Biology subjects

Stone, M. D.

Publications and source records attributed to Stone, M. D..

6 recordsLinked to original sources

bioRxiv2023

Rescue of blood coagulation Factor VIII exon-16 mis-splicing by antisense oligonucleotides

The human Factor VIII (F8) protein is essential for the blood coagulation cascade and specific F8 mutations cause the rare bleeding disorder Hemophilia A (HA). Here, we investigated the impact of HA-causing single-nucleotide mutations on F8 pre-mRNA splicing. We found that 14/97 ([~]14.4%) coding sequence mutations tested in our study induced exon skipping. Splicing patterns of 4/11 ([~]36.4%) F8 exons tested were especially sensitive to the presence of common disease-causing mutations. RNA-chemical probing analyses revealed a three-way junction structure at the 3' end of intron 15 (TWJ-3-15). TWJ-3-15 sequesters the polypyrimidine tract, a key determinant of 3' splice site strength. Using exon-16 of the F8 gene as a model, we designed specific antisense oligonucleotides (ASOs) that target TWJ-3-15 and identified three that promote the splicing of F8 exon-16. Interaction of TWJ-3-15 with ASOs increases accessibility of the polypyrimidine tract and inhibits the binding of hnRNPA1-dependent splicing silencing factors. Moreover, ASOs targeting TWJ-3-15 rescue diverse splicing-sensitive HA-causing mutations, most of which are distal to the 3 splice site being impacted. The TWJ-3-15 structure and its effect on mRNA splicing provide a model for HA etiology in patients harboring specific F8 mutations and provide a framework for precision RNA-based HA therapies.

molecular biology↗

bioRxiv2021

Multiplexed single-molecule analysis of human telomerase synthesizing DNA

Genomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we developed a real-time single-molecule RNA sequencing approach that visualizes telomerase catalysis and structural dynamics at single-nucleotide resolution using FRET and zero-mode waveguides. The method permits direct detection of dynamic steps and structural states throughout the telomerase catalytic cycle and can be generalized to other nucleic acid polymerase systems.

biophysics↗

bioRxiv2021

Single-molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA

Telomeres are essential chromosome end capping structures that safeguard the genome from dangerous DNA processing events. DNA strand invasion occurs during vital transactions at telomeres, including telomere length maintenance by the alternative lengthening of telomeres (ALT) pathway. During telomeric strand invasion, a single stranded guanine-rich (G-rich) DNA invades at a complimentary duplex telomere repeat sequence forming a displacement loop (D-loop) in which the displaced DNA consists of the same G-rich sequence as the invading single stranded DNA. Single stranded G-rich telomeric DNA readily folds into stable, compact, structures called G-quadruplexes (GQ) in vitro, and is anticipated to form within the context of a D-loop; however, evidence supporting this hypothesis is lacking. Here we report a magnetic tweezers assay that permits the controlled formation of telomeric D-loops (TDLs) within uninterrupted duplex human telomere DNA molecules of physiologically relevant lengths. Our results are consistent with a model wherein the displaced single stranded DNA of a TDL folds into a GQ. This study provides new insight into telomere structure and establishes a framework for development of novel therapeutics designed to target GQs at telomeres in cancer cells.

biophysics↗

bioRxiv2019

Folding heterogeneity in the essential human telomerase RNA three-way junction

Telomeres safeguard the genome by suppressing illicit DNA damage responses at chromosome termini. In order to compensate for incomplete DNA replication at telomeres, most continually dividing cells, including many cancers, express the telomerase ribonucleoprotein (RNP) complex. Telomerase maintains telomere length by catalyzing de novo synthesis of short DNA repeats using an internal telomerase RNA (TR) template. TRs from diverse species harbor structurally conserved domains that contribute to RNP biogenesis and function. In vertebrate TRs, the conserved regions 4 and 5 (CR4/5) fold into a three-way junction (3WJ) that binds directly to the telomerase catalytic protein subunit and is required for telomerase function. We have analyzed the structural properties of the human TR (hTR) CR4/5 domain using a combination of in vitro chemical mapping, endogenous RNP assembly assays, and single-molecule structural analysis. Our data suggest that a functionally essential stem loop within CR4/5 is not stably folded in the absence of the telomerase reverse transcriptase protein subunit in vitro. Rather, the hTR CR4/5 domain adopts a heterogeneous ensemble of conformations. RNA structural engineering intended to bias the folding landscape of the hTR CR4/5 demonstrates that a stably folded 3WJ motif is necessary but not sufficient to promote assembly of a functional RNP complex. Finally, single-molecule measurements on the hTR CR4/5 domain show that RNP assembly selects for a conformation that is not the major population in the heterogeneous free RNA ensemble, suggesting that non-canonical hTR folds may be required during telomerase biogenesis.

biochemistry↗

bioRxiv2019

Reevaluation of the RNA binding properties of the Tetrahymena thermophila telomerase reverse transcriptase N-terminal domain

Telomerase restores chromosome-capping telomeric repeats lost with each round of genome replication by DNA-templated DNA polymerases. The telomerase reverse transcriptase (TERT) N-terminal (TEN) domain is a peripheral, telomerase-specific, processivity-stimulatory addition to more conserved domains that encircle the active site cavity. Reports of ciliate, yeast, and mammalian telomerase TEN domain associations with the telomerase RNA subunit (TR) describe low affinity interactions of uncertain specificity. Unfortunately two cryo-EM structures of synthesis-paused telomerase holoenzymes lack sufficient resolution to discriminate molecular specificity of possible TR contact(s) with the TEN domain, and there is no assigned density for the TEN domain termini implicated in RNA binding. Furthermore, studies have revealed alternative secondary structures for TR regions that could interact with TERT prior to TR folding into active conformation. Informed by recent advances in knowledge of telomerase structure, we returned to the investigation of Tetrahymena thermophila TERT TEN domain interaction with TR. Instead of finding specificity for a particular TR sequence or structure, we discovered that the tagged TEN domain used in previous characterizations has trace contamination with a bacterial RNA-interacting protein not detectable by SDS-PAGE. By resolving this interference, we show that the TEN domain binds RNAs dependent on RNA length rather than sequence.

biochemistry↗

bioRxiv2018

Telomere DNA G-quadruplex folding within actively extending human telomerase

Telomerase maintains telomere length by reverse transcribing short G-rich DNA repeat sequences from its internal RNA template. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using K+ versus Li+ salts yield changes in DNA product profiles consistent with formation of GQ structure within the telomerase-DNA complex. Single-molecule FRET experiments reveal complex DNA structural dynamics during real-time catalysis, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we fit telomerase time series data to a global kinetic model that converges to a unique set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis and provide a detailed characterization of GQ modulation of polymerase function.\n\nSIGNIFICANCETelomeres protect the ends of linear chromosomes from illicit DNA processing events that can threaten genome stability. Telomere structure is built upon repetitive G-rich DNA repeat sequences that have the ability to fold into stable secondary structures called G-quadruplexes (GQs). In rapidly dividing cells, including the majority of human cancers, telomeres are maintained by the specialized telomerase enzyme. Thus, telomerase and its telomere DNA substrates represent important targets for developing novel cancer drugs. In this work, we provide evidence for GQ folding within the newly synthesized DNA product of an actively extending telomerase enzyme. Our results highlight the delicate interplay between the structural properties of telomere DNA and telomerase function.

biochemistry↗