Biology subjects

Grzechnik, P.

Publications and source records attributed to Grzechnik, P..

5 recordsLinked to original sources

bioRxiv2026

Antisense oligonucleotides targeting transcription termination windows disrupt mRNA 3' end processing and decrease gene expression

Antisense oligonucleotides (ASOs) are short, synthetic nucleic acids that bind to complementary RNA sequences and alter gene expression, making them versatile therapeutic agents. Here, we identify transcription termination windows of protein-coding genes as previously unrecognized targets for ASOs. We show that ASOs can act on nascent RNA synthesised downstream of the annotated genes, leading to a pronounced decrease in the corresponding mRNA levels. These downstream-of-gene ASOs (DG-ASOs) induce RNase H1-dependent cleavage, impairing mRNA 3 end processing, directing the unprocessed mRNAs for exosome-dependent degradation and creating early entry points in the nascent RNA for the XRN2 exonuclease termination factor. Altogether, we show that termination windows are genuine ASO targets which can be exploited to suppress gene expression. Importantly, we also reveal an underappreciated source of off-target effects which may arise from ASOs binding downstream of genes. Our findings indicate the necessity to expand ASO design and off-target assessment guidelines to include termination window sequences, thereby improving therapeutic efficacy and safety.

molecular biology↗

bioRxiv2022

Ceg1 depletion reveals mechanisms governing degradation of non-capped RNAs

Most functional eukaryotic mRNAs contain a 7-methylguanosine (m7G) cap which serves as a platform that recruits proteins to support essential biological functions such as mRNA processing, nuclear export and translation. Although capping is accomplished during the first steps of transcription the fate and turnover of uncapped transcripts have not been studied extensively. Here, we employed fast nuclear depletion of the capping enzymes in Saccharomyces cerevisiae to uncover the turnover of the transcripts that failed to be capped. We show that the levels of non-capped mRNAs are determined principally by the abundance of their synthesis. Nuclear depletion of the capping enzymes increases the levels of lowly expressed mRNAs and decreases mRNAs that are highly transcribed altogether mimicking the effects observed in cells lacking the predominantly cytoplasmic 5-3 exonuclease Xrn1. The nuclear 5-3 exonuclease Rat1 is not involved in the degradation of cap-defective transcripts and the lack of the capping does not affect the distribution of RNA Polymerase II on the chromatin. Our data indicate that the mRNAs that failed to be capped are not directed to a specific quality-control pathway and that the 5 cap role is associated with the Xrn1-dependent buffering of the cellular mRNA levels, along with protecting from 5-3 degradation.

molecular biology↗

bioRxiv2021

RPRD Proteins Control Transcription in Human Cells

The regulation of transcription is an essential process that allows the cell to respond to various internal and external signals. RNA Polymerase II (Pol II) activity is controlled by a number of factors which bind to the C-terminal domain (CTD) of its largest subunit, RPB1, and stimulate or suppress RNA synthesis. Here, we demonstrate that CTD-interacting proteins, RPRD2, RPRD1B and RPRD1A act as negative regulators of transcription and their levels inversely correlate with the accumulation of nascent and newly transcribed RNA in human cells. We show that the RPRD proteins form mutually exclusive complexes with Pol II to coordinate their roles in transcriptional control. Our data indicate that RPRD2 exerts the most substantial impact on transcription and has the potential to alter key biological processes including the cellular stress response and cell growth.

molecular biology↗

bioRxiv2021

Non-coding RNAs Associated with Prader-Willi Syndrome Regulate Transcription of Neurodevelopmental Genes in Human Induced Pluripotent Stem Cells

Mutations and aberrant gene expression during cellular differentiation lead to neurodevelopmental disorders such as Prader-Willi syndrome (PWS) which results from the deletion of an imprinted locus on chromosome 15. We analysed chromatin-associated RNA in human induced pluripotent cells (iPSCs) upon depletion of hybrid small nucleolar long non-coding RNAs (sno-lncRNAs) and 5 snoRNA capped and polyadenylated long non-coding RNAs (SPA-lncRNAs) transcribed from the locus deleted in PWS. We found that rapid ablation of these lncRNAs affects transcription of specific gene classes. Downregulated genes contribute to neurodevelopment and neuronal maintenance while genes that are upregulated are predominantly involved in the negative regulation of cellular metabolism and apoptotic processes. Our data revealed the importance of SPA-lncRNAs and sno-lncRNAs in controlling gene expression in iPSCs and provided a platform for synthetic experimental approaches in PWS studies. We conclude that ncRNAs transcribed from the PWS locus are critical regulators of a transcriptional signature important for neuronal differentiation and development.

molecular biology↗

bioRxiv2021

Supramolecular cylinders target bulge structures in the 5-prime UTR of the RNA genome of SARS-CoV-2 and inhibit viral replication

The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with Molecular Dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5 UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in the stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel antiviral agents.

biochemistry↗