Biology subjects

Maestri, S.

Publications and source records attributed to Maestri, S..

8 recordsLinked to original sources

bioRxiv2026

Structure-function dissection of huntingtin exon 1 identifies a PRD-driven modifier of neuronal toxicity in Huntington's disease

The Huntingtin gene (HTT) contains a conserved, yet expandable CAG repeat within exon 1. While the pathogenic expansion in Huntingtons Disease (HD) is well studied, the role of surrounding domains remains unclear. Using genome-edited mini-organoids and neurons, we dissected HTT exon 1 and found species-specific toxicity: the human variant caused more severe deficits than the mouse. Swapping the proline-rich domain (PRD) - the most divergent region - revealed its key role: the mouse PRD mitigated, while the human PRD worsened neuronal phenotypes. Omics profiling showed that pathogenic human exon 1 induced broad protein dysregulation, largely reversed by mouse PRD replacement. Bioinformatics implicated the actin cytoskeleton and transcriptional coactivator MKL2/MRTFB. We validated MKL2/MRTFB dysregulation in HD models and showed that restoring its expression rescued neuronal abnormalities. These findings highlight the PRDs contribution to HD toxicity and point to MKL2/MRTFB and the cytoskeleton as candidate mediators.

neuroscience↗

bioRxiv2026

Long-read cross-platform validation reveals novel repeat features in myotonic dystrophy type 2

The broader application of long-read sequencing (LRS) for repeat expansion characterization in myotonic dystrophy type 2 (DM2) and other repeat expansion disorders (REDs) remains limited by the lack of systematic validation and benchmarking of sequencing results and bioinformatic workflows. Here, we performed an orthogonal cross-platform validation of previously generated Oxford Nanopore Technologies (ONT) data by sequencing the same DNA samples with Pacific Biosciences (PacBio) HiFi following amplification-free targeted enrichment in a cohort of 8 DM2 patients. Despite substantial differences in sequencing chemistry and coverage, the two platforms showed high concordance in repeat size estimation, somatic mosaicism, and repeat architecture. This validation confirmed the presence of the (TCTG)n motif and enabled the identification of a previously unreported (CCCG)n motif at the 3' end of expanded alleles, further highlighting the structural complexity of the CNBP expansion. Through this analysis, we also established a bioinformatic workflow that improved ONT-based repeat characterization, addressing limitations in motif resolution and enabling more accurate analysis of CNBP expansions. Overall, this study provides a validated framework for LRS-based CNBP repeat analysis, supporting the integration of these technologies into routine molecular investigation for DM2 and other REDs.

genomics↗

bioRxiv2024

A comprehensive survey of RNA modifications in a human transcriptome

RNA modifications are critical for transcript function and regulation, yet detecting these modifications transcriptome-wide and at isoform-level resolution remains technically challenging. Here, we present a robust in vitro transcription (IVT)-based strategy coupled with direct RNA nanopore sequencing (dRNA-seq) to detect a wide spectrum of endogenous RNA modifications without requiring prior knowledge of modification types or modification-specific biochemical assays. We generated an IVT modification-free reference transcriptome from K562 cells and used Nanocompore to compare it to the native RNA. We detected 26,619 modification sites across 2,520 isoforms from 1,766 genes. We used motif and annotation-based inference to identify at least eight distinct RNA modifications, with m6A and m5C being the most prevalent. Importantly, we uncovered non-random co-occurrence of m6A and m5C on both the same transcripts and a subset of the same molecules, suggesting potential combinatorial regulation. Furthermore, RNA modification patterns were often isoform-specific, pointing to a link between the epitranscriptome and alternative splicing. This approach also revealed previously underexplored modification patterns in mitochondrial mRNAs, suggesting broader regulatory complexity than previously appreciated. Our study provides a survey of RNA modifications across the transcriptome, demonstrating the utility of in vitro transcription coupled with direct RNA nanopore sequencing to simultaneously detect multiple modifications without the need for additional independent biochemical assays, enabling future investigations into the dynamics, coordination, and functional consequences of RNA modifications across different biological contexts.

genomics↗

bioRxiv2023

Cholesterol modulates the human FPN1 iron export function in plasma membrane liquid-ordered microdomains

Ferroportin 1 (FPN1) is the only known mammalian iron efflux transporter. This multi-pass membrane protein, which adopts the Major Facilitator Superfamily fold, is tightly controlled by serum hepcidin to assure maintenance of adequate cellular and systemic iron levels. Earlier studies have shown that cholesterol-lowering drugs can reduce FPN1 expression in liquid-ordered plasma membrane microdomains and its sensitivity to hepcidin. However, the molecular mechanism by which cholesterol depletion regulates the localization of FPN1 at the cell surface remains unknown. In biochemical experiments, we show that cholesterol depletion reduces the iron export function of FPN1. Repletion with cholesterol restores FPN1 activity. This is not observed with the diastereoisomer epicholesterol, suggesting a direct interaction between cholesterol and FPN1. Consistent with this, we demonstrate that mutants affecting the key tyrosine residues of three cholesterol-recognition amino acid consensus (CRAC/CARC) motifs have a negative impact on FPN1 activity, in manner that also decreases its abundance in ordered plasma membrane microdomains. A complementary structural analysis allows us to focus on a conserved CARC motif (CARC-1) located in a deep hydrophobic groove between transmembrane helices 1 and 5. Molecular docking suggests that this groove is well suited to cholesterol binding. All these findings indicate that the interaction between FPN1 and cholesterol is of major importance for the localization of FPN1 in ordered microdomains of the plasma membrane, which is necessary for its optimal activity, and so its responsiveness to hepcidin.

biochemistry↗

bioRxiv2022

Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing

Myotonic dystrophy type 2 (DM2) is caused by CCTG repeat expansions in the CNBP gene, comprising 75 to >11,000 units and featuring extensive mosaicism, making it challenging to sequence fully-expanded alleles. To overcome these limitations, we used PCR-free Cas9-mediated nanopore sequencing to characterize CNBP repeat expansions at the single-nucleotide level in nine DM2 patients. The length of normal and expanded alleles can be assessed precisely using this strategy, agreeing with traditional methods, and revealing the degree of mosaicism. We also sequenced an entire [~]50-kbp expansion, which has not been achieved previously for DM2 or any other repeat-expansion disorders. Our approach precisely counted the repeats and identified the repeat pattern for both short interrupted and uninterrupted alleles. Interestingly, in the expanded alleles, only two DM2 samples featured the expected pure CCTG repeat pattern, while the other seven presented also TCTG blocks at the 3' end, which have not been reported before in DM2 patients, but confirmed hereby with orthogonal methods. The demonstrated approach simultaneously determines repeat length, structure/motif and the extent of somatic mosaicism, promising to improve the molecular diagnosis of DM2 and achieve more accurate genotype- phenotype correlations for the better stratification of DM2 patients in clinical trials.

genomics↗

bioRxiv2020

Genomic structural variation in Nebbiolo grapevines at the individual, clonal and cultivar levels

Structural Variants (SVs) are a widely unexplored source of genetic variation, both due to methodological limitations and because they are generally associated to deleterious effects. However, with the advent of long-range genomic platforms, it has become easier to directly detect SVs. In the same direction, clonally propagated crops provide a unique opportunity to study SVs, offering a suitable genomic environment for their accumulation in heterozygosis. In particular, it has been reported that SVs generate drastic levels of heterozygosity in grapevines. Nebbiolo (Vitis vinifera L.) is a grapevine cultivar typical of north-western Italy, appreciated for its use in producing high-quality red wines. Here, we aimed to analyze the frequency of SVs in Nebbiolo, at three different organizational levels. For this purpose, we generated genomic data based on long-reads, linked-reads and optical mapping. We assembled a reference genome for this cultivar and compared two different clones, including V. vinifera reference genome (PN40024) in our comparisons. Our results indicate that SVs differentially occurring between Nebbiolo clones might be rare, while SVs differentiating haplotypes of the same individual are as abundant as those that occur differentially between cultivars.

genomics↗

bioRxiv2020

RNA secondary structure mediated by Alu insertion as a novel disease-causing mechanism

We previously identified a homozygous Alu insertion variant (Alu_Ins) in the 3-UTR of the SPINK1 gene as the cause of a novel pediatric disease. Although we established that Alu_Ins leads to the complete loss of SPINK1 mRNA expression, the precise mechanisms remained elusive. Here we aimed to elucidate these mechanisms through a hypothesis-driven approach. Initially, we speculated that Alu_Ins could independently disrupt mRNA 3 end formation and/or affect other post-transcriptional processes such as nuclear export and translation, due to its particular location. However, the presence of Alu_Ins in the 3-UTR resulted in only an ~50% reduction in luciferase reporter activity compared to the wild-type, suggesting the involvement of additional mechanisms. Using RepeatMasker, we identified two Alu elements within SPINK1s third intron, both of which resided in an orientation opposite to that of Alu_Ins. Through RNAfold predictions and full-length gene expression assays designed to examine orientation-dependent interactions between Alu repeats, we present evidence linking the detrimental effect of Alu_Ins to extensive double-stranded RNA structures formed between Alu_Ins and pre-existing intronic Alu sequences. Our results reveal a novel pathogenetic mechanism involving an Alu insertion, highlighting the importance of considering interactions between new and pre-existing Alu elements in inverted orientations within disease-associated genes.

genetics↗

bioRxiv2019

A rapid and accurate MinION-based workflow for tracking species biodiversity in the field

Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION platform. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure reliable and robust performance under suboptimal field conditions without increasing costs. Here we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 500 reads per sample. ONTrack-derived consensus barcodes have high accuracy, ranging from 99,8% to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions.

genomics↗