Biology subjects

Cheeseman, I. M.

Publications and source records attributed to Cheeseman, I. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Functional genetics reveals a role for the proteasome in regulating centromere assembly

Centromeres link chromosomes to spindle microtubules to direct their segregation during mitosis. The vertebrate centromere is built on two critical DNA-binding proteins, the histone H3-variant CENP-A and histone fold protein CENP-T. CENP-T is a potent scaffold for kinetochore assembly, such that concentrating it at ectopic sites is sufficient to build kinetochore-like structures. How cells prevent excess CENP-T from acting at non-centromere sites remains unclear. Here, using optical pooled screening to systematically define the requirements for CENP-A and CENP-T localization, we recover the established CENP-A deposition machinery and reveal a specific role for the proteasome in controlling CENP-T levels. We find that non-centromere CENP-T is degraded in a ubiquitin- and proteasome-dependent manner. Whereas centromere-incorporated CENP-T is stable, CENP-T that is not bound to centromeres is rapidly turned over, with the CENP-T histone fold region sufficient to confer proteasome sensitivity. Quiescent cells, which disassemble their centromeres, continue to express CENP-T but eliminate it through continuous degradation. Thus, whereas CENP-A is controlled primarily through regulated deposition, cells additionally control CENP-T by selectively degrading the unincorporated protein.

cell biology↗

bioRxiv2026

Translational isoforms of mammalian heterochromatin protein 1 (HP1) tune its interactions with chromatin effectors

The heterochromatin protein 1 (HP1) protein family maintains heterochromatin by reading H3K9 methylation and recruiting diverse chromatin effectors. Multiple effectors compete for the same binding surface on HP1, requiring cells to regulate the ability of HP1 to engage the right effectors across differing conditions. Here, we show that mammalian cells produce multiple N-terminally extended isoforms of HP1{beta} from conserved, in-frame near-cognate start codons in the CBX1 5' UTR. Each of these isoforms retains the localization to H3K9me3 marks. However, the extended HP1{beta} isoforms shift its binding preference away from proteins with a conserved PxVxL motif and toward the G9a/GLP complex. Structural predictions suggest that the extension blocks the PxVxL interaction surface. Indeed, we find that an extended HP1{beta} isoform fails to bind a model PxVxL-containing substrate in vitro. Together, our data reveal an alternative HP1{beta} isoform that reshapes its effector binding preferences, providing an additional regulatory layer for heterochromatin organization.

molecular biology↗

bioRxiv2026

Systematic functional genetic analysis of cell-substrate adhesion

Cellular adhesion is critical for tissue organization and integrity, but the full complement of proteins required for proper adhesion remains unresolved. Here, we define the requirements for cell-substrate adhesion in cultured human cells using orthogonal, large-scale functional genetic approaches. Using mechanical assays to test the maintenance ("shake-off") or formation of adhesion and parallel large-scale assays of cell morphology, we identify dozens of gene targets with roles in adhesion. Our analyses reveal dynamic requirements for adhesion across timepoints and cell lines. We additionally conduct targeted downstream mechanistic analyses to resolve the molecular basis for altered adhesion. Collectively, we identify established and uncharacterized regulators of adhesion, including genes involved in mitosis, focal adhesions, actin regulation, and membrane trafficking. Unexpectedly, we find that cells that fail cytokinesis display impaired adhesion, with strongly altered actin organization and nuclear dynamics. Together, this work provides a comprehensive view of the genetic requirements for cell-substrate adhesion.

cell biology↗

bioRxiv2026

A machine learning model predicts protein stability of annotated and alternate protein isoforms

The regulation of protein stability is essential for cellular homeostasis and is determined by a combination of intrinsic sequence motifs and extrinsic recognition enzymes. Despite growing knowledge of the protein degradation machinery, the ability to predict a proteins stability from its amino acid sequence remains challenging. Here we develop a machine learning model to predict protein stability from N-terminal amino acid sequences. Using our model and experimental validation, we identify known and novel sequence motifs governing protein stability. We additionally use this model to predict the stability of alternative translational isoforms with distinct N-termini produced from the same mRNA. Despite differing by a limited number of amino acids, we identify N-terminal isoforms with drastically different stabilities relative to their annotated counterparts, highlighting the potential of N-terminal extensions and truncations to regulate protein function. Together, this model provides a valuable tool for evaluating additional protein datasets and protein design strategies.

cell biology↗

bioRxiv2026

The one-week automated genome-wide optical pooled screen

Optical pooled screens (OPS) are bottlenecked by labor-intensive in situ sequencing and analysis protocols. Here we present OttoSeq, an automated OPS platform combining the Otto2 fluid handling system with the Brieflow analysis pipeline. We utilized OttoSeq to complete a genome-wide cell painting screen in eight days, sampling more than 5 million high-quality cells across 21,732 gene knockout perturbations (224 cells per gene) and interpreting 320 functional gene clusters.

genomics↗

bioRxiv2026

5' UTR length regulates alternative N-terminal protein isoform production in health and disease

The 5' untranslated region (5' UTR) of an mRNA is classically viewed as a regulatory region that controls the amount of protein production, but not the resulting protein sequence. Here, we demonstrate that 5' UTR length plays a direct role in alternative N-terminal protein isoform production by controlling start codon selection. We find that very short 5' UTRs enhance leaky ribosome scanning, thereby promoting the production of truncated alternative N-terminal protein isoforms. We also show that endogenous changes in 5' UTR length due to alternative transcription initiation can tune the relative abundance of alternative N-terminal isoforms from the same gene. In addition, we identify mutations in rare genetic diseases that alter 5' UTR length, including a deletion in the VHL 5' UTR in von Hippel-Lindau disease that shifts translation toward the shorter VHLp19 isoform. Together, our results implicate 5' UTR length as a determinant of alternative N-terminal isoform production and reveal an underappreciated mechanism by which noncoding changes can reshape the proteome. HighlightsO_LI5' UTR length affects the landscape of endogenous alternative N-terminal protein isoforms C_LIO_LIGeneration of an alternative truncated AKR7A2 isoform is mediated by short 5' UTR length C_LIO_LIAlternative transcription initiation modulates 5' UTR length to tune N-terminal isoform ratios C_LIO_LIPathogenic VHL 5' UTR variants perturb N-terminal isoform ratios by altering 5' UTR length C_LI

molecular biology↗

bioRxiv2025

The dynamics of centromere assembly and disassembly during quiescence

Quiescence is a state in which cells undergo a prolonged proliferative arrest while maintaining their capacity to reenter the cell cycle. Here, we analyze entry and exit from quiescence, focusing on how cells regulate the centromere, a structure involved in chromosome segregation. Despite the constitutive localization of centromere proteins throughout the cell cycle, we find that cells rapidly disassemble most centromere proteins during quiescence entry, while preserving those required to maintain centromere identity. During quiescence exit, the centromere is reassembled and rapidly regains normal homeostatic levels of centromere proteins. Although the histone variant CENP-A is typically deposited during G1, we find that CENP-A deposition does not occur during the G1 immediately following quiescence exit, and instead occurs after cells complete their first mitosis. In contrast, other centromere proteins relocalize during the first S phase independent of DNA replication. These findings reveal centromere dynamics during quiescence entry and exit and highlight paradigms for the timing and control of centromere protein deposition.

cell biology↗

bioRxiv2025

Functional genetics with a hypomorphic CENP-C mutant reveals a regulatory system for chromosome congression

Chromosome congression is a key process that acts to align chromosomes at the spindle equator via kinetochore-microtubule interactions, with defects in chromosome alignment leading to chromosomal instability. However, defining the mechanisms that underlie chromosome congression is limited due to the multiple factors that act in parallel to regulate chromosome movement. Here, we conducted a genome-wide Cas9-based functional genetics screen using a hypomorphic CENP-C mutant that affects its kinetochore interactions. Our analysis identified KIF18A, whose knockout resulted in synthetic lethality with the CENP-C mutant. Further analysis revealed that the synthetic defect was due to a reduction in CENP-E function in the CENP-C mutant. Our work suggests that KIF18A promotes chromosome alignment in cooperation with CENP-E downstream of CENP-C during early prometaphase. Thus, our analysis enables us to dissect parallel molecular mechanisms for chromosome congression and identify sensitivities and biomarkers that might guide anti-KIF18A chemotherapeutics.

cell biology↗

bioRxiv2025

SRSF12 is a primate-specific splicing factor that induces a tissue-specific gene expression program

Alternative splicing expands proteomic diversity and is tightly regulated by splicing factors, including the serine/arginine-rich (SR) protein family. Here, we analyze the poorly characterized protein SRSF12. Although SRSF12 is conserved across vertebrates, it is poorly expressed in most mammals, and we find that SRSF12 knockout mice do not display overt physiological or transcriptomic alterations. In contrast, SRSF12 is more highly expressed in primates where it is predominantly transcribed in the testes, oocytes, and brain. SRSF12 co-localizes with other splicing components to nuclear speckles and interacts with core splicing factors in cultured human cells. Strikingly, ectopic expression of SRSF12 in human cells induces widespread transcriptional changes, activating meiosis-, testis- and brain-specific genes. SRSF12 overexpression also leads to mitotic arrest and cell death, phenotypes that require both its structured RNA recognition motif and intrinsically disordered arginine/serine-rich C-terminal domain. Together, our results suggest that SRSF12 has evolved primate-specific expression to regulate testis- and brain-specific genes.

cell biology↗

bioRxiv2025

Global inhibition of deadenylation stabilizes the transcriptome in mitotic cells

In the presence of cell division errors, mammalian cells can pause in mitosis for tens of hours with little to no transcription, while still requiring continued translation for viability. These unique aspects of mitosis require substantial adaptations to the core gene expression programs. Indeed, during interphase, the homeostatic control of mRNA levels involves a constant balance of transcription and degradation, with a median mRNA half-life of [~]2-4 hours. If such short mRNA half-lives persisted in mitosis, cells would be expected to quickly deplete their transcriptome in the absence of new transcription. Here, we report that the transcriptome is globally stabilized during prolonged mitotic delays. Typical mRNA half-lives are increased >4-fold in mitosis compared to interphase, thereby buffering mRNA levels in the absence of new synthesis. Moreover, the poly(A)-tail-length profile of mRNAs changes in mitosis, strongly suggesting a mitotic repression of deadenylation. We further show that mRNA stabilization in mitosis is dependent on cytoplasmic poly(A)-binding proteins PABPC1&4. Depletion of PABPC1&4 disrupts the maintenance of mitotic arrest, highlighting the critical physiological role of mitotic transcriptome buffering. HighlightsO_LIThe cellular transcriptome is globally stabilized during prolonged mitotic arrest C_LIO_LIDistinct poly(A)-tail-length profile of mRNAs in mitosis suggests repression of deadenylation C_LIO_LImRNA stabilization in mitosis is dependent on PABPC1 and PABPC4 C_LIO_LIDegradation of mRNAs during mitosis compromises maintenance of mitotic arrest C_LI

cell biology↗

bioRxiv2025

Differential roles for the spindle assembly checkpoint in error surveillance and mitotic timing

The Spindle Assembly Checkpoint (SAC) plays critical roles in regulating mitotic fidelity and progression. Here, we utilized a SAC-deficient cell line lacking the full-length Cdc20 translational protein isoform (Cdc20 {Delta}FL) to define its differential genetic interactions using CRISPR/Cas9-based gene targeting. Cdc20 {Delta}FL cells display synthetic lethal relationships with gene targets required for proper chromosome segregation, highlighting the critical role of the SAC in error surveillance. Surprisingly, we found that the checkpoint component Mad2 becomes dispensable for viability in Cdc20 {Delta}FL cells. Prior work suggested that Mad2 acts as an essential mitotic "timer" to control mitotic duration in unperturbed cells. Instead, our functional analysis indicates that the mitotic timer depends on the interdependent and overlapping actions of: (1) Mad2 inhibition of APC/C-Cdc20, (2) Cdk1-mediated phosphorylation of Cdc20, and (3) total Cdc20 protein levels. Simultaneously perturbing these pathways results in near immediate mitotic exit and catastrophic chromosome mis-segregation.

cell biology↗

bioRxiv2025

Brieflow: An Integrated Computational Pipeline for High-Throughput Analysis of Optical Pooled Screening Data

Optical pooled screening (OPS) has emerged as a powerful technique for functional genomics, enabling researchers to link genetic perturbations with complex cellular morphological phenotypes at unprecedented scale. However, OPS data analysis presents challenges due to massive datasets, complex multi-modal integration requirements, and the absence of standardized frameworks. Here, we present Brieflow, a computational pipeline for end-to-end analysis of fixed-cell optical pooled screening data. We demonstrate Brieflows capabilities through reanalysis of a CRISPR-Cas9 screen encompassing 5,072 fitness-conferring genes, processing more than 70 million cells with multiple phenotypic markers. To accelerate biological interpretation, we additionally present MozzareLLM, a framework leveraging large language models to identify biological processes within phenotypic clusters and prioritize gene candidates for experimental validation. Our combined analysis recovered coherent biological modules missed by existing analytical approaches, including five core mitochondrial sub-programs that were absent from the original study. The modular design and open-source implementation of Brieflow facilitates the integration of novel analytical components while ensuring computational reproducibility and improved performance for the use of high-content phenotypic screening in biological discovery.

cell biology↗

bioRxiv2025

Alternative start codon selection shapes mitochondrial function during evolution, homeostasis, and disease

Mitochondrial endosymbiosis was a pivotal event in eukaryotic evolution, requiring core proteins to adapt to function both within the mitochondria and in the host cell. Here, we systematically profile the localization of protein isoforms generated by alternate start codon selection during translation. We identify hundreds of pairs of differentially-localized protein isoforms, many of which affect mitochondrial targeting and are essential for mitochondrial function. The emergence of dual-localized mitochondrial protein isoforms coincides with mitochondrial acquisition during early eukaryotic evolution. We further reveal that eukaryotes use diverse mechanisms--such as leaky ribosome scanning, alternative transcription, and paralog duplication--to maintain the production of dual-localized isoforms. Finally, we identify multiple isoforms that are specifically dysregulated by rare disease patient mutations and demonstrate how these mutations can help explain unique clinical presentations. Together, our findings illuminate the evolutionary and pathological relevance of alternative translation initiation, offering new insights into the molecular underpinnings of mitochondrial biology.

cell biology↗

bioRxiv2025

Molecular determinants of RNase MRP specificity and function

RNase MRP and RNase P are evolutionarily related complexes that facilitate rRNA and tRNA biogenesis, respectively. The two enzymes share nearly all protein subunits and have evolutionarily related catalytic RNAs. Notably, RNase P includes a unique subunit, Rpp21, whereas no RNase MRP-specific proteins have been found in humans, limiting molecular analyses of RNase MRP function. Here, we identify the RNase MRP-specific protein, C18orf21/RMRPP1. RMRPP1 and Rpp21 display significant structural homology, but we identify specific regions that drive interactions with their respective complexes. Additionally, we reveal that RNase MRP is required for 40S, but not 60S, ribosome biogenesis uncovering an alternative pathway for ribosome assembly. Finally, we identify Nepro as an essential rRNA processing factor that associates with the RNase MRP complex. Together, our findings elucidate the molecular determinants of RNase MRP function and underscore its critical role in ribosome biogenesis.

molecular biology↗

bioRxiv2025

19S proteasome loss causes monopolar spindles through ubiquitin-independent KIF11 degradation

To direct regulated protein degradation, the 26S proteasome recognizes ubiquitinated substrates through its 19S particle and then degrades them in the 20S enzymatic core. Despite this close interdependency between proteasome subunits, we demonstrate that knockouts from different proteasome subcomplexes result in distinct highly cellular phenotypes. In particular, depletion of 19S PSMD lid proteins, but not that of other proteasome subunits, prevents bipolar spindle assembly during mitosis, resulting in a mitotic arrest. We find that the monopolar spindle phenotype is caused by ubiquitin- independent proteasomal degradation of the motor protein KIF11 upon loss of 19S proteins. Thus, negative regulation of 20S-mediated proteasome degradation is essential for mitotic progression and 19S and 20S proteasome components can function independently outside of the canonical 26S structure. This work reveals a role for the proteasome in spindle formation and identifies the effects of ubiquitin- independent degradation on cell cycle control.

cell biology↗

bioRxiv2024

Structural constraints and drivers of molecular evolution in a macromolecular complex; the kinetochore

Evolutionary theory suggests that critical cellular structures should be subject to strong purifying selection as protein changes would result in inviability. However, how this evolutionary principle relates to multi-subunit complexes remains incompletely explored. For example, the macromolecular kinetochore complex, which mediates the faithful segregation of DNA during cell division, violates the expectation of purifying selection as subsets of kinetochore proteins exhibit rapid evolution despite its critical role. Here, we developed a multi-level approach to investigate the evolutionary dynamics of the kinetochore as a model for understanding how an essential multi-protein structure can experience high rates of diversifying selection while maintaining function. Our comprehensive approach analyzed 57 kinetochore genes for signatures of purifying and diversifying selection across 70 mammalian species. Intraspecies comparisons of kinetochore gene evolution showed that members of the order Afrotheria experience higher rates of diversifying selection than other mammalian orders. Among individual loci, genes that serve regulatory functions, such as the mitotic checkpoint genes, are conserved under strong purifying selection. In contrast, the proteins that serve as the structural base of the kinetochore, including the inner and outer kinetochore, evolve rapidly across species. We also demonstrated that diversifying selection is targeted to protein regions that lack clear structural predictions. Finally, we identified sites that exhibit corresponding trends in evolution across different genes, potentially providing evidence of compensatory evolution in this complex. Together, our study of the kinetochore reveals a potential avenue by which selection can alter the genes that comprise an essential cellular complex without compromising its function.

evolutionary biology↗

bioRxiv2024

A conserved germline-specific Dsn1 alternative splice isoform supports oocyte and embryo development

Alternative mRNA splicing can generate distinct protein isoforms to allow for the differential control of cell processes across cell types. However, alternative splice isoforms that differentially modulate distinct cell division programs have remained elusive. Here, we demonstrate that mammalian germ cells express an alternate mRNA splice isoform for the kinetochore component, DSN1, a subunit of the MIS12 complex that links the centromeres to spindle microtubules during chromosome segregation. This germline DSN1 isoform bypasses the requirement for Aurora kinase phosphorylation for its centromere localization due to the absence of a key regulatory region allowing DSN1 to display persistent centromere localization. Expression of the germline DSN1 isoform in somatic cells results in constitutive kinetochore localization, chromosome segregation errors, and growth defects, providing an explanation for its tight cell type-specific expression. Reciprocally, precisely eliminating expression of the germline DSN1 splice isoform in mouse models disrupts oocyte maturation and early embryonic divisions coupled with a reduction in fertility. Together, this work identifies a germline-specific splice isoform for a chromosome segregation component and implicates its role in mammalian fertility.

cell biology↗

bioRxiv2024

Nuclear release of eIF1 globally increases stringency of start-codon selection to preserve mitotic arrest physiology

Regulated start-codon selection has the potential to reshape the proteome through the differential production of uORFs, canonical proteins, and alternative translational isoforms. However, conditions under which start-codon selection is altered remain poorly defined. Here, using transcriptome-wide translation initiation site profiling, we reveal a global increase in the stringency of start-codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This increased stringency of start-codon selection during mitosis results from increased interactions between the key regulator of start-codon selection, eIF1, and the 40S ribosome. We find that increased eIF1-40S ribosome interactions during mitosis are mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the changes to translational stringency during mitosis, resulting in altered mitotic proteome composition. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage following treatment with anti-mitotic chemotherapeutics. Thus, cells globally control translation initiation stringency with critical roles during the mammalian cell cycle to preserve mitotic cell physiology.

molecular biology↗