Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.05.01.591760

PAN deadenylase is required for mitosis in response to microtubule stress in yeast and humans

Verma, J.·He, Z.·Brown, J. A. R.·Dean, P.·Young, B.·Flibotte, S.·Howe, L. J.·Maxwell, C.·Roskelley, C.·Loewen, C.
View original source ↗Export metadata ↓

Abstract

HIGHLIGHTSO_LIPAN2-PAN3 catalytic activity is required for mitotic robustness under MT stress C_LIO_LICatalytic-dead Pan2-D1020A phenocopies pan2{Delta}: spindle defects and G2/M arrest C_LIO_LILoss of Pan2 catalytic activity amplifies nocodazole-induced changes in CLB1 and CLN3 mRNA C_LIO_LIPAN2 depletion in human cells produces multipolar spindles under MT stress C_LI IN BRIEFVerma et al. show that PAN2-PAN3 deadenylase catalytic activity is selectively required for mitotic fidelity under microtubule stress. Yeast pan2{Delta} cells exhibit spindle defects, G2/M arrest, and altered CLB1 and CLN3 mRNA levels under nocodazole. PAN2 knockdown in human cells produces multipolar spindles, demonstrating evolutionary conservation. Cytoplasmic deadenylation by PAN2-PAN3 and CCR4-NOT regulates mRNA stability and translation, but the contribution of individual deadenylases to mitotic progression is poorly defined. Here we show that PAN2-PAN3 catalytic activity is selectively required for mitotic robustness under microtubule stress. In budding yeast, the catalytically inactive Pan2-D1020A mutant fails to complement pan2{Delta} nocodazole sensitivity, leading to G2/M arrest, spindle defects, and increased cell death. pan2{Delta} exhibits synthetic genetic interactions with tubulin and tubulin-folding genes, and RNA-seq reveals dysregulation of cell-cycle transcripts including the cyclins CLB1 and CLN3. The phenotype is stress-conditional: pan2{Delta} cells proliferate normally without drug. In mammalian cells, PAN2 depletion sensitizes HEK293A cells to nocodazole and colchicine and produces prolonged metaphase with multipolar spindles. Together, these findings define a conserved, stress-conditional role for PAN2-PAN3 deadenylase activity in safeguarding mitotic fidelity when microtubules are compromised. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/591760v3_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@19e6662org.highwire.dtl.DTLVardef@53ba18org.highwire.dtl.DTLVardef@de9c99org.highwire.dtl.DTLVardef@fb0f9d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO See accompanying graphical abstract file. C_FIG

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Verma, J., He, Z., Brown, J. A. R., Dean, P., Young, B., Flibotte, S., Howe, L. J., Maxwell, C., Roskelley, C., Loewen, C.. 2024-05-03. PAN deadenylase is required for mitosis in response to microtubule stress in yeast and humans. https://doi.org/10.1101/2024.05.01.591760

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Unraveling the metabolic landscape of alkaptonuria through a human-relevant in vitro liver disease model

Alkaptonuria (AKU) is a rare inherited metabolic disorder of tyrosine catabolism caused by a deficient homogentisate 1,2-dioxygenase (HGD) enzyme. This results in the accumulation of homogentisic acid (HGA), driving a progressive multisystem pathology characterized by debilitating early-onset osteoarthritis due to connective tissue degeneration. While previous in vitro studies have primarily relied on exogenous HGA exposure in osteoarticular cell models, the direct metabolic consequences of endogenous HGD deficiency within its native hepatic context remain poorly understood. Here, we established the first human-relevant HGD knockout hepatic in vitro model using a universal in-house-developed homology-directed repair approach. Integrative multi-omic analysis revealed that HGD deficiency induces widespread metabolic rewiring extending beyond disrupted tyrosine catabolism. HGD-deficient hepatocytes exhibited elevated oxidative stress accompanied by impaired mitochondrial respiration and a pseudohypoxic metabolic adaptation toward increased glycolytic dependency. Despite this glycolytic shift, the cells displayed reduced anabolic and translational activity alongside attenuated proliferation, consistent with a chronic stress-adaptive survival state rather than a proliferative metabolic phenotype. This study provides systems-level insights into the pathophysiology of AKU and establishes a versatile platform for mechanistic and therapeutic investigation.

cell biology↗
bioRxiv2026

P-body sequestration of clock transcripts delays repressor synthesis to set circadian period in Drosophila

Negative-feedback oscillators require a delay between the accumulation of a repressor's mRNA and the action of its protein. In the circadian clock, this delay has been attributed largely to post-translational control of PERIOD (PER) stability and nuclear entry. The RNA-binding proteins shown to regulate per translation, ATAXIN2 and its partners, promote it, leaving open whether any step holds clock transcripts back before they are translated. Here, using time-resolved miniTurbo proximity labeling of endogenous PER across four phases of the circadian cycle in Drosophila clock neurons, we define a 252-protein PER proximitome that partitions into a nuclear arm and a cytoplasmic RNA-metabolism arm. A behavioral RNAi screen identified two P-body components, the DEAD-box helicase Me31B (DDX6) and the 5'-3' exonuclease Pacman (Pcm; XRN1), as strong regulators of circadian rhythms. Using single-molecule RNA-FISH, proximity RNA editing and ribosome profiling, we show that as per and tim transcripts accumulate, they localize to Me31B-labeled P-bodies and are poorly translated, most prominently at ZT12. Me31B knockdown disrupts P-bodies and releases per mRNA from them, causing PER to accumulate earlier and to ~2-fold higher levels, whereas Me31B overexpression delays PER accumulation and lengthens the free-running period by ~2 h. Knockdown of Pcm, in contrast, impairs clearance of per mRNA, sustaining PER and TIM accumulation, prolonging the repression phase and abolishing cycling of ~89% of rhythmic transcripts. Together, these findings identify P-body sequestration as a repressive step that delays repressor synthesis, and Pcm-dependent decay as required to end repression on time. Given the deep conservation of DDX6 and XRN1, RNP compartments may provide a conserved means of generating delay in circadian and other negative-feedback circuits.

cell biology↗
bioRxiv2026

Defining redundancy in the stickers and spacers of the cell-cell junction protein Canoe's intrinsically disordered region

Cell-cell adherens junctions (AJs) and their dynamic cytoskeletal linkage power morphogenesis. AJs are enormous complexes with hundreds of proteins linked by multivalent interactions. Like other biomolecular condensates, intrinsically disordered regions (IDRs) in junctional proteins play important roles in AJ assembly and function, using spacer elements to span distances, and stickers to engage targets. To define molecular mechanisms, we need to define the functional units within IDRs. Drosophila Canoe, homolog of human Afadin, is our model. Canoe mediates morphogenesis and has an extensive IDR, with two conserved F-actin-binding stickers and two poorly conserved spacers. We combined biochemical, genetic and cell biological approaches to define the function of these IDR elements. While no single element is essential, deleting the full IDR essentially eliminates Canoe function. By scrambling the amino acid sequence of the spacers, we find that length and composition are more important than amino acid sequences, though sequences in the C-terminal spacer affect Canoe localization. Finally, we test redundancy of the F-actin-binding stickers. Deleting both reduces but does not eliminate viability, and sensitized assays reveal their redundant roles. These data reveal the robustness of IDRs.

cell biology↗