Biology subjects

Blower, M. D.

Publications and source records attributed to Blower, M. D..

15 recordsLinked to original sources

bioRxiv2026

SAF-A/HNRNPU regulates euchromatin dynamics and is required for nuclear envelope integrity

Defects in nuclear morphology are associated with cancer and premature ageing. Nuclear morphology is established by a balance of external forces produced by actin contractility and resistance provided by chromatin and the nuclear lamina. Euchromatin is decompacted to facilitate transcription, which has been shown to decrease local nucleosome motion. However, the role of transcription and euchromatic chromatin structure in nuclear envelope integrity are not clear. Here, we demonstrate that acute depletion of SAF- A/hnRNPU causes compaction of euchromatic regions and constriction of chromatin mobility, leading to loss of nuclear envelope integrity in a transcription-dependent manner. SAF-A is required in a dose-dependent manner to preserve nuclear shape under conditions of nuclear softening and elevated transcription. Our work identifies SAF-A as a dynamic scaffold that regulates chromatin structure and dynamics at sites of active transcription to preserve nuclear envelope mechanical tension.

cell biology↗

bioRxiv2026

Spatial control of mitochondrial retrograde signaling by nuclear pore-associated contact sites

Mitochondria relay their functional state to the nucleus via retrograde signaling, yet whether the spatial organization of the mitochondrial network plays a role in this process remains unclear. Here, we show that stress-induced clustering of mitochondria around the nucleus is a crucial part of the retrograde response. Perinuclear clustering facilitates the formation of mitochondria- nucleus contact sites (MNCS) and the nuclear entry of GPS2, a key mediator of mitochondrial retrograde signaling essential for activating nuclear-encoded mitochondrial and stress-response genes in response to various mitochondrial stressors. Unexpectedly, TSPO-driven MNCS are dispensable for GPS2-based retrograde signaling. Instead, we identify the mitochondrial import receptor TOMM70 and the nucleoporin RanBP2/NUP358 as components of a stress-induced nuclear pore-associated tethering complex required for promoting GPS2 nuclear translocation and activation of downstream programs. These findings establish MNCS as a functional gateway for mitochondrial retrograde signaling, highlighting that organelle positioning and tethering at the nuclear pore provide an unexpected layer of stress regulation.

cell biology↗

bioRxiv2026

Proteolytic dissection of eIF4G reveals the closed-loop mRNP as an architecture for translation repression.

Formation of a "closed-loop" mRNP, in which the 5' cap and 3' poly(A) tail are bridged by eIF4E-eIF4G-PABP interactions, has long been proposed to drive efficient translation initiation. Direct tests of this model in mammalian cells have remained elusive. Using auxin-inducible degron (AID) technology to acutely deplete eIF4G1, we find that global translation is only partially reduced and recovers without restoration of eIF4G1 levels. We identify eIF4G3 as an underappreciated contributor to basal translation that buffers translational output upon eIF4G1 loss without increased protein expression, explaining the modest defects observed in prior RNAi-based studies. Systematic replacement of eIF4G1 with defined cleavage products and interaction mutants reveals that PABP binding by eIF4G1 is dispensable for bulk translation initiation: the central caspase-3 cleavage fragment of eIF4G1 (casp3-cpM), which lacks the PABP-interaction domain, fully rescues global protein synthesis, and acute depletion of both major cytoplasmic PABP paralogs primarily destabilizes mRNAs rather than impairing initiation. In contrast, the N-terminal enteroviral 2A cleavage product (2A-cpN) is a potent, dominant translational repressor that requires simultaneous eIF4E and PABP engagement to form a dead-end closed-loop mRNP that sequesters initiation factors without enabling 43S recruitment. These findings reveal that the eIF4G-PABP closed-loop architecture is not required for productive initiation but can be actively co-opted for translational silencing. This explains why viral eIF4G cleavage, but not factor depletion, produces near-complete translational shutoff. The modular architecture of eIF4G enables diametrically opposing translational outcomes through selective proteolytic processing.

molecular biology↗

bioRxiv2026

Decreased tRNA abundance contributes to decreased translation elongation rate in a prolonged mitosis

Cells undergo dramatic structural rearrangements upon entering mitosis. In addition, the biochemistry of mitotic cells is dramatically altered, including a significant decrease in protein synthesis. The majority of studies of mitotic translation have used cells synchronized by cell cycle altering drugs in transformed cells and much less is known about mitotic translation in primary cells under native conditions. Previous work has found that mitosis activates the integrated stress response (ISR) to trigger eIF2 phosphorylation, but little is known about the input for this response. In this study, we focus on mitotic translational regulation in an immortalized, non-transformed cell line. We confirm decreased mitotic protein synthesis in primary cells and under native conditions. Additionally, we confirm activation of the ISR by phosphorylation of eIF2 during both normal and prolonged mitosis. Interestingly, we also find that decreased translational elongation during mitosis, as evidenced by increased eEF2 phosphorylation and a slower elongation rate. Analysis of mitotic ribosome profiling data revealed an increase in pausing at Alanine-GCG codons during mitosis and a decreased abundance of its cognate tRNA-AlaCGC by northern blotting. Decreased tRNA-AlaCGC is likely sustained by the inability to synthesize additional tRNA due to RNAPol III inhibition in mitosis, yielding an stronger effect with an increased time in mitosis. These results suggest that decreased translation elongation in mitosis triggers inhibition of initiation to decrease global protein synthesis.

cell biology↗

bioRxiv2025

Role of the SAF-A/HNRNPU ATPase and RGG domains in X chromosome inactivation, nuclear dynamics, transcription, splicing, and cell proliferation

The SAF-A/HNRNPU gene encodes an abundant nuclear protein conserved throughout vertebrates, and is mutated in individuals with HNRNPU syndrome, a neurological human disease. SAF-A is important for maintaining lncRNA localization, splicing, and gene expression state. The mechanistic role of SAF-A in each of these processes is coordinated by one or more of its functional domains, which include an N-terminal SAP domain, a central ATPase domain, and a series of C-terminal RGG repeats embedded in a low-complexity region. The SAP domain and RGG repeats define two nucleic acid interaction domains, with both capable of binding DNA or RNA. Here we use an allelic reconstitution strategy to investigate the role of the SAF-A ATPase domain and RGG repeats. We show that both the ATPase and RGG repeats control SAF-A nuclear dynamics, and present genetic evidence that SAF-A interacts with nascent transcripts through the RGG repeats. The SAF-A ATPase domain and RGG repeats were also required for maintaining XIST RNA and facultative heterochromatin marks on the inactive X chromosome, with distinct effects of mutations that block ATP binding and ATP hydrolysis. Analysis of transcriptome datasets revealed that the SAF-A ATPase domain and RGG repeats are both required for proper mRNA splicing, but not for gene expression. Importantly, we found that like the SAP domain, the SAF-A ATPase domain and RGG repeats are required for cell proliferation. Collectively, our findings highlight the importance of the SAF-A SAP, ATPase and RGG domains in essential functions of nuclear biology. These analyses will therefore inform our understanding of the disease state in HNRNPU syndrome.

cell biology↗

bioRxiv2025

Reductive death is averted by an ancient metabolic switch.

Biguanides, including metformin, the worlds most prescribed oral hypoglycemic, extend health-span and lifespan in vertebrates and invertebrates. Given the widespread use and apparent safety of metformin, it is assumed that its effects are not associated with toxicity, except when in marked excess. Here we determine that accumulation of damaging reducing equivalents is an unanticipated toxicity associated with biguanides, the defense against which requires post-transcriptional protection of de novo fatty acid biosynthesis. We demonstrate that biguanide treatment during impaired fatty acid biosynthesis drives NADPH toxicity, leading to catastrophic elevation of NADH/GSH reducing equivalents and accelerated death across metazoans. Multiple NADPH-generating interventions require fatty acid biosynthesis to prevent markedly shortened survival, indicating that this defense mechanism is broadly leveraged. We propose that fatty acid biosynthesis is a tunable rheostat which can minimize biguanide-induced reductive stress whilst maximizing its pro-longevity outcomes and serve as an exploitable vulnerability in reductive stress sensitive cancers. HIGHLIGHTSO_LIBiguanides inhibit cytosolic mRNA translation to extend lifespan in C. elegans. C_LIO_LIFatty acid synthesis is translationally protected by eIF3 complex subunits. C_LIO_LIpod-2/fasn-1 inactivation amplifies biguanide-induced reductive stress and death. C_LIO_LINADPH-generating insults require fatty acid synthesis to buffer reductive stress. C_LI

molecular biology↗

bioRxiv2025

A minimum module for positioning the Chromosomal Passenger Complex at the cell center for cytokinesis

The Aurora B kinase-containing Chromosomal Passenger Complex (CPC) is an essential regulator of cytokinesis, the final and irreversible step of cell division. During anaphase, the CPC concentrates at the spindle midzone - a bundle of overlapping antiparallel microtubules organized at the cell center. How CPC is selectively enriched at the midzone within the dense, heterogeneous, and dynamic spindle microtubule network is unknown. Here, we define a minimal CPC midzone enrichment module. We show that the maximal enrichment of CPC at antiparallel microtubule overlaps requires PRC1-crosslinked microtubules and the interaction of CPC with two mitotic kinesins, KIF4A and KIF20A. We find that the two motors exhibit a division of labor: KIF20A delivers CPC from non-overlapping microtubules, and KIF4A retains CPC at PRC1-crosslinked overlaps. Conditional depletion of KIF4A in mitotic cells reveals that it is required for CPC localization at the spindle midzone in anaphase. Taken together, our findings reveal how the collective activity of two kinesins enables navigation through the complex microtubule network of the spindle to organize kinase signaling at the cell center for cytokinesis.

cell biology↗

bioRxiv2025

Metal stress uncouples early pre-rRNA processing from ISR activation and reveals flexible checkpoints in human ribosome biogenesis

Ribosome synthesis is one of the most energy-intensive processes in a growing cell, consuming more than 60% of cellular energy reserves. As such, ribosome biogenesis is highly sensitive to stress to prevent costly expenditures under adverse conditions. Moreover, successful assembly requires precise stoichiometric balance between ribosomal proteins and ribosomal RNAs. Here, we define novel regulatory mechanisms of ribosome biogenesis under stress that reveal previously unrecognized aspects of rRNA maturation. We demonstrate that early pre-rRNA processing is particularly sensitive to stress induced by environmentally relevant heavy metals. Surprisingly, our analysis shows that 5' and 3' end processing can be uncoupled in human cells, with 3' end cleavage occurring independently of 5' end processing. We further show that classical inducers of endoplasmic reticulum stress suppress ribosomal protein synthesis without inhibiting rRNA transcription, leading to an imbalance between these essential components of ribosome assembly. This imbalance may exacerbate cellular stress and compromise proteostasis. Together, our findings uncover stress-specific checkpoints in ribosome biogenesis that link environmental exposures to disrupted nucleolar function and highlight new layers of regulation in human rRNA maturation.

biochemistry↗

bioRxiv2025

Robust CENP-A incorporation in human cells is independent of transcription and cohesin components

Centromeres are essential chromosomal components that ensure proper cell division by serving as assembly sites for kinetochores, which connect chromosomes to spindle microtubules. Centromeres are marked by the evolutionarily conserved centromere-specific histone H3 variant, CENP-A, which is deposited into centromere nucleosomes during G1 in human cells. Centromeres retain cohesin, a ring-like protein complex during mitosis, protecting sister chromatid cohesion and centromere transcription to prevent chromosome missegregation. Previous work in Drosophila has suggested that centromere transcription and centromeric RNAs are important for CENP-A deposition in chromatin. During mitosis centromeric cohesin is critical for centromere transcription. However, it is not clear how or if centromeric transcription and cohesin contribute to CENP-A deposition in G1 in human cells. To address these questions, we combined a cell synchronization strategy with the Auxin Inducible Degron technology and transcription inhibition in human cells. In contrast to Drosophila cells, our results demonstrated that neither centromeric transcription nor cohesin is required for CENP-A deposition in human cells. Our data demonstrate clear differences in the CENP-A deposition mechanism between human and Drosophila cells. These findings provide deeper insights into the plasticity underlying centromere maintenance and highlight evolutionary divergence in centromere maintenance systems across species.

cell biology↗

bioRxiv2024

Role of the SAF-A SAP domain in X inactivation, transcription, splicing, and cell proliferation

SAF-A is conserved throughout vertebrates and has emerged as an important factor regulating a multitude of nuclear functions, including lncRNA localization, gene expression, and splicing. SAF-A has several functional domains, including an N-terminal SAP domain that binds directly to DNA. Phosphorylation of SAP domain serines S14 and S26 are important for SAF-A localization and function during mitosis, however whether these serines are involved in interphase functions of SAF-A is not known. In this study we tested for the role of the SAP domain, and SAP domain serines S14 and S26 in X chromosome inactivation, protein dynamics, gene expression, splicing, and cell proliferation. Here we show that the SAP domain serines S14 and S26 are required to maintain XIST RNA localization and polycomb-dependent histone modifications on the inactive X chromosome in female cells. In addition, we present evidence that an Xi localization signal resides in the SAP domain. We found that that the SAP domain is not required to maintain gene expression and plays only a minor role in mRNA splicing. In contrast, the SAF-A SAP domain, in particular serines S14 and S26, are required for normal protein dynamics, and to maintain normal cell proliferation. We propose a model whereby dynamic phosphorylation of SAF-A serines S14 and S26 mediates rapid turnover of SAF-A interactions with DNA during interphase.

cell biology↗

bioRxiv2022

The midbody and midbody remnant are assembly sites for RNA and active translation

The midbody (MB) is a transient structure at the spindle midzone that is required for cytokinesis, the terminal stage of cell division. Long ignored as a vestigial remnant of cytokinesis, we now know MBs are released post-abscission as extracellular vesicles called MB remnants (MBRs) and can modulate cell proliferation, fate decisions, tissue polarity, neuronal architecture, and tumorigenic behavior. Here, we demonstrate that the MB matrix--the structurally amorphous MB core of unknown composition--is the site of ribonucleoprotein assembly and is enriched in mRNAs that encode proteins involved in cell fate, oncogenesis, and pluripotency, that we are calling the MB granule. Using a quantitative transcriptomic approach, we identified a population of mRNAs enriched in mitotic MBs and confirmed their presence in signaling MBR vesicles released by abscission. The MB granule is unique in that it is translationally active, contains both small and large ribosomal subunits, and has both membrane-less and membrane-bound states. Both MBs and post-abscission MBRs are sites of spatiotemporally regulated translation, which is initiated when nascent daughter cells re-enter G1 and continues after extracellular release. We demonstrate that the MB is the assembly site of an RNP granule. MKLP1 and ARC are necessary for the localization and translation of RNA in the MB dark zone, whereas ESCRT-III was necessary to maintain translation levels in the MB. Our data suggest a model in which the MB functions as a novel RNA-based organelle with a uniquely complex life cycle. We present a model in which the assembly and transfer of RNP complexes are central to post-mitotic MBR function and suggest the MBR serves as a novel mode of RNA-based intercellular communication with a defined biogenesis that is coupled to abscission, and inherently links cell division status with signaling capacity. To our knowledge, this is the first example of an autonomous extracellular vesicle with active translation activity. HighlightsO_LIThe MB, the center region of the intercellular bridge, is the assembly site of a ribonucleoprotein granule, we call the MB granule C_LIO_LIDistinct oncogenic and pluripotent transcription factor RNAs, including Jun/Fos and KLF4, are packaged in MBs and MBRs C_LIO_LIThe MB granule is coincident with the MB matrix, or dark zone, of the MB C_LIO_LIThe Kif23/MKLP1 kinesin is a core hexanediol-sensitive MB granule component C_LIO_LIThe MB and MBR are site of active translation that begins in early G1 and continues post-mitotically C_LIO_LIMKLP1 and ARC are necessary for RNA targeting/maintenance and translation at the MB C_LIO_LIDepletion of ESCRT-III increases the levels of translation during abscission C_LIO_LIAbscission releases MBRs as MB granule-harboring, translating extracellular vesicles C_LIO_LIMultiple cell types including cancer, stem, neural stem, all have actively translating MBRs C_LIO_LIMBRs are proposed as a novel mode of intercellular communication by extracellular vesicle-mediated direct transfer of RNA C_LI

cell biology↗

bioRxiv2022

Differential nuclear import determines lncRNA inheritance following mitosis

Mitosis results in a dramatic reorganization of chromatin structure in order to promote chromatin compaction and segregation to daughter cells. Consequently, mitotic entry is accompanied by transcriptional silencing and removal of most chromatin-bound RNAs from chromosomes. As cells exit mitosis, chromatin rapidly decondenses and transcription restarts as waves of differential gene expression. However, little is known about the fate of chromatin-bound RNAs following cell division. Here we explored whether nuclear RNA from the previous cell cycle is present in G1 cells following mitosis. We found that half of all nuclear RNAs are inherited in a transcriptionindependent manner following mitosis. Interestingly, the snRNA U2 is efficiently inherited by G1 cells while lncRNAs NEAT1 and MALAT1 show no inheritance following mitosis. We found that the nuclear protein SAF-A, which is hypothesized to tether RNA to DNA, did not play a prominent role in nuclear RNA inheritance, indicating the mechanism for RNA inheritance may not involve RNA chaperones that have chromatin binding activity. Instead, we observe that the timing of RNA inheritance indicates a select group of nuclear RNAs are reimported into the nucleus after the nuclear envelope has reassembled. Taken together, our work demonstrates that there is a fraction of nuclear RNA from the previous cell cycle that is reimported following mitosis and suggest that mitosis may serve as a time to reset the interaction of lncRNAs with chromatin.

cell biology↗

bioRxiv2019

Prophase removal of chromosome-associated RNAs facilitates anaphase chromosome segregation

During mitosis, the genome is transformed from a decondensed, transcriptionally active state to a highly condensed, transcriptionally inactive state. Mitotic chromosome reorganization is marked by the general attenuation of transcription on chromosome arms, yet how the cell regulates nuclear and chromatin-associated RNAs after chromosome condensation and nuclear envelope breakdown is unknown. SAF-A/hnRNPU is an abundant nuclear protein with RNA-to-DNA tethering activity, coordinated by two spatially distinct nucleic acid binding domains. Here we show that RNA is evicted from prophase chromosomes through Aurora-B-dependent phosphorylation of the SAF-A DNA-binding domain; failure to execute this pathway leads to accumulation of SAF-A:RNA complexes on mitotic chromosomes and elevated rates of anaphase segregation defects. This work reveals a role for Aurora-B in removing chromatin-associated RNAs during prophase, and demonstrates that Aurora-B dependent relocalization of SAF-A during cell division contributes to the fidelity of chromosome segregation.

cell biology↗

bioRxiv2019

Genome-wide analysis identifies cis-acting elements regulating mRNA polyadenylation and translation during vertebrate oocyte maturation

Changes in gene expression are required to orchestrate changes in cell state during development. Most cells change patterns of gene expression through transcriptional regulation. In contrast, oocytes are transcriptionally silent and use changes in mRNA poly-A tail length to control protein production. Poly-A tail length is positively correlated with translation activation during early development. However, it is not clear how poly-A tail changes affect mRNA translation at a during vertebrate oocyte maturation. We used Tail-seq and polyribosome analysis to measure poly-A tail and translational changes during oocyte maturation in Xenopus laevis. We identified large-scale poly-A and translational changes during oocyte maturation and found that poly-A tail changes precede translation changes. Additionally, we identified a family of U-rich sequence elements that are enriched near the polyadenylation signal of polyadenylated and translationally activated mRNAs. A modest density of U-rich elements was correlated with polyadenylation while a high density of U-rich elements was required to activate translation, showing that polyadenylation and translation activation can be uncoupled. Collectively, our data show that changes in mRNA polyadenylation are a key mechanism regulating protein expression during vertebrate oocyte maturation and that these changes are controlled by a spatial code of cis-acting sequence elements. Our results provide insight into mechanisms of translational control in oocytes and identify novel proteins important for the completion of meiosis.

genomics↗

bioRxiv2019

Cohesin removal reprograms gene expression upon mitotic entry

Entering mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA Polymerase II (RNAPII) in mitosis. Here, we demonstrate that chromatin-bound cohesin is sufficient to retain RNAPII at centromeres while WAPL-mediated removal of cohesin during prophase is required for RNAPII dissociation from chromosome arms. Failure to remove cohesin from chromosome arms results in a failure to release elongating RNAPII and nascent transcripts from mitotic chromosomes and dramatically alters gene expression. We propose that prophase cohesin removal is the key step in reprogramming gene expression as cells transition from G2 to mitosis, and is temporally coupled with chromosome condensation to coordinate chromosome segregation with changes in gene expression.\n\nHighlightsO_LIMitotic centromere transcription requires cohesin\nC_LIO_LICohesin removal releases elongating RNA Pol II and nascent RNA from chromatin\nC_LIO_LIThe prophase pathway reprograms gene expression during mitosis\nC_LI

cell biology↗