Julian König

Alternative splicing of pre-mRNA is a fundamental step in human gene regulation, and aberrant splicing is frequently linked to complex diseases, including cancer. All splicing events, such as skipping or inclusion of an alternative exon (AE) and intron retention (IR), are regulated by a common molecular machinery, the spliceosome. However, it remains elusive how the spliceosome coordinates different splicing decisions. Here, we analyzed a large-scale mutagenesis screen and transcriptome-wide RNA sequencing data to show that intron retention adjacent to alternative exons most commonly occurs at intermediate inclusion levels. Using data-driven mathematical modeling, we revealed that multistep exon recognition mediated by spliceosome assembly and maturation explains the observed AE-IR dependency for cis-acting sequence mutations and upon knockdown of trans-acting RNA-binding proteins. Furthermore, we found that multistep exon recognition is commonly perturbed in a transcriptome-wide manner in cancer cells, which leads to the coordinated deregulation of IR and AE decisions. In total, our work suggests that stepwise alternative exon recognition by the spliceosome coordinates AE inclusion and the retention of flanking introns, which may facilitate the search for common molecular mechanisms for mis-splicing in cancer.

SUMMARY: UV-C crosslinking and immunoprecipitation (CLIP)–based methods are the gold standard for identifying direct RNA binding protein (RBP) interaction sites on cellular RNA in vivo . Here, we describe individual-nucleotide resolution CLIP version 3 (iCLIP3), an optimized protocol for generating transcriptome-wide maps of RBP–RNA interaction sites at single-nucleotide resolution from low-input material. iCLIP3 introduces several key improvements over previous iCLIP variants, including rapid and safe infrared-based visualization of RBP–RNA complexes, silica column-based RNA isolation, and the incorporation of TruSeq adapter sequences with unique dual indexing. These modifications streamline library preparation, facilitate multiplexing, and enable concurrent sequencing of iCLIP3 libraries alongside unrelated RNA-seq libraries. In addition, we provide a detailed bioinformatics workflow for identifying crosslinking events and defining RBP binding sites. The complete protocol can be performed within 4–5 days, offering a robust and efficient approach for high-resolution mapping of RBP–RNA interactions. Highlights: 3′ RNA labeling with pCp-IR750 enables rapid infrared visualization of RBP–RNA complexes Silica column-based RNA isolation improves efficiency and reproducibility TruSeq adapters with unique dual indexing enable flexible and scalable multiplexing iCLIP3 generates high-quality libraries from ultra-low amounts of cellular material

Alternative splicing, regulated by RNA-binding proteins (RBPs), enables the generation of diverse transcript isoforms critical for cellular function. However, how RNA secondary structure impacts RBP binding and function remains poorly understood. Here, we unravel how RNA G-quadruplexes (rG4s) facilitate cooperativity in splicing regulation by the RBP heterogeneous nuclear ribonucleoprotein H (HNRNPH). Through high-throughput in vivo and in vitro studies combined with theoretical modeling, we dissect how rG4s mediate cooperative HNRNPH binding to RNA, ultimately modulating the splicing of hundreds of exons. rG4 unfolding by HNRNPH exposes multiple G-rich binding sites, thereby establishing indirect cooperativity, which is further amplified to achieve switch-like splicing regulation. HNRNPH-mediated regulation is evident in breast cancer patients, with tumors showing rG4-disrupting variants and global HNRNPH alterations, driving distinct splicing patterns that distinguish tumor subtypes. Overall, our findings offer valuable insights into the mechanistic role of RNA secondary structures in cooperative RBP binding and splicing regulation and highlight the clinical relevance of HNRNPH-dependent splicing in cancer.

Liquid–liquid phase separation (LLPS) of polymers underlies the formation of biomolecular condensates and offers a versatile route to functional soft materials. Traditionally, LLPS is attributed to changes in solvent quality or associative coacervation, but here a purely entropic connectivity‐driven mechanism is demonstrated: reversible crosslinking. Using coarse‐grained simulations of a minimal bead–spring model in good solvent, it is shown that transient, pairwise crosslinks alone can drive phase separation at ultralow polymer densities, yielding highly swollen, water‐rich condensates. The phase behavior exhibits closed‐loop coexistence and re‐entrant percolation. This is captured quantitatively by a mean‐field Semenov–Rubinstein theory with a single fit parameter, the effective repulsion parameter. Notably, phase boundaries are largely robust to rearrangements of crosslinkable domains along the sequence; only highly blocky sequences appreciably reduce the phase separation region and can even convert condensates into micelles or connected micelle networks. These results establish an entropy‐enabled mechanism for mesoscale organization and suggest routes to programmable, membraneless materials in synthetic and RNA‐protein contexts.

Replication-dependent (RD) histones are crucial for packaging newly replicated DNA into chromatin, ensuring genome stability. In metazoans, the mRNA of RD histones is uniquely regulated through a conserved 3′ stem–loop bound by stem–loop binding protein (SLBP). This allows cell cycle-coupled regulation of these important transcripts. However, oocytes must stabilise histone mRNAs independently of the cell cycle to ensure maternal loading to support the first embryonic divisions. Using Caenorhabditis elegans as a model system, we discovered an SLBP-independent mechanism that ensures RD histone transcript stability during oogenesis. This is mediated by the protein complex PETISCO, bound to the effector protein TOST-1, which directly binds the histone stem–loop region and maintains maternal histone mRNA levels during oogenesis and early embryogenesis. Loss of this mechanism disrupts histone homeostasis, leading to premature genome activation, mitotic defects, and embryonic lethality. Interestingly, the same complex, PETISCO, acts in piRNA biogenesis when bound to the effector PID-1, revealing an intriguing co-option of this histone mRNA homeostasis mechanism by the piRNA pathway. Our findings reveal a unique SLBP-independent mechanism of histone mRNA regulation, that served as a basis for the evolution of a novel piRNA biogenesis mechanism.

The mRNA metabolism passively shapes the levels of an mRNA modification called m6A within a steady-state cell and upon stress.

Ribonucleoprotein complexes are dynamic assemblies of RNA with RNA-binding proteins (RBPs), which can modulate the fate of the RNA molecules from transcription to degradation. Vice versa, RNA can regulate the interactions and functions of the associated proteins. Dysregulation of RBPs is linked to diseases such as cancer and neurological disorders. RNA and RBPs are present in mitotic structures like the centrosomes and spindle microtubules, but their influence on mitotic spindle integrity remains unknown. Thus, we applied the R-DeeP strategy for the proteome-wide identification of RNA-dependent proteins and complexes to cells synchronized in mitosis versus interphase. The resulting atlas of RNA-dependent proteins in cell division can be accessed through the R-DeeP 3.0 database (R-DeeP3.dkfz.de). It revealed key mitotic factors as RNA-dependent such as AURKA, KIFC1 and TPX2 that were linked to RNA despite their lack of canonical RNA-binding domains. KIFC1 was identified as a new interaction partner and phosphorylation substrate of AURKA at S ³⁴⁹ and T ³⁵⁹ . In addition, KIFC1 interacted with both, AURKA and TPX2, in an RNA-dependent manner. Our data suggest a riboregulation of mitotic protein-protein interactions during spindle assembly, offering new perspectives on the control of cell division processes by RNA-protein complexes. Highlights: Differential R-DeeP screens in mitosis and interphase are provided as a resource in a user-friendly database at R-DeeP3.dkfz.de An atlas of RNA-dependent proteins in cell division identifies a substantial number of unconventional RNA-binding proteins among mitotic factors Investigation of protein-protein interactions reveals KIFC1 as a new AURKA and TPX2 interaction partner during spindle assembly KIFC1, AURKA and TPX2 interact with each other in an RNA-dependent manner and directly bind to RNA AURKA phosphorylates KIFC1 at residues S ³⁴⁹ and T ³⁵⁹