Biology subjects

Van Haute, L.

Publications and source records attributed to Van Haute, L..

5 recordsLinked to original sources

bioRxiv2026

Development of the Mitochondrial Base Editor Analysis Package (MitoBEAP).

For many years, the genetic manipulation of mitochondrial DNA was largely hampered by inefficient delivery of nucleic acids to mitochondria. However, the development of mitoCBEs, such as mitochondrial cytosine base editors (DdCBEs), which catalyse C*G-to-T*A conversions, and more recently, mitoABEs, such as transcription-activator-like effector (TALE)-linked deaminases (TALEDs) enabling A*T-to-G*C conversion, has transformed this field. Generally, mitochondrial base editors exhibit high on-target efficiency and are straightforward to design and use. Nonetheless, unintended off-target effects cannot be overlooked and should be assessed consistently with each experiment, which can be challenging without specialised bioinformatic expertise. Here, we introduce Mitochondrial Base Editor Analysis Package (MitoBEAP), which, to our knowledge, is the first R package specifically designed to analyse next-generation sequencing data from base-edited mtDNA samples. The package facilitates the analysis of potential off-target effects, offers multiple visualisation options, and allows customisation of graphics and thresholds for calculations. As a proof of concept, this study demonstrates how MitoBEAP can be utilised to measure the efficiency of DdCBE treatment targeting human 12S rRNA, as well as to identify potentially harmful off-target conversions across the mtDNA.

bioinformatics↗

bioRxiv2024

Mitochondrial adenine base editing of mouse somatic tissues via adeno-associated viral delivery

The development of adenine base editing in mitochondria, alongside cytidine base editing, has significantly expanded the genome engineering capabilities of the mitochondrial DNA. We tested the recent advancements in adenine base editing technology using optimised TALEs targeting genes Mt-Cytb, Mt-CoII and Mt-Atp6 in mouse cells, and observed successful A:T to G:C conversions within the target windows of each gene. We then used the best-performing Mt-Atp6 pairs for systemic AAV9 delivery to neonatal mice and quantified editing in somatic tissues after 4 weeks and 6 months. Adenine editing was low at 4 weeks and increased only modestly after 6 months, indicating that prolonged exposure alone is insufficient to overcome the limited activity of the editor architectures tested here in vivo. These findings establish the feasibility of AAV-delivered mitochondrial A-to-G editing while defining important limitations that require further optimisation.

bioengineering↗

bioRxiv2021

A late-stage assembly checkpoint of the human mitochondrial ribosome large subunit

The epitranscriptome plays a key regulatory role in cellular processes in health and disease, including ribosome biogenesis. Here, analysis of the human mitochondrial transcriptome shows that 2-O-methylation is limited to residues of the mitoribosomal large subunit (mtLSU) 16S mt-rRNA, modified by MRM1, MRM2, and MRM3. Ablation of MRM2 leads to a severe impairment of the oxidative phosphorylation system, caused by defective mitochondrial translation and accumulation of mtLSU assembly intermediates. Structures of these particles (2.58 [A]) present disordered RNA domains, partial occupancy of bL36m and bound MALSU1:L0R8F8:mtACP anti-association module. Additionally, we present five mtLSU assembly states with different intersubunit interface configurations. Complementation studies demonstrate that the methyltransferase activity of MRM2 is dispensable for mitoribosome biogenesis. The Drosophila melanogaster orthologue, DmMRM2, is an essential gene, with its knock-down leading to developmental arrest. This work identifies a key late-stage quality control step during mtLSU assembly, ultimately contributing to the maintenance of mitochondrial homeostasis.

molecular biology↗

bioRxiv2019

YbeY is required for ribosome small subunit assembly and tRNA processing in human mitochondria

Mitochondria contain their own translation apparatus which enables them to produce the polypeptides encoded in their genome. The mitochondrially-encoded RNA components of the mitochondrial ribosome require various post-transcriptional processing steps. Additional protein factors are required to facilitate the biogenesis of the functional mitoribosome. We have characterised a mitochondrially-localized protein, YbeY, which interacts with the assembling mitoribosome through the small subunit. Loss of YbeY leads to a severe reduction in mitochondrial translation and a loss of cell viability, caused by less accurate mitochondrial mt-tRNASer(AGY) processing from the primary transcript and an accumulation of immature mitochondrial small subunit. Our results suggest that YbeY performs a dual function in mitochondria coupling tRNA processing to mitoribosome biogenesis. Issue SectionNucleic Acid Enzymes O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/874305v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@12f69cborg.highwire.dtl.DTLVardef@1cac774org.highwire.dtl.DTLVardef@1ec99ccorg.highwire.dtl.DTLVardef@652c59_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2019

NSUN2 introduces 5-methylcytosines in mammalian mitochondrial tRNAs

Maintenance and expression of mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. Post-transcriptional modification of mitochondrial RNA has emerged as one of the key regulatory steps of human mitochondrial gene expression. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterised as an RNA methyltransferase that introduces 5-methylcytosine (m5C) in nuclear-encoded tRNAs, mRNAs, microRNA and noncoding RNAs. In these roles, NSUN2 has been associated with cell proliferation and differentiation. Pathogenic variants in NSUN2 have been linked with neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation - knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells - we show that NSUN2 in necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease.

biochemistry↗