Nuclear stress bodies (nSBs) are stress-inducible membraneless organelles formed on HSATIII long noncoding RNAs (lncRNAs) that regulate pre-mRNA splicing during thermal stress recovery. During stress, dephosphorylated serine/arginine-rich splicing factors (SRSFs) accumulate in nSBs, whereas upon stress removal, their kinase CLK1 is recruited to rephosphorylate SRSFs, thereby promoting target intron detention. However, the mechanism underlying CLK1 localization to nSBs has remained unclear. Using HeLa cells and cell-free reconstitution, we identify CLK1 Ser341 phosphorylation as a critical determinant of its nSB localization and define its regulatory mechanism. Ser341 is phosphorylated under normal conditions, dephosphorylated by protein phosphatase 1(PP1) during stress, and rephosphorylated by RIOK2 during recovery, thus enabling CLK1 localization to nSBs specifically during recovery. We further identify PPP1R2, an entirely intrinsically disordered PP1 inhibitory subunit, as a reversible thermosensor that dissociates under stress to activate PP1. Together, our findings reveal multilayered thermosensing mechanisms that coordinate the staged localization of SRSFs and CLK1 to nSBs, thereby regulating temperature-dependent pre-mRNA splicing.

In a recent issue of Nature, Scholz et al.[1] apply the RNA-triggered DNA shredding activity of CRISPR-Cas12a2 in eukaryotic cells to enable programmable elimination of yeast and human cells expressing target transcripts with single-nucleotide resolution specificity and non-detectable off-target activity.

In this issue of Molecular Cell, Budinich et al. reveal a role for the local production of RNA in mutant ENL condensate formation, chromatin occupancy, transcriptional regulation, and leukemogenesis.

In this issue, Shu et al.[1] identify the RNA-binding protein PCBP1 as a PARP1 interactor and negative regulator, a function relieved by SIRT7 deacetylation of PCBP1 upon DNA damage. Tumor studies indicated clinical relevance of this pathway, especially in response to radiotherapy, in modulating PARP1 activity as a therapeutic strategy.

Sun et al.[1] present biochemical data challenging the proposed cut-and-paste mechanistic model of insertion sequence 110 transposition, arguing instead for a dual-pathway mechanism relying on top-strand excision and a direct RNA-guided strand transfer.

The hierarchical, multiphase organization of the nucleolus underlies ribosome biogenesis. Ribonucleoprotein particles that regulate ribosomal subunit assembly are heterogeneously distributed in the nucleolar granular component (GC). However, the molecular origins of the GC's spatial heterogeneity and their link to ribosome subunit assembly remain poorly understood. Here, using super-resolution microscopy in DLD-1 cells, we uncover that key GC biomolecules-NPM1, SURF6, and ribosomal RNA (rRNA)-are heterogeneously localized within GC sub-phases. In vitro reconstitution with E. coli- and human-derived rRNA revealed that these GC biomolecules form multiphase condensates with a SURF6/rRNA-rich core and NPM1-rich shell, providing a mechanistic basis for this heterogeneity. SURF6's association with rRNA weakens upon ribosome subunit assembly, enabling NPM1 to extract assembled subunits from condensates, suggesting an assembly-line-like mechanism of subunit efflux from the GC. Our results establish a framework for understanding the GC's heterogeneous structure and reveal how its distinct sub-phases facilitate ribosome subunit assembly.

Pseudouridines (ψ) in mRNA are linked to alternative splicing, but their regulatory mechanisms remain unclear due to the lack of known reader proteins. Here, we identify SMD2, a core spliceosomal component, as a direct ψ reader. Through in vitro and ex vivo assays, we show that SMD2 preferentially binds to ψ-modified RNA over unmodified uridines in human cells. Specifically, SMD2 collaborates with ψ synthase (PUS) family enzymes to regulate alternative splicing by recognizing ψ residues near exon-intron junctions. Notably, SNRPD2, the gene encoding SMD2, is overexpressed in multiple cancers and is essential for tumor cell proliferation through modulating mRNA maturation. These findings establish a direct mechanistic link between ψ and spliceosomal function, which positions SMD2 as a key regulator of ψ-mediated splicing and a promising therapeutic target in cancer.

Speckle-type POZ protein (SPOP), a substrate receptor for the Cullin-3-RING (CRL3) ubiquitin ligase, is mutated in different cancers. Both activating and inactivating mutations in SPOP drive oncogenesis, underscoring the need for precise regulation. Among substrate receptors, SPOP uniquely assembles into filaments that are multivalent for substrate binding. Conversely, many substrates contain multiple SPOP-binding motifs. How this unusual reciprocal multivalent architecture regulates ubiquitination and how mutations activate SPOP are unclear. Gain-of-function mechanisms are also generally poorly understood. Here, we reveal that SPOP assemblies exist in an equilibrium between an active filament and a large, autoinhibited, circular "double-donut" state. Activating mutations shift the equilibrium, resulting in aberrant substrate turnover. Combinations of activating and inactivating mutations can produce intermediate activities, thus uncovering a tunable regulatory axis with implications for targeted cancer therapies. Therefore, SPOP's ability to assemble into long filaments is required for its regulation in human cells and underlies a gain-of-function mechanism.

The nuclear pore complex (NPC) serves as the central transport gateway between nucleus and cytoplasm. NPC biogenesis requires the assembly of over 500 proteins culminating in the fusion of the inner and outer nuclear membranes. The mechanism of membrane fusion is unknown. Here, we elucidate how Brl1 and Brr6 mediate membrane fusion in S. cerevisiae. Our data suggest that both proteins form ring-shaped complexes with membrane-remodeling activity. Brl1 localizes to NPC assembly sites via a nuclear export sequence and interacts with Brr6 across the nuclear envelope through conserved hydrophobic loops. Disrupting this interaction blocks fusion and halts NPC assembly. Molecular dynamics simulations suggest that the Brl1-Brr6 complex drives membrane fusion by forming a channel across the bilayers enabling lipid exchange. Phylogenetic analyses and functional experiments in human cells and D. melanogaster establish CLCC1 as the NPC fusogen in metazoans. Together, our results uncover a conserved membrane fusion mechanism in eukaryotes.

The ubiquitin-fold modifier 1 (UFM1) pathway is essential for endoplasmic-reticulum-associated ribosome quality control (ER-RQC) through UFMylation of the 60S ribosomal protein RPL26, but the regulation and physiological significance of UFM1 deconjugation remain poorly understood. Here, we identify the ER-anchored UFSP2-ODR4 complex as a spatially confined deUFMylation module critical for neuronal proteostasis. Structural modeling and biochemical analyses show that ODR4 recruits UFSP2 to the ER, enabling efficient deUFMylation of RPL26. Disruption of the UFSP2-ODR4 interaction causes the accumulation of UFMylated RPL26 and defective ER-RQC. Neural progenitor-specific knockin mice expressing a catalytically inactive UFSP2 mutant exhibit perinatal lethality, microcephaly, and neuronal apoptosis. We also identify a patient with biallelic UFC1 mutations that enhance UFL1 binding and induce hyper-UFMylation of RPL26 in patient-derived neurons. These findings establish spatially confined deUFMylation as a critical mechanism for safeguarding neuronal proteostasis.

Short interspersed nuclear elements (SINEs) are abundant non-autonomous transposable elements derived from RNA polymerase III (POL III)-transcribed short non-coding RNAs. SINEs retain sequence features recognized by the POL III machinery and constitute a substantial portion of vertebrate genomes. Despite their impact on genome stability and evolution, the mechanisms governing SINE transcription remain poorly understood. Although DNA methylation and heterochromatin formation have been implicated in their repression, we find that these pathways play only a minor role in mouse embryonic stem cells. Instead, we identify the ChAHP complex as a key repressor of SINE B2 elements. ChAHP directly inhibits POL III transcription by blocking TFIIIB recruitment without affecting TFIIIC binding. This selective interference prevents transcription initiation and highlights a distinct regulatory mechanism. Our findings establish ChAHP as a non-canonical repressor of POL III-dependent SINE transcription, thus offering new insights into the control of this pervasive class of non-coding genomic elements.

Transcription in eukaryotes is regulated by chromatin-based mechanisms that control nucleosome occupancy, chromatin modifications, and transcription factor binding. We have previously shown that the transcription factor ADNP forms the ChAHP complex with the chromatin remodeler CHD4 and HP1 proteins, acting as a site-specific regulator of transcription and an antagonist of CTCF binding. However, the molecular basis of these functions remained unclear. Here, we demonstrate in mouse embryonic stem cells that the CHD4 subunit is essential for antagonizing CTCF and silencing the transcription of transposons, whereas HP1 proteins are dispensable. Although the remodeling activity of CHD4 is not required for ChAHP chromatin association, it is critical for both transposon repression and CTCF antagonism. Our findings support a model in which ADNP recruits chromatin-remodeling activity in a sequence-specific manner, enabling transcriptional control and local modulation of chromatin architecture.

Bridge-like lipid transfer proteins (BLTPs) play fundamental roles in cellular lipid redistribution between organellar membranes. They comprise bridge domains spanning organelles at contact sites that allow lipids to transit through the cytosol between adjacent membranes. The assembly of BLTPs into complexes with adaptor proteins enables lipid transfer. To address the mechanisms underlying the assembly and regulation of BLTP complexes, we used cryo-EM to resolve the structure of one such BLTP, the Parkinson's disease protein VPS13C, at near-atomic resolution. The structure identifies a lipid-transfer-nonpermissive conformation, in which the built-in C-terminal VAB adaptor module blocks the end of the lipid transfer bridge, interfering with lipid delivery. We also identify calmodulin (CaM), central to calcium signaling, as a constitutive VPS13C interactor. Calcium induces conformational changes in the VPS13C-CaM complex, suggesting calcium regulation of VPS13 function. Altogether, this structure of intact VPS13C serves as a starting point for understanding its regulation and that of other VPS13 proteins.

The biological significance of forkhead box A1 (FOXA1) in non-steroid-driven malignancies, such as pancreatic ductal adenocarcinoma (PDAC), has garnered increasing recognition. It assumes a pivotal role in regulating critical processes such as PDAC cell lineage, metabolism, and metastasis. However, its regulatory mechanisms remain elusive. Here, we demonstrate that histone deacetylase 5 (HDAC5) mediates the deacetylation of FOXA1 at lysine residue 270 (K270), leading to repression of FOXA1's global chromatin occupancy. In HDAC5-loss PDAC, K270 hyper-acetylated FOXA1 is reprogrammed to the transcription start sites (TSSs) of HIF1α-targeted genes, functioning as a pioneer factor of HIF1α signaling. Additionally, we show that HDAC5 antagonizes LSD1-mediated FOXA1 activation by converging on the dynamic equilibrium of acetylation-methylation transition at K270. Pharmacological inhibition of HIF1α/LSD1 suppresses the growth and progression of HDAC5-deficient PDAC in in vivo and in vitro models. Our study reveals the role of FOXA1 as a pioneer factor of HIF1α in PDAC, providing potential therapeutic strategies for HDAC5-deficient PDAC.