Paula Armina V. Buco

RECQL4, a RecQ family helicase, is essential for DNA replication and genome stability. Mutations in RECQL4 cause severe human disorders yet we do not fully understand its functions, particularly regarding ATP-dependent helicase activity. To understand RECQL4’s functions further, we performed a genome-wide forward genetic screen using a murine model harbouring patient-like RECQL4 mutations. We identify KLHDC3, a substrate-binding subunit of the Cullin-RING ligase E3 complex, loss as the most significant rescue allele. KLHDC3 loss restores proliferation and replication in RECQL4-deficient cells by stabilizing trace levels of a truncated RECQL4 fragment containing the N-terminal 480 amino acids, lacking the helicase and C-terminal regions. This RECQL4 fragment forms after Cre-mediated recombination of the Recql4 ^fl allele and contains a neo-degron sequence specific for KLHDC3. Although this mechanism does not apply to human mutations, it demonstrates that minimal RECQL4 levels, without any ATPase domain/activity, are sufficient to support DNA replication. This demonstrates that RECQL4 is an essential and non-redundant regulator of DNA replication and cell viability and that this activity does not require its ATP-dependent helicase activity.

Background: Protein ubiquitination is a key post-translational modification that governs protein stability and cellular homeostasis. KLHDC3 is a substrate recognition receptor in the recently identified C-terminal degron-mediated DesCEND ubiquitination pathway. It selectively binds proteins with C-terminal RxxxG motifs, targeting them for degradation. While N-terminal degron pathways are well-characterized, the physiological roles of C-terminal degrons remain poorly understood. To explore KLHDC3’s function in a physiological context, we generated mice deficient in the Klhdc3 gene. Results: Klhdc3 -deficient mice exhibited sub-Mendelian birth rates and progressive postnatal lethality, with a median survival of 136 days and a maximum lifespan of approximately one year. Surviving mice showed early growth retardation followed by normalization of body mass, and later developed pronounced obesity, with some individuals reaching fat mass levels exceeding 50% of total body weight. Transcriptomic and proteomic analyses of Klhdc3 ^−/− embryonic fibroblasts revealed significant changes in protein expression with minimal alterations in transcript levels, consistent with KLHDC3’s role in post-translational regulation. Among the upregulated proteins, HINT1 was identified as a novel KLHDC3 substrate containing a C-terminal degron motif. Protein stability assays and immunoblotting confirmed HINT1 as a candidate target of KLHDC3. Conclusions: This study establishes a physiological role for the DesCEND pathway in vivo and identifies KLHDC3 as a critical regulator of development, survival, and adiposity in mice. The identification of HINT1 as a putative KLHDC3 substrate expands our understanding of C-terminal degron-mediated protein regulation and suggests broader implications for developmental and metabolic processes.

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 KLHDC3’s 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.

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 RECQL4’s 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 Recql4 ^fl 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.

The Kelch protein superfamily is an evolutionary conserved family containing 63 alternate protein coding members. The superfamily is split into three subfamilies: Kelch like (KLHL), Kelch‐repeat and bric‐a‐bracs (BTB) domain containing (KBTBD) and Kelch domain containing protein (KLHDC). The KLHDC subfamily is one of the smallest within the Kelch superfamily, containing 10 primary members. There is little known about the structures and functions of the subfamily; however, they are thought to be involved in several cellular and molecular processes. Recently, there have been significant structural and biochemical advances for KLHDC2, which has aided our understanding of other KLHDC family members. Furthermore, small molecules directly targeting KLHDC2 have been identified, which act as tools for targeted protein degradation. This review utilises this information, in conjunction with a thorough exploration of the structural aspects and potential biological functions to summarise the relationship between KLHDCs and human disease.