A SAM-key domain required for enzymatic activity of the Fun30 nucleosome remodeler.

Fun30 is the prototype of the Fun30-SMARCAD1-ETL subfamily of nucleosome remodelers involved in DNA repair and gene silencing. These proteins appear to act as single-subunit nucleosome remodelers, but their molecular mechanisms are, at this point, poorly understood. Using multiple sequence alignment and structure prediction, we identify an evolutionarily conserved domain that is modeled to contain a SAM-like fold with one long, protruding helix, which we term SAM-key. Deletion of the SAM-key within budding yeast Fun30 leads to a defect in DNA repair and gene silencing similar to that of the fun30Δ mutant. In vitro, Fun30 protein lacking the SAM-key is able to bind nucleosomes but is deficient in DNA-stimulated ATPase activity and nucleosome sliding and eviction. A structural model based on AlphaFold2 prediction and verified by crosslinking-MS indicates an interaction of the long SAM-key helix with protrusion I, a subdomain located between the two ATPase lobes that is critical for control of enzymatic activity. Mutation of the interaction interface phenocopies the domain deletion with a lack of DNA-stimulated ATPase activation and a nucleosome-remodeling defect, thereby confirming a role of the SAM-key helix in regulating ATPase activity. Our data thereby demonstrate a central role of the SAM-key domain in mediating the activation of Fun30 catalytic activity, thus highlighting the importance of allosteric activation for this class of enzymes.

RNA-DNA hybrids prevent resection at dysfunctional telomeres.

At critically short telomeres, stabilized TERRA RNA-DNA hybrids drive homology-directed repair (HDR) to delay replicative senescence. However, even at long- and intermediate-length telomeres, not subject to HDR, transient TERRA RNA-DNA hybrids form, suggestive of additional roles. We report that telomeric RNA-DNA hybrids prevent Exo1-mediated resection when telomeres become non-functional. We used the well-characterized cdc13-1 allele, where telomere resection can be induced in a temperature-dependent manner, to demonstrate that ssDNA generation at telomeres is either prevented or augmented when RNA-DNA hybrids are stabilized or destabilized, respectively. The viability of cdc13-1 cells is affected by the presence or absence of hybrids accordingly. Telomeric hybrids do not affect the shortening rate of bulk telomeres. We suggest that TERRA hybrids require dynamic regulation to drive HDR at short telomeres; hybrid presence may initiate HDR through replication stress, whereby their removal allows strand resection.

RNase H1 and H2 Are Differentially Regulated to Process RNA-DNA Hybrids.

RNA-DNA hybrids are tightly regulated to ensure genome integrity. The RNase H enzymes RNase H1 and H2 contribute to chromosomal stability through the removal of RNA-DNA hybrids. Loss of RNase H2 function is implicated in human diseases of the nervous system and cancer. To better understand RNA-DNA hybrid dynamics, we focused on elucidating the regulation of the RNase H enzymes themselves. Using yeast as a model system, we demonstrate that RNase H1 and H2 are controlled in different manners. RNase H2 has strict cell cycle requirements, in that it has an essential function in G2/M for both R-loop processing and ribonucleotide excision repair. RNase H1, however, can function independently of the cell cycle to remove R-loops and appears to become activated in response to high R-loop loads. These results provide us with a more complete understanding of how and when RNA-DNA hybrids are acted upon by the RNase H enzymes.

The role of Xist-mediated Polycomb recruitment in the initiation of X-chromosome inactivation.

Xist RNA has been established as the master regulator of X-chromosome inactivation (XCI) in female eutherian mammals, but its mechanism of action remains unclear. By creating novel Xist-inducible mutants at the endogenous locus in male mouse embryonic stem (ES) cells, we dissect the role of the conserved A-B-C-F repeats in the initiation of XCI. We find that transcriptional silencing can be largely uncoupled from Polycomb repressive complex 1 and complex 2 (PRC1/2) recruitment, which requires B and C repeats. Xist ΔB+C RNA specifically loses interaction with PCGF3/5 subunits of PRC1, while binding of other Xist partners is largely unaffected. However, a slight relaxation of transcriptional silencing in Xist ΔB+C indicates a role for PRC1/2 proteins in early stabilization of gene repression. Distinct modules within the Xist RNA are therefore involved in the convergence of independent chromatin modification and gene repression pathways. In this context, Polycomb recruitment seems to be of moderate relevance in the initiation of silencing.

Short (16-mer) locked nucleic acid splice-switching oligonucleotides restore dystrophin production in Duchenne Muscular Dystrophy myotubes.

Splice-switching antisense oligonucleotides (SSOs) offer great potential for RNA-targeting therapies, and two SSO drugs have been recently approved for treating Duchenne Muscular Dystrophy (DMD) and Spinal Muscular Atrophy (SMA). Despite promising results, new developments are still needed for more efficient chemistries and delivery systems. Locked nucleic acid (LNA) is a chemically modified nucleic acid that presents several attractive properties, such as high melting temperature when bound to RNA, potent biological activity, high stability and low toxicity in vivo. Here, we designed a series of LNA-based SSOs complementary to two sequences of the human dystrophin exon 51 that are most evolutionary conserved and evaluated their ability to induce exon skipping upon transfection into myoblasts derived from a DMD patient. We show that 16-mers with 60% of LNA modification efficiently induce exon skipping and restore synthesis of a truncated dystrophin isoform that localizes to the plasma membrane of patient-derived myotubes differentiated in culture. In sum, this study underscores the value of short LNA-modified SSOs for therapeutic applications.