Sommerkamp, P. | bioRxiv Science

bioRxiv2026

The cancer-promoting enzyme PKM2 binds RNA via a positively charged regulatory patch

Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that coordinates energy production with biosynthetic demands in physiologically proliferating and cancer cells. PKM2 has also emerged as a non-canonical RNA-binding protein. Here, we combine targeted PKM2 mutagenesis with RNA-protein interaction assays, biochemical and biophysical analyses to define how RNA binding relates to PKM2 allosteric control and oligomeric state. We show that the allosteric activator fructose-1,6-bisphosphate (FBP) strongly reduces PKM2 binding to RNA. Mutants with impaired oligomerization show reduced RNA binding, and RNA association is favored by the tetrameric state. A positively charged surface patch in the FBP-binding region is essential for RNA binding and displays emergent properties. Single-residue variants further link RNA association to FBP-responsive tetrameric conformations. Our data integrate RNA binding with PKM2 allosteric regulation.

molecular biology↗

bioRxiv2026

Identification of RNA Targets of Classical and Non-Canonical RNA-binding Proteins by soniCLIP

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/745202v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@52e3edorg.highwire.dtl.DTLVardef@1f21637org.highwire.dtl.DTLVardef@909ee8org.highwire.dtl.DTLVardef@b0c907_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG Crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) is widely used to identify the RNA targets of RNA-binding proteins (RBPs). However, its application to non-canonical RBPs lacking canonical RNA-binding domains and frequently displaying low or transient RNA occupancy, is limited by low signal-to-noise ratios, high input requirements and error-prone ligation steps during library preparation. To overcome these limitations, we developed soniCLIP, a streamlined CLIP-seq workflow that replaces RNase-mediated RNA fragmentation with sonication and uses a ligation-free strategy for library construction. soniCLIP is optimized for reproducible identification of enriched RBP-associated RNA regions from limited starting material. We benchmarked soniCLIP against the widespread eCLIP approach and observed reproducible recovery of known RBP-associated regions and target recovery comparable to ENCODE eCLIP, while requiring only 10% (500 {micro}g) of protein input. We further applied soniCLIP to the glycolytic enzyme and non-canonical RBP pyruvate kinase M2 (PKM2). We identified 197 significantly enriched RNA regions and validated selected targets by RIP-qRT-PCR and in vitro binding assays. By combining reduced input requirements, high reproducibility, a shortened 3.5-day workflow and the elimination of gel-based purification, soniCLIP provides an efficient and robust approach for the identification of RNA targets of canonical and non-canonical RBPs.

molecular biology↗