Biology subjects

Mosbach, V.

Publications and source records attributed to Mosbach, V..

3 recordsLinked to original sources

bioRxiv2025

Partial Bypass of Frataxin Deficiency by ISCU M141I Restores Cytosolic and Nuclear FeS Cluster Assembly

Iron-sulfur (Fe-S) clusters are essential cofactors required for the activity of numerous proteins involved in fundamental cellular processes, including DNA replication, metabolism and mitochondrial respiration. In eukaryotes, Fe-S cluster biogenesis is initiated in mitochondria by the ISC machinery, which assembles iron and sulfur, delivered by a cysteine desulfurase, onto the scaffold protein ISCU. Frataxin (FXN), a key regulator of this pathway, enhances Fe-S production by accelerating persulfide transfer to ISCU. FXN is essential in eukaryotes, and its loss results in "petite" phenotype in yeast, senescence in dividing mammalian cells and embryonic lethality in mice. Interestingly, in yeast, a methionine to isoleucine substitution at position 141 of the scaffold protein Isu1 can bypass the requirement of FXN. To test whether this bypass mechanism is conserved in mammals, we introduced the equivalent M141I substitution into the endogenous Iscu gene in murine fibroblasts carrying a conditional Fxn allele using CRISPR-Cas9. We show that the ISCU M141I variant enables cell survival in the absence of FXN, preventing cell cycle arrest and decreasing baseline DNA damage. However, these FXN-null survivor clones exhibit slower proliferation, persistent mitochondrial dysfunction and defective mitochondrial Fe-S cluster proteins. In contrast, nuclear and cytosolic Fe-S proteins are preserved, as is cellular iron homeostasis. Importantly, the ISCU M141I variant delays, but does not fully rescue, embryonic lethality in Fxn-deficient mice. Altogether, our results reveal a previously unrecognized compartment-specific rescue of Fe-S cluster dependent processes by the ISCU M141I variant in mammalian cells, raising for the first time the possibility of compartmental regulation of Fe-S cluster biogenesis.

cell biology↗

bioRxiv2023

TALEN-induced contraction of CTG trinucleotide repeats in myotonic dystrophy type 1 cells

Trinucleotide repeat expansions are the cause of two dozen neurodegenerative and developmental disorders. One of these, myotonic dystrophy type 1 (Steinert disease, or DM1) is due to the expansion of a CTG triplet in the 3 UTR of the DMPK gene. We used highly specific DNA endonucleases to induce a double-strand break in the repeat tract to contract it below pathological length. Expression of a TALE Nuclease (TALEN) in human DM1 cells induced moderate CTG repeat contractions in 27% of the clones analyzed. These clones exhibited large internal deletions within the TALEN, occurring by homologous recombination between internal TALE repeats, inactivating the nuclease, and explaining its reduced efficacy. Taking advantage of the degeneracy of the genetic code, we recoded the TALEN sequence, to decrease internal redundancy and optimize codon usage. The new recoded TALEN showed increased efficacy in DM1 cells, with 68% of clones exhibiting a moderate to large contraction of the CTG repeat tract. In contrast, Staphylococcus aureus Cas9 (SaCas9) was unable to contract the CTG repeat tract. In parallel, we completely sequenced to very high coverage the DM1 genome using the PacBio technology. Several clones in which the TALEN was induced were also totally sequenced. In some of them, length changes of other long CTG repeats were detected, possibly corresponding to off-target effects, all of them in introns or intergenic regions. Repeat contractions were never associated with recombination of flanking markers, suggesting that contractions most probably occur by an intra-allelic mechanism such as single-strand annealing. TALENs should now be considered as a promising gene therapy approach, not only for DM1 but also for many other microsatellite expansion disorders.

genomics↗

bioRxiv2019

Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal rearrangements

Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent [~]2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal rearrangements around the repeat tract. These rearrangements depended on RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50{Delta} strain, whereas they were impaired in a sae2{Delta} mutant, only on the DSB end containing most of the repeat tract. This proved that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end.\n\nIn addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfer with gene conversion too. Altogether, these data show that SpCas9 is probably not a good choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome.

genetics↗