Biology subjects

Olejniczak, M.

Publications and source records attributed to Olejniczak, M..

10 recordsLinked to original sources

bioRxiv2026

Modeling ATN1 mutations in human iPSCs reveals early developmental roles of atrophin-1

Atrophin-1 (ATN1), encoded by the ATN1 gene, is an evolutionarily conserved transcriptional corepressor implicated in neuronal development and severe incurable disorders, including the polyglutamine (polyQ) disease - dentatorubral-pallidoluysian atrophy (DRPLA), and the neurodevelopmental syndrome CHEDDA. However, the cellular function of ATN1 and its role during early human development remain largely uncharacterized. Here, we generated an isogenic panel of human induced pluripotent stem cell (iPSC) lines, establishing a novel resource for comprehensive studies of ATN1 function and dysfunction. This panel includes a DRPLA patient-derived line (wt/mut), CRISPR-Cas9-corrected wild-type controls (wt/wt), complete ATN1 knock-out clones (-/-), and heterozygous lines expressing either a wild-type or a mutant allele (wt/- and -/mut). While pluripotency markers remained unaffected across all variants, further characterization revealed marked proliferation deficits in ATN1 knock-out lines and heterozygous iPSCs without wild-type allele expression. Moreover, the formation of punctate ATN1 assemblies was enhanced in iPSCs with -/mut variants. The pronounced phenotype observed in cells expressing only the mutant ATN1 allele may result from a combination of loss-of-function and toxic gain-of-function mechanisms. Overall, the generated panel of iPSC lines provides a unique cellular platform for investigating ATN1-dependent molecular mechanisms and their contribution to neuronal development and disease.

cell biology↗

bioRxiv2026

Machine learning-guided design of artificial microRNAs for targeted gene silencing

Artificial microRNAs (amiRNAs) offer a powerful strategy for targeted gene silencing, but their rational design is limited by complex sequence-structure-processing relationships and the lack of tools capable of optimizing efficacy and specificity. To address this need, we developed miRarchitect, a web-based platform that uses machine learning to support the customizable design of amiRNAs. miRarchitect integrates neural network-guided target-site selection, siRNA insert design, and scaffold choice, utilizing large-scale data from human primary microRNAs (pri-miRNAs) and next-generation sequencing. The platform generates molecules that closely resemble endogenous pri-miRNAs and includes comprehensive off-target analysis to enhance specificity. Experimental validation targeting TMPRSS2 and ACE-2 confirmed precise processing, robust knockdown, and high specificity of miRarchitect-designed amiRNAs. In comparative benchmarking, miRarchitect consistently produced functional amiRNAs, whereas only half of the top candidates generated by other tools showed measurable activity. miRarchitect is freely available at https://rnadrug.ichb.pl/mirarchitect and provides an intuitive interface with an automated workflow for generating, ranking, and selecting candidate amiRNAs for research and therapeutic applications.

bioinformatics↗

bioRxiv2026

Targeted 3'-end RNA sequencing uncovers cryptic polyadenylation in Huntington's disease linked to somatic instability and CAG repeat purity

Huntingtons disease (HD) is a progressive neurodegenerative disorder caused by expanded CAG repeats in the first exon of the HTT gene, which encodes for huntingtin (HTT) protein. Full-penetrance is established at 40 repeats, but beyond, somatic repeat instability in the brain and CAG repeat purity modulate disease onset and severity. Previous studies have described that expanded repeats induce the incomplete splicing of HTT intron 1 to express the most pathogenic HTT isoform, known as HTT1a. Yet, the lack of a robust and sensitive method to evaluate HTT RNA-misprocessing has limited our understanding of HTT1a expression in HD pathophysiology. Here we describe a targeted RNA sequencing approach, known as 3-end targeted RNA sequencing or 3TRS, to simultaneously quantify multiple HTT transcripts generated by canonical and cryptic polyadenylation in several HD models. We show that activation of HTT cryptic polyadenylation is highly selective and requires long and uninterrupted CAG repeat expansions. In HD knock-in mice and human postmortem brain, cryptic HTT expression strongly correlates with brain-specific somatic repeat instability, supporting a model where ultralong and unstable CAG repeats drive toxicity by activating HTT RNA-misprocessing. Overall, 3TRS provides a robust framework to investigate HTT1a biogenesis and expression and to evaluate HTT-lowering therapeutic strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/697463v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@168d8e1org.highwire.dtl.DTLVardef@76ba3forg.highwire.dtl.DTLVardef@bdaf93org.highwire.dtl.DTLVardef@151e581_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

DCLRE1A orchestrates CAG repeat contraction following Cas12a-induced DNA breaks

Despite their well-defined genetic background, repeat expansion diseases (REDs) still represent an unmet medical need, with no causative therapy offered to patients. The strategy of repeat shortening using genome editing tools is very attractive because a single intervention can result in permanent repair of the disease-causing mutation. However, a limited understanding of DNA repair mechanisms in repetitive sequences complicates the prediction and control of the editing effects. Using a CRISPR interference (CRISPRi) screen, we identified pathways and factors responsible for the repair of staggered cuts generated by Cas12a within CAG repeat tracts. This analysis revealed a central role for interstrand crosslink (ICL) repair factors in mediating CAG repeat contraction, with DCLRE1A emerging as a key effector. We demonstrated that DCLRE1A recognizes and binds structures generated by Cas12a. Moreover, DCLRE1A interacts with SLX4, promoting the generation of pure contractions, and with POLI, leading to the formation of inverted repeats at the break site during template switching mechanisms. We then used this knowledge to increase the contribution of pure contractions to the pool of editing outcomes using fusions of Cas12a with DNA repair proteins. Our study indicates that Cas12a can be used as an effective tool for generating repeat contractions. Although the mechanisms leading to repeat shortening are complex, understanding them can help researchers develop more precise therapeutic strategies with greater control of the editing process.

cell biology↗

bioRxiv2024

Different RNA recognition by ProQ and FinO depends on the sequence surrounding intrinsic terminator hairpins

Escherichia coli ProQ and FinO proteins both have RNA-binding FinO domains, which bind to intrinsic transcription terminators, but each protein recognizes distinct RNAs. To explore how ProQ and FinO discriminate between RNAs we transplanted sequences surrounding terminator hairpins between RNAs specific for each protein, and compared their binding to ProQ, the isolated FinO domain of ProQ (ProQNTD), and FinO. The results showed that the binding specificity of chimeric RNAs towards ProQ, ProQNTD, or FinO was determined by the origin of the transplanted sequence. Further analysis showed that the sequence surrounding the terminator hairpin, including a purine-purine mismatch, in natural RNA ligands of FinO and in chimeric RNAs weakened their binding by ProQNTD. Overall, our studies suggest that the discrimination between RNAs by ProQ and FinO is determined by RNA sequence elements surrounding the intrinsic terminator hairpin.

biochemistry↗

bioRxiv2024

RNA recognition by minimal ProQ from Neisseria meningitidis

Neisseria meningitidis minimal ProQ is a global RNA binding protein belonging to the family of FinO-domain proteins. The N. meningitidis ProQ consists only of the FinO domain accompanied by short N- and C-terminal extensions. To better understand how this minimal FinO-domain protein recognizes RNAs, we compared its binding to seven different natural RNA ligands of this protein. Next, two of these RNAs, rpmG-3 and AniS, were subject to further mutational studies. The data showed that N. meningitidis ProQ binds the lower part of the intrinsic transcription terminator hairpin, and that the single-stranded sequences on the 5 and 3' side of terminator stem are required for tight binding. However, the specific lengths of 5 and 3' RNA sequences required for optimal binding differed between the two RNAs. Additionally, our data show that the 2'-OH and 3'-OH groups of the 3' terminal ribose contribute to RNA binding by N. meningitidis ProQ. In summary, the minimal ProQ protein from N. meningitidis has generally similar requirements for RNA binding as the isolated FinO domains of other proteins of this family, but differs from them in detailed RNA features that are optimal for specific RNA recognition.

biochemistry↗

bioRxiv2023

CRISPR/Cas9-induced double-strand breaks in huntingtin locus lead to CAG repeat contraction through the extensive DNA end resection and homology-mediated repair

Expansion of the CAG/CTG repeats in functionally unrelated genes is a causative factor in many inherited neurodegenerative disorders, including Huntingtons disease (HD), spinocerebellar ataxias (SCAs) and myotonic dystrophy type 1 (DM1). Despite many years of research, the mechanism responsible for repeat instability is unknown, and recent findings indicate the key role of DNA repair in this process. The repair of DSBs induced by genome editing tools results in the shortening of long CAG repeats in yeast models. Understanding this mechanism is the first step to developing a therapeutic strategy based on controlled shortening of repeats. The aim of this study was to characterize Cas9-induced DSB repair products in the endogenous HTT locus in human cells and to identify factors affecting the formation of specific types of sequences. The location of the cleavage site and the surrounding sequence influence the outcome of DNA repair. DSBs within CAG repeats result in shortening of the repeats in frame in [~]90% of products. The mechanism of this contraction involves MRE11-CTIP and RAD51 activity and extensive DNA end resection reaching [~]5000 bp. We demonstrated that a DSB located upstream of CAG repeats induces polymerase theta-mediated end joining, resulting in deletion of the entire CAG tract. Furthermore, using unbiased proteomic analysis, we identified novel factors that may be involved in CAG sequence repair. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/568568v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@7ecb6dorg.highwire.dtl.DTLVardef@5d71c5org.highwire.dtl.DTLVardef@d86ffdorg.highwire.dtl.DTLVardef@1a0651f_HPS_FORMAT_FIGEXP M_FIG C_FIG DNA double-strand break repair in the CAG repeat locus engages numerous factors from various repair pathways and depends mainly on DNA end resection, leading to tract shortening.

molecular biology↗

bioRxiv2023

Biochemical and genetic dissection of the RNA-binding surface of the FinO domain of Escherichia coli ProQ

RNA-binding proteins play important roles in bacterial gene regulation through interactions with both coding and non-coding RNAs. ProQ is a FinO-domain protein that binds a large set of RNAs in Escherichia coli, though the details of how ProQ binds these RNAs remain unclear. In this study, we used a combination of in vivo and in vitro binding assays to confirm key structural features of E. coli ProQs FinO domain and explore its mechanism of RNA interactions. Using a bacterial three-hybrid assay, we performed forward genetic screens to confirm the importance of the concave face of ProQ in RNA binding. Using gel shift assays, we directly probed the contributions of ten amino acids on ProQ binding to seven RNA targets. Certain residues (R58, Y70, and R80) were found to be essential for binding of all seven RNAs, while substitutions of other residues (K54 and R62) caused more moderate binding defects. Interestingly, substitutions of two amino acids (K35, R69), which are evolutionarily variable but adjacent to conserved residues, showed varied effects on the binding of different RNAs; these may arise from the differing sequence context around each RNAs terminator hairpin. Together, this work confirms many of the essential RNA-binding residues in ProQ initially identified in vivo and supports a model in which residues on the conserved concave face of the FinO domain such as R58, Y70 and R80 form the main RNA-binding site of E. coli ProQ, while additional contacts contribute to the binding of certain RNAs.

molecular biology↗

bioRxiv2021

Bacterial chaperone protein Hfq facilitates the annealing of sponge RNAs to small regulatory RNAs

Bacterial small RNAs (sRNAs) in association with the chaperone protein Hfq regulate the expression of many target mRNAs. Since sRNAs action is crucial to engender a response to changing environmental conditions, their activity needs to be regulated. One such mechanism occurs at posttranscriptional level and involves sponge RNAs (or anti-sRNAs) which sequester sRNAs affecting their regulatory output. Both types of RNAs were identified on Hfq, but it is not known how Hfq interacts with RNA sponges and stimulates their base-pairing with sRNAs. Here, we used biochemical methods to demonstrate that anti-sRNAs resemble sRNAs by their structure and their modes of Hfq binding. Hfq facilitates sponge RNA annealing to sRNA, and each surface of the protein plays a particular role in the process. Moreover, we found that the efficiency of sponge RNA interactions with sRNAs can be improved, therefore, we propose that natural RNA sponges might not sequester sRNAs optimally.

molecular biology↗

bioRxiv2020

Determinants of RNA recognition by the FinO domain of the Escherichia coli ProQ protein

The regulation of gene expression by small RNAs in Escherichia coli depends on RNA binding proteins Hfq and ProQ, which bind mostly distinct RNA pools. To understand how ProQ discriminates between RNA substrates, we compared its binding to six different RNA molecules. Full-length ProQ bound all six RNAs similarly, while the isolated N-terminal FinO domain (NTD) of ProQ specifically recognized RNAs with Rho-independent terminators. Analysis of malM 3-UTR mutants showed that tight RNA binding by the ProQ NTD required a terminator hairpin of at least two base pairs preceding an 3 oligoU tail of at least four uridine residues. Substitution of an A-rich sequence on the 5 side of the terminator to uridines strengthened the binding of several ProQ-specific RNAs to the Hfq protein, but not to the ProQ NTD. Substitution of the motif in the malM-3 and cspE-3 RNAs also conferred the ability to bind Hfq in E. coli cells, as measured using a three-hybrid assay. In summary, these data suggest that the ProQ NTD specifically recognizes 3 intrinsic terminators of RNA substrates, and that the discrimination between RNA ligands by E. coli ProQ and Hfq depends both on positive determinants for binding to ProQ and negative determinants against binding to Hfq.

molecular biology↗