Biology subjects

Wojciechowska, M.

Publications and source records attributed to Wojciechowska, M..

3 recordsLinked to original sources

bioRxiv2025

Modulating backbone flexibility in hydroxamate siderophores for improved iron chelation and peptide nucleic acid (PNA) delivery into bacteria

Peptide nucleic acid (PNA) is a synthetic oligonucleotide analog with a peptide-based backbone that selectively binds with high affinity to natural nucleic acids. PNA is a valuable tool in antisense technology with potential antibacterial applications. However, PNA cannot penetrate bacterial cells alone. To address this, we explored iron chelators - siderophores - as PNA carriers. Bacteria acquire iron through siderophores, which are transported via specific TonB-dependent receptors in the bacterial envelope. Previously, we demonstrated that a synthetic hydroxamate-type siderophore (SL) exploited this transport system to deliver PNA into bacterial cells, achieving a gene-silencing effect. However, this transport was limited to an Escherichia coli mutant with continuous iron uptake, and was not observed in wild-type E. coli. In this study, we developed a new synthetic siderophore (SGLY) with glycine spacers between modified ornithine residues for enhanced flexibility and iron-binding. We also synthesized marine siderophore analogs (MGLY and MALA) inspired by natural moanachelins. Using circular dichroism spectroscopy, spectrophotometric assays, and molecular dynamics simulations, we confirmed iron binding. Growth recovery experiments showed SGLY recognition and internalization via the TonB-dependent transport system, likely using hydroxamate siderophore pathways. The MGLY and MALA siderophores showed lower growth promotion than SGLY, indicating less efficient internalization. Molecular docking revealed high affinity of SGLY for E. coli receptors involved in the uptake of hydroxamate siderophores. However, upon conjugation to PNA, all three siderophores effectively delivered PNA into E. coli cells. We confirmed PNA-mediated gene silencing using fluorescence measurements and confocal microscopy.

biochemistry↗

bioRxiv2023

Regulation of Toxic RNA Foci and Repeat Expansion DMPK Transcripts: Role of MBNL Proteins and RNA Decay Pathways

Myotonic dystrophy type 1 (DM1) is a progressive, multisystemic disorder caused by an expansion of CTG repeats in the 3 untranslated region of the DMPK gene. When transcribed the mutant RNAs accumulate in affected tissues appearing as distinct foci when visualised by in situ hybridisation. The RNA foci are aggregates of CUG repeat-containing RNAs that sequester RNA-binding proteins, particularly muscleblind-like (MBNL) proteins, leading to their dysfunction and causing downstream molecular and cellular defects. Here we show the double knock-out of MBNL1 and 2 prevents RNA foci formation and nuclear retention of mutant DMPK mRNA in DM1 cells as well as promoting their degradation and nuclear export. Using stochastic optical reconstruction microscopy (STORM), we find the presence of both large foci and micro foci in DM1 cells. Large foci consist of multiple DMPK transcripts, while many micro foci are (CUG)n fragments. The absence of MBNL proteins not only prevents the aggregation of multiple DMPK transcripts into large foci, but also promotes their degradation and nuclear processing. However, although a substantial amount of MBNL1 proteins are bound to the mutant transcripts, the pools of free MBNL1 proteins are similar in DM1 nuclei to those in controls. Furthermore, we have identified several factors that are involved in the control of mutant DMPK mRNA turnover, including XRN2, EXOSC10, UPF1 and STAU1. Our study indicates that these factors are implicated in the RNA foci accumulation and the degradation of mutant DMPK mRNA. UPF1 and STAU1 may have additional roles beyond degradation, impacting the nuclear processing of mutant DMPK mRNA. Our study also highlights the critical role of MBNL proteins in regulating mutant DMPK mRNA metabolism: the absence of MBNLs in DM1 appears to expedite the processing of mutant DMPK mRNA mediated by these RNA decay factors. Significance statementOur investigations uncovered valuable data on the RNA foci dynamics in DM1, revealing the intricate mechanisms that underlie their formation, stability, and turnover. Our findings also contributed to delineate the complex pathways involved in the transportation and degradation of the mutant mRNA and provided insights into the critical role played by MBNL proteins in these processes. Studying the degradation mechanism of mutant DMPK mRNA in myotonic dystrophy may provide a foundation for comprehending the mechanisms of RNA degradation in other diseases caused by short tandem repeat (STR) mutations, such as Huntingtons disease, Fragile X syndrome, and several types of ataxia. Additionally, the use of cutting-edge STORM technology can provide a valuable tool for investigating RNA foci in other STR expansion disorders.

genetics↗

bioRxiv2018

Global increase in circRNA levels in myotonic dystrophy

Splicing aberrations induced as a consequence of the sequestration of MBNL splicing factors on the DMPK transcript, which contains expanded CUG repeats, present a major pathomechanism of myotonic dystrophy type 1 (DM1). As MBNLs may also be important factors involved in the biogenesis of circular RNAs (circRNAs), we hypothesized that the level of circRNAs would be decreased in DM1. To test this hypothesis, we selected twenty well-validated circRNAs and analyzed their levels in several experimental systems (e.g., cell lines, DM muscle tissues, and a mouse model of DM1) using droplet digital PCR assays. We also explored the global level of circRNAs using two RNA-Seq datasets of DM1 muscle samples. Contrary to our original hypothesis, our results consistently showed a global increase in circRNA levels in DM1 and we identified numerous circRNAs that were increased in DM1. We also identified many genes (including muscle-specific genes) giving rise to numerous (>10) circRNAs. Thus, this study is the first to show an increase in global circRNA levels in DM1. We also provided preliminary results showing the association of circRNA level with muscle weakness and alternative splicing changes that are biomarkers of DM1 severity. Author SummaryRecently, a great deal of interest has been focused on a new class of RNA molecules called circular RNAs (circRNAs). To date, thousands of circRNAs have been found in different human cells/tissues. Although the function of circRNAs remains mostly unknown, circRNAs have emerged as an important component of the RNA-RNA and RNA-protein interactome. Thus, intensive efforts are being made to fully understand the biology and function of circRNAs, especially their role in human diseases. As an important role in the biogenesis of circRNA may be played by MBNL splicing factors, in this study we used DM1 (to a lesser extent, DM2) as a natural model in which the level of MBNLs is decreased. In contrast to the expected effect, our results consistently showed a global increase in circRNA levels in DM1. As a consequence, whole genome transcriptome analysis revealed dozens of circRNAs with significantly altered (mostly increased) levels in DM1. Furthermore, we observed that the circRNA levels were in many cases strongly associated with DM1 severity.

molecular biology↗