Biology subjects

Smeets, M.

Publications and source records attributed to Smeets, M..

4 recordsLinked to original sources

bioRxiv2025

ADAR1p150 RNA binding, independent of A-to-I RNA editing, buffers immunogenicity from the tonic type I IFN induced transcriptome in vivo.

ADAR1 edits adenosine to inosine (A-to-I) in double-stranded RNA to prevent MDA5 sensing of cellular transcripts, its key physiological role. However, editing-independent functions of ADAR1 remain poorly understood. Using a series of Adar1 mutant mice rescued by loss of MDA5 and PKR, we investigated isoform-specific, editing-independent roles of ADAR1 in vivo. We found that the cytoplasmic ADAR1p150 isoform is essential for maintaining peripheral T cell numbers and differentiation of hematopoietic stem and progenitor cells (HSPCs). In bone marrow transplants, ADAR1p150 protein, but not its editing activity, was crucial for T cell regeneration and HSPC repopulation, demonstrating a cell-intrinsic function in hematopoiesis. Experiments with IFN{beta}-treatment of purified HSPCs in vitro and in vivo IFNAR1 neutralization revealed hypersensitivity to tonic type I interferon (IFN) in the absence of ADAR1p150. Using cell lines, we demonstrate that type I IFN activates the OAS-RNaseL pathway, leading to cell death. This study shows that tonic type I IFN induces immunogenic cellular RNAs in sterile conditions. ADAR1p150 suppresses immune sensing of self-dsRNAs through both editing-dependent (MDA5) and editing-independent (PKR, OAS-RNaseL) mechanisms. Thus, ADAR1p150 protein levels and activity combine to set the threshold for tolerance to self-derived dsRNAs.

immunology↗

bioRxiv2025

Deletion of KLHDC3, an E3 ubiquitin ligase complex substrate receptor, leads to obesity in mice.

Protein ubiquitination is a critical post-translational modification that regulates protein stability and cellular homeostasis. KLHDC3 is a substrate recognition receptor within the recently identified C-terminal degron-mediated DesCEND ubiquitination pathway. It has been characterised to selectively bind proteins with C-terminal RxxxG motifs, targeting them for degradation. Unlike the well-characterised N-terminal degron pathway, the physiological roles of C-terminal degrons remain poorly understood. To investigate KLHDC3 function in vivo, we generated Klhdc3-deficient (Klhdc3-/-) mice. These mice exhibited sub-Mendelian birth rates and progressive postnatal lethality, with a median survival of 136 days and maximum survival observed of [~]1 year of age. Early growth retardation was apparent, followed by a normalisation of body mass with age. However, as Klhdc3-/- mice aged they developed pronounced obesity at the expense of lean mass, with some individuals reaching fat mass exceeding 50% of total body weight. Combined transcriptomic and proteomic analyses of Klhdc3-/- embryonic fibroblasts revealed significant changes in protein expression with minimal impact on transcript levels, consistent with KLHDC3s role in post-translational regulation. Among the upregulated proteins, HINT1 was identified as a novel KLHDC3 substrate, possessing a C-terminal degron motif. Protein stability assays and immunoblotting confirmed HINT1 as a direct target of KLHDC3. These findings establish a new in vivo physiological role for the DesCEND pathway and highlight KLHDC3 as a key regulator of development, survival, and adiposity in mice.

developmental biology↗

bioRxiv2025

Minute amounts of helicase-deficient truncated RECQL4 are sufficient for DNA replication.

RECQL4 is a member of the RecQ family of helicases, playing essential roles in DNA replication and maintaining genome integrity. Mutations in RECQL4 are linked to severe human diseases, including Rothmund-Thomson Syndrome, RAPIDALINO Syndrome, and Baller-Gerold Syndrome. However, we still do not fully understand its functions and genetic interactions. The role of the ATP-dependent helicase activity in RECQL4 remains controversial. To understand RECQL4s functions further, we conducted a genome-wide forward genetic screen using murine models that closely mimic the RECQL4 mutations found in patients with Rothmund-Thomson syndrome. Our goal was to identify loss-of-function alleles that could rescue the proliferation and viability defects associated with RECQL4 mutation. From our screening we identified the loss of KLHDC3, a substrate-binding subunit of the Cullin-RING ligase (CRL) E3, as the most significant rescue allele. KLHDC3 facilitates the ubiquitin-mediated destruction of proteins with specific C-terminal degron motifs. Its loss normalized cell proliferation and DNA replication rates in cells with mutated RECQL4. Further analysis revealed that the loss of KLHDC3 led to the stabilization of minute levels of a truncated RECQL4 protein. This RECQL4 fragment contained a neo-degron sequence specific for KLHDC3, formed after Cre-mediated recombination of the Recql4fl allele. Although this rescue mechanism does not apply to human RECQL4 mutations, it shows that very low chromatin-bound levels of a truncated RECQL4 protein--comprising only the N-terminal 480 amino acids, including its Sld2-like domain but lacking the ATP-dependent helicase domain and the entire C-terminal portion--are sufficient to support DNA replication in mammalian cells. These results demonstrate that the ATPase activity and helicase domain of RECQL4 are not essential for DNA replication in mammals. Furthermore, our findings suggest that there are unlikely to be monogenic loss-of-function alleles that can rescue RECQL4 mutations. This demonstrates that RECQL4 is an essential and non-redundant regulator of DNA replication and cell viability and that this activity does not require the ATP dependent helicase activity.

cell biology↗

bioRxiv2020

Rothmund-Thomson Syndrome-like RECQL4 truncating mutations cause a haploinsufficient low bone mass phenotype in mice

Rothmund-Thomson Syndrome (RTS) is an autosomal recessive disorder characterized by poikiloderma, sparse or absent hair, and defects in the skeletal system such as bone hypoplasia, short stature, low bone mass, and an increased incidence of osteosarcoma. RTS type 2 patients typically present with germline compound bi-allelic protein-truncating mutations of RECQL4. As existing murine models predominantly employ Recql4 null alleles, we have here attempted to more accurately model the mutational spectrum of RTS by generating mice with patient-mimicking truncating Recql4 mutations. We found that truncating mutations impaired stability and subcellular localization of RECQL4, which translated to a homozygous embryonic lethality and haploinsufficient low bone mass and reduced cortical bone thickness phenotypes. Combination of a truncating mutation with a conditional Recql4 null allele demonstrated that these defects were intrinsic to the osteoblast lineage. However, the truncating mutations did not promote tumorigenesis, even after exposure to irradiation. We also utilized murine Recql4 null cells to assess the impact of a wider range of human RECQL4 mutations using an in vitro complementation assay. We found differential effects of distinct RECQL4 mutations. While some created unstable protein products, others altered subcellular localization of the protein. Interestingly, the severity of the phenotypes correlated with the extent of protein truncation. Collectively, our results reveal that truncating RECQL4 mutations lead to the development of an osteoporosis-like phenotype through defects in early osteoblast progenitors in mice and identify RECQL4 gene dosage as a novel regulator of bone mass.

cancer biology↗