POT1 recruits and regulates CST-Polα/primase at human telomeres.

Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.

Human vascular organoids with a mosaic AKT1 mutation recapitulate Proteus syndrome.

Vascular malformation, a key clinical phenotype of Proteus syndrome, lacks effective models for pathophysiological study and drug development due to limited patient sample access. To bridge this gap, we built a human vascular organoid model replicating Proteus syndrome's vasculature. Using CRISPR/Cas9 genome editing and gene overexpression, we created induced pluripotent stem cells (iPSCs) embodying the Proteus syndrome-specific AKTE17K point mutation for organoid generation. Our findings revealed that AKT overactivation in these organoids resulted in smaller sizes yet increased vascular connectivity, although with less stable connections. This could be due to the significant vasculogenesis induced by AKT overactivation. This phenomenon likely stems from boosted vasculogenesis triggered by AKT overactivation, leading to increased vascular sprouting. Additionally, a notable increase in dysfunctional PDGFRß+ mural cells, impaired in matrix secretion, was observed in these AKT-overactivated organoids. The application of AKT inhibitors (ARQ092, AZD5363, or GDC0068) reversed the vascular malformations; the inhibitors' effectiveness was directly linked to reduced connectivity in the organoids. In summary, our study introduces an innovative in vitro model combining organoid technology and gene editing to explore vascular pathophysiology in Proteus syndrome. This model not only simulates Proteus syndrome vasculature but also holds potential for mimicking vasculatures of other genetically driven diseases. It represents an advance in drug development for rare diseases, historically plagued by slow progress.

CST-Polα/Primase: the second telomere maintenance machine.

It has been known for decades that telomerase extends the 3' end of linear eukaryotic chromosomes and dictates the telomeric repeat sequence based on the template in its RNA. However, telomerase does not mitigate sequence loss at the 5' ends of chromosomes, which results from lagging strand DNA synthesis and nucleolytic processing. Therefore, a second enzyme is needed to keep telomeres intact: DNA polymerase α/Primase bound to Ctc1-Stn1-Ten1 (CST). CST-Polα/Primase maintains telomeres through a fill-in reaction that replenishes the lost sequences at the 5' ends. CST not only serves to maintain telomeres but also determines their length by keeping telomerase from overelongating telomeres. Here we discuss recent data on the evolution, structure, function, and recruitment of mammalian CST-Polα/Primase, highlighting the role of this complex and telomere length control in human disease.

POT1 recruits and regulates CST-Polα/Primase at human telomeres.

Telomere maintenance requires extension of the G-rich telomeric repeat strand by telomerase and fill-in synthesis of the C-rich strand by Polα/Primase. Telomeric Polα/Primase is bound to Ctc1-Stn1-Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/Primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Phosphorylation of POT1 is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/Primase in an inactive auto-inhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/Primase into an active state that completes telomere replication through fill-in synthesis.

Focused ultrasound-mediated brain genome editing.

Gene editing in the mammalian brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain.

CST/Polα/primase-mediated fill-in synthesis at DSBs.

DNA double-strand breaks (DSBs) pose a major threat to the genome, so the efficient repair of such breaks is essential. DSB processing and repair is affected by 53BP1, which has been proposed to determine repair pathway choice and/or promote repair fidelity. 53BP1 and its downstream effectors, RIF1 and shieldin, control 3' overhang length, and the mechanism has been a topic of intensive research. Here, we highlight recent evidence that 3' overhang control by 53BP1 occurs through fill-in synthesis of resected DSBs by CST/Polα/primase. We focus on the crucial role of fill-in synthesis in BRCA1-deficient cells treated with PARPi and discuss the notion of fill-in synthesis in other specialized settings and in the repair of random DSBs. We argue that - in addition to other determinants - repair pathway choice may be influenced by the DNA sequence at the break which can impact CST binding and therefore the deployment of Polα/primase fill-in.

Cryo-EM structure of the human CST-Polα/primase complex in a recruitment state.

The CST-Polα/primase complex is essential for telomere maintenance and functions to counteract resection at double-strand breaks. We report a 4.6-Å resolution cryo-EM structure of human CST-Polα/primase, captured prior to catalysis in a recruitment state stabilized by chemical cross-linking. Our structure reveals an evolutionarily conserved interaction between the C-terminal domain of the catalytic POLA1 subunit and an N-terminal expansion in metazoan CTC1. Cross-linking mass spectrometry and negative-stain EM analysis provide insight into CST binding by the flexible POLA1 N-terminus. Finally, Coats plus syndrome disease mutations previously characterized to disrupt formation of the CST-Polα/primase complex map to protein-protein interfaces observed in the recruitment state. Together, our results shed light on the architecture and stoichiometry of the metazoan fill-in machinery.

The evolution of metazoan shelterin.

The mammalian telomeric shelterin complex-comprised of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-blocks the DNA damage response at chromosome ends and interacts with telomerase and the CST complex to regulate telomere length. The evolutionary origins of shelterin are unclear, partly because unicellular organisms have distinct telomeric proteins. Here, we describe the evolution of metazoan shelterin, showing that TRF1 emerged in vertebrates upon duplication of a TRF2-like ancestor. TRF1 and TRF2 diverged rapidly during vertebrate evolution through the acquisition of new domains and interacting factors. Vertebrate shelterin is also distinguished by the presence of an HJRL domain in the split C-terminal OB fold of POT1, whereas invertebrate POT1s carry inserts of variable nature. Importantly, the data reveal that, apart from the primate and rodent POT1 orthologs, all metazoan POT1s are predicted to have a fourth OB fold at their N termini. Therefore, we propose that POT1 arose from a four-OB-fold ancestor, most likely an RPA70-like protein. This analysis provides insights into the biology of shelterin and its evolution from ancestral telomeric DNA-binding proteins.

Structural and functional analysis of mRNA export regulation by the nuclear pore complex.

The nuclear pore complex (NPC) controls the passage of macromolecules between the nucleus and cytoplasm, but how the NPC directly participates in macromolecular transport remains poorly understood. In the final step of mRNA export, the DEAD-box helicase DDX19 is activated by the nucleoporins Gle1, Nup214, and Nup42 to remove Nxf1•Nxt1 from mRNAs. Here, we report crystal structures of Gle1•Nup42 from three organisms that reveal an evolutionarily conserved binding mode. Biochemical reconstitution of the DDX19 ATPase cycle establishes that human DDX19 activation does not require IP6, unlike its fungal homologs, and that Gle1 stability affects DDX19 activation. Mutations linked to motor neuron diseases cause decreased Gle1 thermostability, implicating nucleoporin misfolding as a disease determinant. Crystal structures of human Gle1•Nup42•DDX19 reveal the structural rearrangements in DDX19 from an auto-inhibited to an RNA-binding competent state. Together, our results provide the foundation for further mechanistic analyses of mRNA export in humans.