Biology subjects

Lienhard, M.

Publications and source records attributed to Lienhard, M..

4 recordsLinked to original sources

bioRxiv2024

Long-read transcriptome sequencing of CLL and MDS patients uncovers molecular effects of SF3B1 mutations

BackgroundMutations in splicing factor 3B subunit 1 (SF3B1) frequently occur in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS). These mutations have a different effect on the disease prognosis with beneficial effect in MDS and worse prognosis in CLL patients. A full-length transcriptome approach can expand our knowledge on SF3B1 mutation effects on RNA splicing and its contribution to patient survival and treatment options. ResultsWe applied long-read transcriptome sequencing to 44 MDS and CLL patients with and without SF3B1 mutations and found > 60% of novel isoforms. Splicing alterations were largely shared between cancer types and specifically affected the usage of introns and 3 splice sites. Our data highlighted a constrained window at canonical 3 splice sites in which dynamic splice site switches occurred in SF3B1-mutated patients. Using transcriptome-wide RNA binding maps and molecular dynamics simulations, we showed multimodal SF3B1 binding at 3 splice sites and predicted reduced RNA binding at the second binding pocket of SF3B1K700E. ConclusionsOur work presents the hitherto most complete long-read transcriptome sequencing study in CLL and MDS and provides a resource to study aberrant splicing in cancer. Moreover, we showed that different disease prognosis results most likely from the different cell types expanded during cancerogenesis rather than different mechanism of action of the mutated SF3B1. These results have important implications for understanding the role of SF3B1 mutations in hematological malignancies and other related diseases. HighlightsO_LILong-read transcriptome sequencing data enables the identification of > 60% of novel isoforms in the transcriptomes of CLL and MDS patients and isogenic cell lines. C_LIO_LISF3B1 mutations trigger common splicing alterations upon SF3B1 mutations across patient cohorts, most frequently decreased intron retention and increased alternative 3 splice site usage. C_LIO_LIMutation effect depends on alternative 3 splice site and branch point positioning that coincide with bimodal SF3B1 binding at these sites C_LIO_LIMolecular dynamics simulations predict reduced binding of SF3B1K700E to mRNA at the second binding pocket harboring the polypyrimidine tract. C_LI

cancer biology↗

bioRxiv2023

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification

AbstractThe Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

genomics↗

bioRxiv2021

Neurofibromin 1 controls metabolic balance and Notch-dependent quiescence of juvenile myogenic progenitors

Patients affected by neurofibromatosis type 1 (NF1) frequently show muscle weakness with unknown etiology. Here we show that Neurofibromin-1 (Nf1) is not required in muscle fibers, but specifically in early postnatal myogenic progenitors (MPs), where Nf1 loss led to cell cycle exit and differentiation blockade, depleting the MP pool resulting in reduced myonuclear accrual as well as reduced muscle stem cell numbers. This was caused by precocious induction of stem cell quiescence coupled to metabolic reprogramming of MPs impinging on glycolytic shutdown, which was conserved in muscle fibers. We show that a Mek/Erk/NOS pathway hypersensitizes Nf1-deficient MPs to Notch signaling, consequently, early postnatal Notch pathway inhibition ameliorated premature quiescence, metabolic reprogramming and muscle growth. This reveals an unexpected role of Ras/Mek/Erk signaling supporting postnatal MP quiescence in concert with Notch signaling, which is controlled by Nf1 safeguarding coordinated muscle growth and muscle stem cell pool establishment. Furthermore, our data suggest transmission of metabolic reprogramming across cellular differentiation, affecting fiber metabolism and function in NF1.

cell biology↗

bioRxiv2021

Long-read transcriptome sequencing analysis with IsoTools

Long-read transcriptome sequencing (LRTS) holds the promise to boost our understanding of alternative splicing. Recent advances in accuracy and throughput have diminished the major limitations and enabled the direct quantification of isoforms. Considering the complexity of the data and the broad range of potential applications, it is clear that highly flexible, accurate analysis tools are crucial. Here, we present IsoTools, a comprehensive Python-based analysis package, for the improvement of alternative and differential splicing analysis. Iso-Tools provides a comprehensive data structure that integrates genomic information from LRTS transcripts together with the reference annotation, and enables broad functionality to quality control, visualize and analyze the data. Additionally, we implemented a graph-based method for the identification of alternative splicing events and a statistical approach based on the beta binomial distribution for the detection of differential events. To demonstrate our methods, we generated PacBio Iso-Seq data of human hepatocytes treated with the HDAC inhibitor valproic acid, a compound known to induce widespread transcriptional changes. Contrasted with short read RNA-Seq of the same samples, this analysis shows that LRTS provides valuable additional insights for a better understanding of alternative splicing, in particular with respect to complex novel and differential splicing events. IsoTools is made available for the community along with extensive documentation at https://github.com/MatthiasLienhard/isotools.

genetics↗