Biology subjects

Smith, M. J.

Publications and source records attributed to Smith, M. J..

18 recordsLinked to original sources

bioRxiv2026

The flavin-containing monooxygenases OSGIN1 and OSGIN2 antagonize each other to favor cytokinesis completion

Cytokinesis is a fundamental process that bisects a mother cell into two distinct daughter cells via an ingressing ring of actomyosin. It initiates after anaphase chromosome separation by the mitotic spindle, which coordinates the activation of the small GTPase RhoA at the cell equator. We previously identified OSGIN1 as a flavin-containing monooxygenase enzyme required for cytokinesis completion via the regulation of RhoA activity. Here we show that the OSGIN1 paralog OSGIN2 is also a flavin-containing monooxygenase implicated in cytokinetic regulation. Cells lacking OSGIN2 have reduced RhoA activity and increased rates of cytokinesis failure, similar to OSGIN1-deleted cells. Strikingly, these phenotypes are suppressed in cells lacking both OSGIN1 and OSGIN2, indicating that the two proteins antagonize each other. Consistent with this, we find that OSGIN1 and OSGIN2 form both homo- and heteromeric complexes in vitro and in cells. Together, our results support a model in which OSGIN1 and OSGIN2 both function as negative regulators of RhoA signaling and that their heteromeric interaction during mitosis antagonizes their activity to favor cytokinesis completion.

cell biology↗

bioRxiv2025

Development of a genetically encoded and potent PDE6D inhibitor

PDE6D is a trafficking chaperone of prenylated proteins, such as small GTPases. Several small molecule inhibitors have been developed against it, given that the oncogene K-Ras is one of the cargo proteins. Inhibitor development suffered from the fact that inhibitors against the hydrophobic pocket of PDE6D were typically poorly water-soluble. Here we describe the development of genetically encoded inhibitors that are inspired by high-affinity natural cargo of PDE6D. Our most potent inhibitor, SNAP-STI, encodes merely a farnesylated tetra-peptide, which efficiently blocks PDE6D binding of farnesylated cargo. Direct comparison with small molecule PDE6D inhibitors suggests its higher potency. We show that inhibition of K-Ras membrane anchorage and K-RasG12C-dependent MAPK-signaling by SNAP-STI is weak, consistent with what is observed after PDE6D knockdown. Our data therefore further support that PDE6D is not a suitable surrogate target for efficient inhibition of K-Ras membrane anchorage and MAPK-activity. Nonetheless, by exploiting contacts at the pocket entry, we established a generalizable strategy to design high-affinity PDE6D inhibitors, providing powerful tools for PDE6D biology and target validation.

biochemistry↗

bioRxiv2025

Argonaute-siRNA loading via the RNA-binding protein RDE-4 in C. elegans

Small RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), associate with Argonaute proteins to control gene expression, impacting a wide range of cellular processes, such as antiviral defense, transposon silencing, and development1. Plants and animals typically have several classes of small RNAs, along with multiple Argonautes2. These Argonautes often confer distinct functionality to the various classes of small RNAs3. But how small RNAs are selectively loaded into the appropriate Argonaute is not well understood. miRNAs and siRNAs are typically generated from double-stranded RNA (dsRNA) precursors by the endoribonuclease Dicer4. siRNAs are often processed from fully base-paired precursors derived from various endogenous and exogenous sources, whereas miRNAs typically originate from genetically encoded partially base-paired hairpins1. In C. elegans, Dicer/DCR-1 processing of siRNAs and a related small RNA class, known as 26G- RNAs, is mediated by the dsRNA-binding protein RDE-45-7. Here, we show that RDE-4 also facilitates loading of siRNAs (but not miRNAs) into the Argonaute RDE-1, but not into ALG-1, and loading of 26G-RNAs into the Argonaute ERGO-1. Although we do not find evidence that ALG-3/4 associated 26G-RNAs require RDE-4 for Argonaute loading, their levels are strongly reduced in rde-4 mutants indicating that RDE-4 is broadly required for their formation or stability. Our findings reveal a role for RDE-4 as a critical determinant of small RNA loading specificity and provide insight into the mechanisms by which small RNAs are selectively paired with their corresponding Argonautes.

biochemistry↗

bioRxiv2025

Contemporary seasonal human coronaviruses display differences in cellular tropism compared to laboratory-adapted reference strains

Seasonal human coronaviruses (sHCoVs) cause 15-30% of common colds. The reference strains used for research were isolated decades ago and have been passaged extensively but contemporary sHCoVs have been challenging to study as they are notoriously difficult to grow in standard immortalized cell lines. Here we addressed these issues by utilizing primary human nasal epithelial cells (HNECs) and immortalized human bronchial epithelial cells (BCi) differentiated at an air-liquid interface, as well as human embryonic stem cell-derived alveolar type II (AT2) cells to recover contemporary sHCoVs from human nasopharyngeal specimens. From 21 specimens we recovered four 229E, three NL63 and eight OC43 viruses. All contemporary sHCoVs showed sequence differences from lab-adapted CoVs, particularly within the spike gene. Evidence of nucleotide changes in the receptor binding domains within 229E and detection of recombination for both 229E and OC43 isolates was also observed. Importantly, we developed methods for the amplification of high titre stocks of NL63 and 229E, that maintained sequence identity, and we established methods for the titration of contemporary sHCoV isolates. Comparison of lab-adapted and contemporary strains in immortalised cell lines and airway epithelial cells revealed differences in cell tropism, growth kinetics and cytokine production between lab-adapted and contemporary sHCoV strains. These data confirm that contemporary sHCoVs differ from lab-adapted reference strains and, using the methods established here, should be used for study of CoV biology and evaluation of medical countermeasures. ImportanceZoonotic coronaviruses have caused significant public health emergencies. The occurrence of a similar spillover event in the future is likely and efforts to further understand coronavirus biology should be a high priority. Several seasonal coronaviruses circulate within the human population. Efforts to study these viruses have been limited to reference strains isolated decades ago due to the difficulty in isolating clinical isolates. Here, we use human airway and alveolar epithelial cultures to recover contemporary isolates of NL63, 229E and OC43. We establish methods to make high titre stocks and titrate 229E and NL63 isolates. We show that contemporary isolates of NL63 and OC43 have a different tropism within the respiratory epithelium compared to lab-adapted strains. Although 229E clinical and lab-adapted strains similarly infect the respiratory epithelium, differences in host response and replication kinetics are observed. Using the methods developed here, future research should include contemporary isolates when studying coronavirus biology.

microbiology↗

bioRxiv2024

Islet-antigen reactive B cells display a unique phenotype and BCR repertoire in autoantibody positive and recent-onset type 1 diabetes patients

Autoreactive B cells play an important but ill-defined role in autoimmune type 1 diabetes (T1D). To better understand their contribution, we performed single cell gene and BCR-seq analysis on pancreatic islet antigen-reactive (IAR) B cells from the peripheral blood of nondiabetic (ND), autoantibody positive prediabetic (AAB), and recent-onset T1D individuals. We found that the frequency of IAR B cells was increased in AAB and T1D. IAR B cells from these donors had altered expression of B cell signaling, pro-inflammatory, infection, and antigen processing and presentation genes. Both AAB and T1D donors demonstrated a significant increase in certain heavy and light chain V genes, and these V genes were enriched in islet-reactivity. Public clones of IAR B cells were restricted almost entirely to AAB and T1D donors. IAR B cells were clonally expanded in the autoimmune donors, particularly the AAB group. Notably, a substantial fraction of IAR B cells in AAB and T1D donors appeared to be polyreactive, which was corroborated by analysis of recombinant monoclonal antibodies. These results expand our understanding of autoreactive B cell activation during T1D and identify unique BCR repertoire changes that may serve as biomarkers for increased disease risk. One Sentence SummaryPancreatic islet antigen-reactive B cells from individuals with prediabetes and recently diagnosed with type 1 diabetes display a unique phenotype and BCR repertoire compared to non-diabetic donors.

immunology↗

bioRxiv2024

Regulation of Microprocessor assembly and localization via Pasha's WW domain in C. elegans

Primary microRNA (pri-miRNA) transcripts are processed by the Microprocessor, a protein complex that includes the ribonuclease Drosha and its RNA binding partner DGCR8/Pasha. We developed a live, whole animal, fluorescence-based sensor that reliably monitors pri-miRNA processing with high sensitivity in C. elegans. Through a forward genetic selection for alleles that desilence the sensor, we identified a mutation in the conserved G residue adjacent to the namesake W residue of Pashas WW domain. Using genome editing we also mutated the W residue and reveal that both the G and W residue are required for dimerization of Pasha and proper assembly of the Microprocessor. Surprisingly, we find that the WW domain also facilitates nuclear localization of Pasha, which in turn promotes nuclear import or retention of Drosha. Furthermore, depletion of Pasha or Drosha causes both components of the Microprocessor to mislocalize to the cytoplasm. Thus, Pasha and Drosha mutually regulate each others spatial expression in C. elegans.

molecular biology↗

bioRxiv2024

High-Dimensional Imaging of Vestibular Schwannoma Reveals Distinctive Immunological Networks Across Histomorphic Niches in NF2-related Schwannomatosis

NF2-related Schwannomatosis (NF2 SWN) is a rare tumour-predisposition syndrome characterised by the growth of multiple central and peripheral nervous system neoplasms. The drivers of NF2 SWN are pathogenic variants in the tumour suppressor gene NF2, encoding the protein Merlin, leading to development of bilateral vestibular schwannoma (VS) in >95% of patients. VS tumours are characterised by infiltration of myeloid cells and lymphocytes, highlighting the potential of immunotherapy for VS. However, the immunological landscape in VS and the spatial determinants within the tumour microenvironment that shape the trajectory of disease are presently unknown. In this study, to elucidate the complex immunological networks across VS, we performed imaging mass cytometry (IMC) on clinically annotated VS samples from NF2 SWN patients. We reveal the heterogeneity in neoplastic cell, myeloid cell and T cell populations that co-exist within VS, determining that the cellular composition of VS tumours is independent of NF2-SWN genetic severity. We show that distinct myeloid cell and Schwann cell populations exist within varied spatial contextures across characteristic Antoni A and B histomorphic niches. Interestingly, we show that T-cell populations associate with tumour-associated macrophages (TAMs) in Antoni A regions, seemingly limiting their ability to interact with tumorigenic Schwann cells. This spatial landscape is altered in Antoni B regions, where T-cell populations appear to interact with PD-L1+ Schwann cells. We also demonstrate that prior bevacizumab treatment (VEGF-A antagonist) preferentially reduces alternatively-activated TAMs, whilst enhancing CD44 expression, in bevacizumab-treated tumours. Together, we describe niche-dependent modes of T-cell regulation in NF2 SWN VS, indicating the potential for microenvironment-altering therapies for VS. TeaserImaging mass cytometry and spatial omic analyses illustrate spatially-distinct regions of T-cell regulation in vestibular schwannoma.

immunology↗

bioRxiv2023

Mapping the MOB proteins' proximity network reveals a unique interaction between human MOB3C and the RNase P complex

Distinct functions mediated by members of the monopolar spindle-one-binder (MOB) family of proteins remain elusive beyond the evolutionary conserved and well-established roles of MOB1A and B in regulating the Hippo pathway. Since MOB proteins are adaptors, understanding how they engage in protein-protein interactions and complexes assembly is essential to define the full scope of their biological functions. To address this, we undertook a proximity-dependent biotin identification (BioID) approach to define the interactomes of all seven human MOB proteins in HeLa and HEK293 cell lines. We uncovered > 200 interactions, of which at least 70% are unreported on BioGrid. The generated dataset reliably recalled the bona fide interactors of the well-studied MOBs. We further defined the common and differential interactome between different MOBs on a subfamily and an individual level. We discovered a unique interaction between MOB3C and 7 out of 10 protein subunits of the RNase P complex, an endonuclease that catalyzes tRNA 5 maturation. As a proof-of-principle for the robustness of the generated dataset, we validated the specific interaction of MOB3C with catalytically active RNase P by using affinity purification-mass spectrometry and pre-tRNA cleavage assays of MOB3C pulldowns. In summary, our data provide novel insights into the biology of MOB proteins and reveal the first interactors of MOB3C, components of the RNase P complex, and hence an exciting nexus with RNA biology.

biochemistry↗

bioRxiv2023

ARF-family global interactome mapping uncovers spatial organization of cellular signaling pathways

The ADP-ribosylation factors (ARFs) and ARF-like (ARLs) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we utilized proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified [~]3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling. SUMMARY STATEMENTGeneration of the ARF family interactome allowed the attribution of potential localizations and functions to previously understudied members. We found that ARL14 activates PLD1 and contributes to ESCPE-1-mediated trafficking.

molecular biology↗

bioRxiv2022

High Quality Phasing Using Linked-Read Whole Genome Sequencing of Patient Cohorts Informs Genetic Understanding of Complex Traits

Phasing of heterozygous alleles is critical for interpretation of cis-effects of disease-relevant variation. For population studies, phase is often inferred from external data but read-based phasing approaches that span long genomic distances would be more accurate because they enable both genotype and phase to be obtained from a single dataset. To demonstrate how read-based phasing can provide functional insights, we sequenced 477 individuals with Cystic Fibrosis (CF) using linked-read sequencing. We benchmark read-based phasing with different short- and long-read sequencing technologies, prioritize linked-read technology as the most informative and produce a benchmark phase call set from reference sample HG002 for the community. The 477 samples display an average phase block N50 of 4.39 Mb. We use these samples to construct a graph representation of CFTR haplotypes, which facilitates understanding of complex CF alleles. Fine-mapping and phasing of the chr7q35 trypsinogen locus associated with CF meconium ileus demonstrates a 20 kb deletion and a PRSS2 missense variant p.Thr8Ile (rs62473563) independently contribute to meconium ileus risk (p=0.0028, p=0.011, respectively) and are PRSS2 pancreas eQTLs (p=9.5e-7 and p=1.4e-4, respectively), explaining the mechanism by which these polymorphisms contribute to CF. Phase enables access to haplotypes that can be used for genome graph or reference panel construction, identification of cis-effects, and for understanding disease associated loci. The phase information from linked-reads provides a causal explanation for variation at a CF-relevant locus which also has implications for the genetic basis of non-CF pancreatitis to which this locus has been reported to contribute.

genetics↗

bioRxiv2021

A mechanistic model of malignant transformation in vestibular schwannoma

A vestibular schwannoma (VS) is a relatively rare, benign tumour of the eighth cranial nerve, often involving alterations to the gene NF2. Previous mathematical models of schwannoma incidence have not attempted to account for alterations in specific genes, and could not distinguish between point mutations and loss of heterozygosity (LOH). Here, we present a mechanistic approach to modelling initiation and malignant transformation in Schwannoma. Each parameter is associated with a specific gene or mechanism operative in Schwann cells, and can be determined by combining incidence data with empirical frequencies of pathogenic variants and LOH. This results in new estimates for the base pair mutation rate u = 4.48 x 10-10 and the rate of LOH = 2.03 x 10-6/yr in Schwann cells. In addition to new parameter estimates, we extend the approach to estimate the risk of both spontaneous and radiation-induced malignant transformation. We conclude that radiotherapy is likely to have a negligible excess risk of malignancy for sporadic VS, with a possible exception of rapidly growing tumours.

cancer biology↗

bioRxiv2020

RASSF effectors couple diverse RAS subfamily GTPases to the Hippo pathway

Activated RAS GTPases signal by directly binding effector proteins. Effectors have a folded RAS association (RA) domain that binds exclusively to GTP-loaded RAS, but the specificity of most RA domains for >150 RAS superfamily GTPases is unknown. Ten RAS-association domain family (RASSF) proteins comprise the largest group of effectors, proposed to couple RAS to the pro-apoptotic Hippo pathway. We show that RASSF1-6 complex with Hippo kinase, while RASSF7-10 are a separate family related to p53-regulatory ASPP effectors. Only RASSF5 directly binds activated HRAS and KRAS. Structural modelling reveals that expansion of RASSFs in vertebrates included amino acid substitutions that alter their GTPase binding specificity. We demonstrate that the tumour suppressor RASSF1A complexes with the GTPases GEM, REM1, REM2 and the enigmatic RASL12. Interplay between RASSFs and RAS GTPases can drastically restrict YAP1 nuclear localization. Thus, these simple scaffolds can link activation of diverse RAS proteins to Hippo or p53 regulation.

biochemistry↗

bioRxiv2020

Single-molecule imaging of telomerase RNA reveals a Recruitment-Retention model for telomere elongation

Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatio-temporal coordination of telomerase and telomeres, and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combined CRISPR genome editing and MS2 RNA-tagging to image single-molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, then base-pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life-cycle of telomerase RNA and provides a framework to understand how cancer-associated mutations mechanistically drive defects in telomere homeostasis.

molecular biology↗

bioRxiv2019

Conformational resolution of nucleotide cycling and effector interactions for multiple small GTPases in parallel

Small GTPase proteins alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes. Numerous GTPases are implicated in oncogenesis, particularly three RAS isoforms HRAS, KRAS and NRAS, and the RHO family GTPase RAC1. Signaling networks comprising small GTPases are highly connected, and there is evidence of direct biochemical crosstalk between the functional G-domains of these proteins. The activation potential of a given GTPase is contingent on a co-dependent interaction with nucleotide and a Mg2+ ion, which bind to individual variants via distinct affinities coordinated by residues in the nucleotide binding pocket. Here, we utilize a selective-labelling strategy coupled with real-time nuclear magnetic resonance (NMR) spectroscopy to monitor nucleotide exchange, GTP hydrolysis and effector interactions of multiple small GTPases in a single complex system. We provide new insight on nucleotide preference and the role of Mg2+ in activating both wild-type and oncogenic mutant enzymes. Multiplexing reveals GEF, GAP and effector binding specificity in mixtures of GTPases and establishes the complete biochemical equivalence of the three related RAS isoforms. This work establishes that direct quantitation of the nucleotide-bound conformation is required to accurately resolve GTPase activation potential, as GTPases such as RALA or the G12C mutant of KRAS demonstrate fast exchange kinetics but have a high affinity for GDP. Further, we propose that the G-domains of small GTPases behave autonomously in solution and nucleotide cycling proceeds independent of protein concentration but is highly impacted by Mg2+ abundance.

biochemistry↗

bioRxiv2019

Characterising the loss-of-function impact of 5' untranslated region variants in whole genome sequence data from 15,708 individuals

Upstream open reading frames (uORFs) are important tissue-specific cis-regulators of protein translation. Although isolated case reports have shown that variants that create or disrupt uORFs can cause disease, genetic sequencing approaches typically focus on protein-coding regions and ignore these variants. Here, we describe a systematic genome-wide study of variants that create and disrupt human uORFs, and explore their role in human disease using 15,708 whole genome sequences collected by the Genome Aggregation Database (gnomAD) project. We show that 14,897 variants that create new start codons upstream of the canonical coding sequence (CDS), and 2,406 variants disrupting the stop site of existing uORFs, are under strong negative selection. Furthermore, variants creating uORFs that overlap the CDS show signals of selection equivalent to coding loss-of-function variants, and uORF-perturbing variants are under strong selection when arising upstream of known disease genes and genes intolerant to loss-of-function variants. Finally, we identify specific genes where perturbation of uORFs is likely to represent an important disease mechanism, and report a novel uORF frameshift variant upstream of NF2 in families with neurofibromatosis. Our results highlight uORF-perturbing variants as an important and under-recognised functional class that can contribute to penetrant human disease, and demonstrate the power of large-scale population sequencing data to study the deleteriousness of specific classes of non-coding variants.

genomics↗

bioRxiv2018

The adequacy of tissue microarrays in the assessment of inter- and intra-tumoural heterogeneity of infiltrating lymphocyte burden in leiomyosarcoma

The characterisation and clinical relevance of tumour-infiltrating lymphocytes (TILs) in leiomyosarcoma (LMS), a subtype of soft tissue sarcoma that exhibits histological heterogeneity, is not established. The use of tissue microarrays (TMA) in studies that profile TIL burden is attractive but given the potential for intra-tumoural heterogeneity to introduce sampling errors, the adequacy of this approach is undetermined. In this study, we assessed the histological inter-and intra-tumoural heterogeneity in TIL burden within a retrospective cohort of primary LMS specimens. Using a virtual TMA approach, we also analysed the optimal number of TMA cores required to provide an accurate representation of TIL burden in a full tissue section. We establish that LMS have generally low and spatially homogenous TIL burdens, although a small proportion exhibit higher levels and more heterogeneous distribution of TILs. We show that a conventional and practical number (1-3) of TMA cores is adequate for correct ordinal categorisation of tumours with high or low TIL burden, but that many more cores ([≥] 11) is required to accurately estimate absolute TIL numbers. Our findings provide a benchmark for the design of future studies aiming to define the clinical relevance of the immune microenvironments of LMS and other sarcoma subtypes.

cancer biology↗

bioRxiv2018

Inherited BRCA1 epimutation as a novel cause of breast and ovarian cancer

BackgroundPathogenic variants in BRCA1 or BRCA2 are identified in ~20% of families with multiple individuals with early-onset breast/ovarian cancer. Extensive searches for additional highly penetrant genes or alternative mutational mechanisms altering BRCA1/2 have not explained the missing heritability. For the first time, we report transgenerational epigenetic silencing of BRCA1 due to promoter hypermethylation in two families with breast/ovarian cancer.\n\nMethodsBRCA1 promoter methylation of ten CpG dinucleotides in breast/ovarian cancer families without germline BRCA1/2 pathogenic variants was assessed by pyrosequencing and clonal bisulfite sequencing. RNA and DNA sequencing of BRCA1 from lymphocytes was undertaken to establish allelic expression and the presence of germline variants.\n\nFindingsBRCA1 promoter hypermethylation was identified in two of 49 families with multiple women affected with grade 3 breast/high grade serous ovarian cancer. Soma-wide BRCA1 promoter hypermethylation was confirmed in blood, buccal mucosa and hair follicles. Methylation levels were ~50%, consistent with the silencing of one allele and confirmed by clonal bisulfite sequencing. RNA sequencing revealed allelic loss of BRCA1 expression in both families and this segregated with a novel heterozygous variant c.-107A>T in the BRCA1 5UTR.\n\nInterpretationOur results indicate a novel mechanism for familial breast/ovarian cancer, caused by epigenetic silencing of the BRCA1 promoter, segregating with an in cis 5UTR variant in two independent families. We propose that methylation analyses are indicated in all families affected by early onset breast/ovarian cancer without a BRCA1/2 pathogenic variant.\n\nFundingFunded by Prevent Breast Cancer (GA 12-006 and GA 15-002) and the Manchester NIHR Biomedical Research Centre (IS-BRC-1215-20007).

genetics↗

bioRxiv2017

Observation of histone nuclear import in living cells: implications in the processing of newly synthesised H3.1 & H4

Highlights- Small-molecule-gated tether-and-release system for rapid pulse-chase of nuclear proteins\n- Tracking nuclear import of histone H3.1 and H4 and their incorporation at sites of active replication\n- Tethered H3.1 and H4 are monomeric and do not associate with ASF1, NASP, RbAp46 or HAT1 in the cytosol\n- Importin-{beta} proteins as cytosolic binders of monomeric histones\n\n\nSummaryWe present here a cytosolic tether-and-release system to study the import and dynamics of newly synthesised nuclear proteins. Release is gated by rapamycin-induced recruitment and activation of a viral protease, with cleavage of a peptide linker releasing the tethered cargo. We use this system to investigate nucleo-cytoplasmic divisions in the histone H3.1 & H4 deposition pathway, revealing that, contrary to previous analyses, H3.1 and H4 are predominantly monomeric in the cytosol, and only associate with the core histone chaperoning machinery after translocation to the nucleus. Whilst we do not detect interaction with known H3-H4 chaperones in the cytosol we do detect interaction with a number of importin-{beta} proteins, that may serve a dual import and chaperoning function, preventing aggregation of histones until they are handed-off to the core histone chaperoning machinery in the nucleus.

molecular biology↗