics during translational arrest

Trendel J, Schwarzl T, Horos R, Prakash A, Bateman A, Hentze MW, Krijgsveld J. Cell 176, 391–403 (2019).

• XRNAX purifies photo-crosslinked protein-RNA complexes from UV-irradiated cells and captures proteins bound to all RNA biotypes, not only polyadenylated mRNA.
• RNA sequencing showed that XRNAX recovers RNA of all biotypes, depleting ribosomal RNA and enriching protein-coding and long non-coding transcripts.
• In several human cell lines it uncovered many previously unknown RNA-binding proteins, including more than 700 that interact with non-polyadenylated RNA and nuclear proteins missed by poly(A)-based methods.
• Enriched peptide-RNA crosslinks located an RNA contact site in a domain of unknown function, now annotated as the WKF RNA-binding domain.
• XRNAX quantified changes in RNA binding during arsenite-induced translational arrest, and as CLIP-seq input its purified complexes revealed RNA targets of LMNB1 and EXOSC2.

pepR-SALT: sequencing protein and RNA on both sides of the molecular interface

Sha S, Kuster B, Trendel J. bioRxiv 2025.09.05.674461 (2025).

• pepR-MS isolates the photo-crosslinked protein-RNA interface represented by peptide-RNA hybrids whose nucleotide moiety can be tuned to different lengths by nuclease choice.
• Removing free RNA by SCX cleanup unmasks peptide signals, raises crosslink identifications and strongly improves chromatography and robustness.
• Over 34,000 unique crosslinks in 589 proteins pinpointed RNA contacts in domains and intrinsically disordered regions (IDRs), revealing crosslinking hotspots at aromatic residues of the RRM β-sheet.
• Over 400 sites in disordered regions clustered in short contact patches rich in aromatic residues and glycine, and poor in acidic residues.
• Analyzing crosslinks with up to five-nucleotide RNA chains, SALT, which builds on MSFragger, assigned peptide and RNA sequence together for over 1,600 crosslinks in one MS run.

PEPseq: transcriptome-wide protein occupancy

Trendel J, Boileau E, Jochem M, Dieterich C, Krijgsveld J. Nucleic Acids Research 51, e79 (2023).

• PEPseq enriches RNA fragments crosslinked to proteins with NHS chemistry and sequences them, mapping the occupancy of the entire RNA-interacting proteome across the transcriptome.
• As the counterpart to methods for the RNA-interacting proteome, it studies the protein-interacting transcriptome without prior knowledge of the proteins involved.
• During arsenite-induced translational arrest in breast cancer cells, protein occupancy rose sharply within the coding regions of mRNAs encoding ribosomal proteins.
• These mRNAs were depleted from stress granules, and nascent-protein proteomics showed they stayed translationally repressed for more than two hours after the arrest was released.
• mRNAs in stress granules restarted translation quickly, suggesting that ribosomes stalled in the coding region keep ribosomal-protein mRNAs out of stress granules.

Rapid photo-crosslinking in living cells

Trendel J, Prokofeva P, Chen ZA, Horn L, Trendel S, Mema M, Stuiver M, Rappsilber J, Kuster B. Nucleic Acids Research 54, gkag339 (2026).

• We introduce UVEN, a high-intensity 365 nm irradiation device that delivers about 1,000 times more energy to cells than conventional bulb-based devices while protecting them from heat and desiccation.
• Rapid irradiation strongly improved protein-drug, protein-protein, protein-DNA and protein-RNA crosslinking in living cells.
• Photo-affinity labeling (PAL) with a diazirine kinase-inhibitor probe was effective after a single second, with cells kept in warm medium, instead of 10 minutes of irradiation on ice.
• Quantifying protein-RNA interactions during acute exposure to RNA-binding drugs revealed a fast response of SRSF proteins to the splicing modulators risdiplam and branaplam.
• Quantifying cytosolic protein-RNA interactions after UV damage uncovered known and new candidate proteins of the RNA damage response.