Human nucleoli are multivalent, involving contributions from up to 10 NOR-bearing acrocentric chromosome p-arms. Precision megabase-scale chromosome engineering defines the requirements for this major genome organizational event. NOR deletions reveal that p-arm clustering can occur independent of rDNA. Deletion of all NOR-distal or NOR-proximal sequences individually have only a marginal effect on nucleolar association. Finally, deletion of an entire p-arm, while leaving centromere function intact, destroys the nucleolar association potential of that acrocentric. We propose that formation of multivalent nucleoli is not a nucleolar fusion event per se; rather it is driven by the surrounding chromosomal context of NORs.

The precise regulation of the replication-dependent histone genes during S phase is essential for packaging newly synthesized DNA and maintaining genome integrity. Identifying the roles of specialized factors that regulate histone biosynthesis is crucial for a comprehensive understanding of this tightly regulated process. In this issue of Genes & Development, Geisler and colleagues (doi:10.1101/gad.353837.126) investigate a previously identified repressor, Mute, and determine that it prevents the accumulation of histone mRNAs outside of S phase by antagonizing Mxc phosphorylation. They also demonstrate a novel mechanism where Mute differentially regulates histone transcript levels. Beyond the repressive mechanism of Mute, Geisler and colleagues highlight how factors can integrate multiple, distinct functions to meet the critical regulatory needs of the histone genes.

A new study by Ma and colleagues (doi:10.1101/gad.353690.126) identifies distinct quality control systems that degrade snRNA variants. Variants expressed from snRNA pseudogenes, deficient in 3' end processing or RNP assembly, are degraded by the nuclear RNA exosome or by cytoplasmic decay initiated by TUT4/7 enzymes. When variant snRNAs are stabilized, they can assemble into spliceosomes and alter splicing. Moreover, pathogenic mutations in canonical snRNA genes can trigger aberrant degradation by these quality control mechanisms, which may contribute to disease pathology. In summary, this work reveals how cells maintain a functional pool of snRNP complexes and links their dysregulation to human diseases.

Genomic rearrangements can drive cancer through mechanisms that extend beyond classical oncogenic fusions such as BCR-ABL A substantial fraction of these events involve long noncoding RNAs (lncRNAs), yet their functional impact on tumorigenesis has remained largely opaque. The lncRNA plasmacytoma variant translocation 1 (PVT1), positioned adjacent to MYC at chromosome 8q24, is among the most frequently altered loci in MYC-driven (MYC[+]) cancers. We recently showed that PVT1 translocations produce a characteristic asymmetric architecture that preserves the 5'-PVT1 region, generating a circular RNA (CircPVT1) that encodes Firefox (FFX), a novel oncoprotein that activates AKT-mTORC1 signaling and cooperates with MYC. Here, we uncover a complementary and opposing function for the deleted 3'-PVT1 segment, which encodes a tumor-suppressive micropeptide we term Honeybadger (HNB). HNB binds KRAS and dampens RAS-MAPK signaling, and its loss derepresses this pathway and stabilizes MYC via Ser[62] phosphorylation. Thus, a single class of structural alterations at PVT1 simultaneously installs FFX-mediated AKT-mTORC1 activation and removes HNB-mediated RAS-MAPK regulation, creating a dual mechanism that synergistically amplifies MYC output. This oncoprotein gain coupled with tumor-suppressor loss provides a mechanistic explanation for the particularly poor prognosis of PVT1-rearranged cancers and establishes PVT1 as a central regulatory hub in MYC[+] malignancies.

MYC-driven (MYC[+]) cancers are highly aggressive and often fatal. MYC dysregulation is a key event in these cancers, yet MYC overexpression alone is often insufficient to initiate or sustain tumorigenesis. Plasmocytoma variant translocation 1 (PVT1), a long noncoding RNA (lncRNA) adjacent to MYC on chromosome 8q24, is frequently co-amplified with MYC in many of these cancers. Our prior work showed that PVT1 potentiates MYC function, although the underlying mechanism has remained unclear. Here we show that, in addition to amplification with MYC, genomic rearrangements at PVT1 frequently involve unbalanced translocations that asymmetrically enrich 5'-PVT1 while depleting 3'-PVT1 The retained 5'-PVT1 region generates a circular RNA, CircPVT1, that encodes a novel protein we have named Firefox (FFX). We show that FFX is essential for MYC-mediated oncogenic signaling, as its depletion markedly reduces MYC protein abundance and transcriptional output. Mechanistically, FFX stimulates AKT-mTORC1 signaling and enhances cap-dependent translational and biosynthetic capacity, thereby establishing a self-reinforcing oncogenic circuit that amplifies MYC activity. Inducible depletion of FFX in vivo significantly impairs tumor growth in MYC[+] xenograft models. These findings define FFX as a critical effector within the MYC-PVT1 locus and reveal a therapeutically actionable vulnerability in MYC[+] cancers.

Coupling histone gene expression to S phase of the cell cycle is essential for genome duplication and stability. Activation of Cyclin E/Cdk2 at the G1-S transition stimulates high-level expression of histone genes during S phase, but how histone genes are turned off at the end of S phase is not understood. Here we demonstrate that the essential Drosophila gene mute functions to repress inappropriate histone mRNA accumulation outside of S phase by counteracting Cyclin E/Cdk2-dependent phosphorylation of Mxc, which activates histone gene expression. Additionally, Mute plays contrasting roles in histone gene expression during S phase by promoting high levels of H1, H2a, and H2b expression but not H3 and H4 Although Mute is present only at replication-dependent histone genes, its loss leads to 801 differentially expressed genes, primarily those involved in muscle related processes in late-stage embryos. Thus, disruptions of histone gene expression control alter the transcriptome resulting in developmental defects.

Prior studies have largely focused on transcriptional and translational control during stress, but how regulated nuclear mRNA export contributes to the stress response remains unresolved. We show that nuclear mRNA export is progressively inhibited during arsenite and heat stress in human cells. In contrast to previous work largely in yeast that suggests nuclear export of stress-induced transcripts is prioritized through sequence-specific mechanisms, we found that mRNA export is governed by temporal gating, in which the timing of mRNA biogenesis determines the nucleocytoplasmic distribution of mRNAs during stress. Using single-molecule mRNA imaging and transcriptome-wide analyses, we observe the majority of stress-induced mRNAs, including heat shock protein transcripts, accumulate in the nucleus during stress. However, a subset of stress-induced mRNAs, notably HMOX1, JUN, and FOS, escape nuclear retention. mRNAs transcribed early during stress, including those encoding immediate early genes, redox mediators, and protein chaperones, are exported from the nucleus prior to the global inhibition of mRNA export. In contrast, mRNAs transcribed later are retained in the nucleus until stress is resolved. Reporter RNA assays confirm that transcriptional timing determines mRNA export competence. This work reveals that the timing of mRNA production, rather than transcript-specific sequence features, is the major determinant of nuclear export efficiency of stress-induced transcripts in human cells.

The eukaryotic nucleolus is a highly organized, multilayered structure essential for ribosomal RNA (rRNA) processing and ribosome assembly. While rRNA transcription is known to drive nucleolar assembly, the contribution of downstream processing steps to nucleolar organization remains less well defined. Here, we show that disruption of endonucleolytic cleavage of the 5' external transcribed spacer (5'ETS), a key early step mediated by the SSU processome, leads to pronounced changes in nucleolar structure and organization. These altered nucleoli display reduced dynamic exchange behavior consistent with altered material properties and exhibit changes in the NPM1-associated nucleolar interactome. In parallel, we observe redistribution of heterochromatin markers, indicating broader effects on nuclear organization. Together, our findings support a model in which progression of pre-rRNA processing contributes to maintaining nucleolar compartmentalization and links early steps of ribosome biogenesis to nuclear organization. The nucleolus is a defining feature of eukaryotic cells, yet the principles that maintain its multilayered organization remain incompletely understood. While rRNA transcription is known to initiate nucleolar assembly, we show that a specific downstream processing step, the cleavage of the 5' external transcribed spacer (5'ETS), plays a key role in maintaining nucleolar organization. By functionally uncoupling rRNA transcription from processing, we demonstrate that defects in 5'ETS maturation are associated with alteration of the NPM1-associated interactome and changes in nucleolar dynamics. These findings support a model in which the progression of rRNA processing contributes to the material organization of the nucleolus, linking enzymatic steps in ribosome biogenesis to nuclear architecture.

ELAV/Hu RNA-binding proteins (RBPs) are key regulators of neuronal alternative splicing and polyadenylation programs across animals. How ELAV/Hu RBPs achieve gene-specific regulation by recognizing spaced U-rich motifs through multimerization, remains uncertain. We determined X-ray crystal structures of ELAV RNA recognition motif 3 (RRM3) to reveal that multimerization is mediated by two evolutionarily conserved interfaces in non-RNA-binding parts of the RRM to form a tetramer and RNA binding is not required for multimerization. Mutational probing of these two interfaces in Drosophila photoreceptor neurons shows that both interfaces contribute to ELAV function in development. Notably, multimerization defective Drosophila elav mutants are embryonic lethal. Genomic profiling demonstrates that multimerization is required to direct neuronal alternative splicing and polyadenylation programs of some, but not all ELAV target genes. Our study provides a structural basis for a mechanistic understanding how ELAV/Hu proteins can extract gene-specific regulation from a landscape of redundant sequence motifs.

Ribosome biogenesis is a resource-consuming process that facilitates rapid growth and feeds uncontrolled, cancerous traits. Constraining ribosome biogenesis and protein translation has become a tenable therapeutic strategy for cancer. Yet, we do not know how cells that rely on high metabolic activity adapt and sustain their growth when deprived of their translational capacity. Conversely, stem cells and treatment-resistant cells persist under low metabolic states challenging their eradication. These are critical questions in cancer therapies. To delineate survival mechanisms that allow cancer cells to adapt to ribosome biogenesis defects, we conducted functional genomics screens during inhibition of RNA polymerase I. We identified that inactivation of mTOR enabled cell survival despite severe translational suppression. This was paradoxical as activation of mTOR is considered oncogenic by boosting ribosome biogenesis and cellular translational programs. We show that mTORC1 inhibition does neither restore rRNA synthesis nor ribosome biogenesis, but redistributes limited ribosomes from highly translated 5'TOP mRNAs to survival-essential transcripts. This mTOR inactivation-mediated prioritization of translational resources represents a minimal requirement for cell survival when translational capacity is compromised, which we term "translational fitness." Our findings redefine the role of mTOR in cell survival and highlight the need for strategic targeting of translation regulation in cancer therapy.

PIF1 family helicases promote genome stability during DNA replication and repair, yet the biochemical activity of human PIF1 (hPIF1) remains poorly understood. Here, we directly compare full-length hPIF1 and budding yeast Pif1 (yPif1) and show that the two enzymes operate differently. In bulk assays, hPIF1 displays weak net DNA unwinding because its robust intrinsic single-stranded DNA (ssDNA) annealing activity rapidly reforms duplex DNA behind the helicase motor. Single-molecule magnetic tweezers experiments reveal that hPIF1 is only modestly slower than yPif1 and similarly processive, indicating that poor apparent unwinding primarily reflects rapid reannealing rather than impaired motor activity. Mutagenesis and structural modeling indicate that this functional divergence depends on a yeast-specific insertion within the catalytic domain. Unlike yPif1, hPIF1 fails to stimulate Polδ-dependent displacement-loop (D-loop) extension, arguing that it does not promote DNA unwinding ahead of the migrating bubble. However, similarly to yPif1, hPIF1 likely unwinds newly synthesized DNA coupled with DNA reannealing behind the migrating D-loop. hPIF1 efficiently remodels intramolecular G-quadruplex DNA into fully base-paired dsDNA, whereas yPif1 generates ssDNA products. Together, our data support a model in which hPIF1 acts primarily as a DNA translocase that couples local unwinding with rapid rewinding to clear secondary DNA structures without generating extensive ssDNA.

Splicing factors are frequently mutated in myeloid cancers, causing splicing aberrations that derail the expression of tumor suppressor genes. In SRSF2 mutated cancers, a key oncogenic splicing event is the inclusion of a "poison" exon that introduces an early stop codon in EZH2 mRNA, causing its destabilization. In this issue of Genes & Development, Islam et al. (doi:10.1101/gad.353628.126) define how mutant SRSF2 binding to the poison exon mediates its inclusion and identify an antisense oligonucleotide that represses the exon to restore EZH2 function and rescues hematopoietic defects. Thus, targeting of poison exons, many of which show protumorigenic and antitumorigenic properties, is a promising new avenue to treat cancer.

Pediatric sarcomas are a heterogeneous group of rare mesodermal malignancies. These cancers, which affect children from infancy through adolescence and young adulthood, are in general challenging to treat with currently available therapies. Biologically, many are characterized by quiet genomes, fusion oncoproteins, immune "cold" microenvironments, and vast epigenetic deregulation that contributes to diverse and complex mechanistic drivers. Multifaceted advancements in research strategies, including high-throughput screening, new model systems, surfaceome profiling, and study of oncogenic fusion condensates have led to new opportunities for understanding the biology of pediatric sarcomas. To continue to make progress for these difficult to treat cancers, it will be critical to continue to improve access to bioinformatic data, approach patient care using innovative clinical trial frameworks, and foster interdisciplinary partnerships among medicinal chemists, scientists, clinicians, advocates, and industry partners.

best4 [+] cells are a recently described vertebrate intestinal epithelial cell type. best4 [+] cells are altered in inflammatory bowel disease and colorectal cancer, suggesting that stimulation of their homeostatic replenishment may have therapeutic potential. However, the development and function of best4 [+] cells remain unclear. Since mice lack best4 [+] cells, we established zebrafish as a tractable in vivo model to observe, manipulate, and remove best4 [+] cells in an organismal context. We dissected best4 [+] cell developmental regulation in vivo from birth to differentiation and specialization, focusing on factors conserved in best4 [+] cells across vertebrates. Lineage tracing demonstrated that best4 [+] cells arise from secretory progenitors, where Notch/Dll4 signaling mediates a decision between best4 [+] and enterochromaffin cells by triggering meis1b expression. Following specification by meis1b, pbx3a spatially diversifies best4 [+] cells, which develop regional heterogeneity in gene expression, intracellular pH, and function. In vivo live imaging and removal of best4 [+] cells showed that best4+ cells sense luminal pH changes and extend dynamic luminal and stromal projections, but are not required to restore global luminal pH after challenge. Altogether, this study experimentally delineates best4 [+] cell developmental regulation and develops a genetic toolkit to examine their function in vivo, both of which will aid investigating how best4 [+] cells are altered or can be restored during disease.

The unfolded protein response (UPR) preserves endoplasmic reticulum proteostasis through coordinated signaling pathways, including the IRE1α-XBP1 axis, which promotes adaptive transcriptional programs via noncanonical XBP1 mRNA splicing. However, upstream mechanisms regulating this pathway remain incompletely defined. Here, we apply CRASP-seq, a scalable RNA-coupled CRISPR screening platform, to systematically identify regulators of XBP1 splicing. We uncovered the U2 snRNP auxiliary factor RBM39 as a critical positive regulator of this process. Perturbation of RBM39 or U2 snRNP components induces alternative splicing of ERN1, leading to exon-18 skipping and the production of an unstable transcript subject to nonsense-mediated decay, as well as a truncated IRE1α isoform that acts in a dominant-negative manner to suppress XBP1 splicing. Mechanistically, we show that heat shock reduces RBM39 functional activity and promotes ERN1 exon-18 skipping, thereby attenuating IRE1α-XBP1 signaling. Functionally, hyperactivation of this pathway is detrimental under proteotoxic stress, suggesting that exon-18 skipping serves as a stress-adaptive mechanism to limit UPR output. Together, our findings reveal a previously unrecognized regulatory axis linking the canonical splicing machinery to UPR signaling and establish alternative splicing of ERN1 as a key modulator of cellular stress responses.