Biology subjects

Ntini, E.

Publications and source records attributed to Ntini, E..

8 recordsLinked to original sources

bioRxiv2025

PVT1 splicing activity predicts genome-wide gene expression with miRNA regulatory signatures

Long non-coding RNAs (lncRNAs) have emerged as key regulators of gene expression, yet the functional impact of their splicing activity remains poorly understood. Here, we focus on the chromatin-associated lncRNA PVT1, whose splicing efficiency at specific 3 splice sites predicts expression of distal genes in breast cancer. Using elastic net modeling across hundreds of breast cancer samples, we identify PVT1 splicing activity as a predictor of gene expression signatures enriched in miR-200/205 targets. Causal inference analysis using tumor-specific SNVs confirms that perturbation of splicing at specific sites alters the expression of distal gene sets enriched in miR-200/205 targets. We suggest a mechanistic model in which inefficient splicing and intron retention expose intronic miR-200 seed sites, perfect 7-mers, found exclusively within PVT1 introns, enabling PVT1 to function as a chromatin-associated lncRNA with intron-retained competing endogenous (ceRNA) activity. Artificial splicing enhancement of PVT1 in vitro with CASFx altered the expression of miR-200 target genes supporting this model. A genome-wide screen for intronic miR-200 seed sites, and splicing-based modeling across additional lncRNAs and protein-coding genes, identified PVT1 as a top candidate for splicing-modulated intron-retained ceRNA activity. Cross-tissue model generalization to prostate cancer supports the broader relevance of PVT1 splicing-based regulation. Our results highlight the regulatory potential of lncRNA splicing activity in trans, suggesting that chromatin-tethered, intron-retained lncRNAs such as PVT1 may serve as key post-transcriptional regulators in cancer.

bioinformatics↗

bioRxiv2023

Hypoxia-driven deSUMOylation of EXOSC10 promotes adaptive changes in the transcriptome profile

Reduced oxygen availability (hypoxia) triggers adaptive cellular responses via hypoxia-inducible factor (HIF)-dependent transcriptional activation. Adaptation to hypoxia also involves transcription-independent processes like post-translational modifications, however these mechanisms are poorly characterized. Investigating the involvement of protein SUMOylation in response to hypoxia, we discovered that hypoxia strongly decreases the SUMOylation of Exosome subunit 10 (EXOSC10), the catalytic subunit of the RNA exosome, in a HIF-independent manner. EXOSC10 is a multifunctional exoribonuclease enriched in the nucleolus that mediates the processing and degradation of various RNA species. We demonstrate that the Ubiquitin-specific protease 36 (USP36) SUMOylates EXOSC10 and we reveal SUMO1/sentrin-specific peptidase 3 (SENP3) as the enzyme mediating deSUMOylation of EXOSC10. Under hypoxia, EXOSC10 dissociates from USP36 and translocates from the nucleolus to the nucleoplasm concomitant with its deSUMOylation. Loss of EXOSC10 SUMOylation does not detectably affect rRNA maturation but affects the mRNA transcriptome by modulating the expression levels of hypoxia-related genes. Our data suggest that dynamic modulation of EXOSC10 SUMOylation and localization under hypoxia regulates the RNA degradation machinery to facilitate cellular adaptation to low oxygen conditions.

molecular biology↗

bioRxiv2021

Distal and proximal cis-regulatory elements sense X-chromosomal dosage and developmental state at the Xist locus

Developmental genes such as Xist, the master regulator of X-chromosome inactivation (XCI), are controlled by complex cis-regulatory landscapes, which decode multiple signals to establish specific spatio-temporal expression patterns. Xist integrates information on X-chromosomal dosage and developmental stage to trigger XCI at the primed pluripotent state in females only. Through a pooled CRISPR interference screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist during the onset of random XCI. By quantifying how enhancer activity is modulated by X-dosage and differentiation, we find that X-dosage controls the promoter-proximal region in a binary switch-like manner. By contrast, differentiation cues activate a series of distal elements and bring them into closer spatial proximity of the Xist promoter. The strongest distal element is part of an enhancer cluster [~]200 kb upstream of the Xist gene which is associated with a previously unannotated Xist-enhancing regulatory transcript, we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. Our study is the first step to disentangle how multiple, functionally distinct regulatory regions interact to generate complex expression patterns in mammals.

genomics↗

bioRxiv2020

Distinctive features of long non-coding RNA chromatin (dis-)association

Long non-coding RNAs (lncRNAs) are involved in gene expression regulation in cis and trans. Although enriched in the chromatin cell fraction, to what degree this defines their broad range of functions remains unclear. In addition, the factors that contribute to lncRNA chromatin tethering, as well as the molecular basis of efficient lncRNA chromatin dissociation and its functional impact on enhancer activity and target gene expression, remain to be resolved. Here, we combine pulse-chase metabolic labeling of nascent RNA with chromatin fractionation and transient transcriptome sequencing to follow nascent RNA transcripts from their co-transcriptional state to their release into the nucleoplasm. By incorporating functional and physical characteristics in machine learning models, we find that parameters like co-transcriptional splicing contributes to efficient lncRNA chromatin dissociation. Intriguingly, lncRNAs transcribed from enhancer-like regions display reduced chromatin retention, suggesting that, in addition to splicing, lncRNA chromatin dissociation may contribute to enhancer activity and target gene expression. HighlightsO_LIChromatin (dis-)association of lncRNAs can be modeled using nascent RNA sequencing from pulse-chase chromatin fractionation C_LIO_LIDistinct physical and functional characteristics contribute to lncRNA chromatin (dis-)association C_LIO_LIlncRNAs transcribed from enhancers display increased degree of chromatin dissociation C_LIO_LIlncRNAs of distinct degrees of chromatin association display differential binding probabilities for RNA-binding proteins (RBPs) C_LI

molecular biology↗

bioRxiv2020

Determination of primary microRNA processing in clinicalsamples by targeted pri-miR-sequencing

MicroRNA expression is important for gene regulation and deregulated microRNA expression is often observed in disease such as cancer. The processing of primary microRNA transcripts is an important regulatory step in microRNA biogenesis. Due to low expression level and association with chromatin primary microRNAs are challenging to study in clinical samples where input material is limited. Here, we present a high-sensitivity targeted method to determine processing efficiency of several hundred primary microRNAs from total RNA using as little as 500 thousand Illumina HiSeq sequencing reads. We validate the method using RNA from HeLa cells and show the applicability to clinical samples by analyzing RNA from normal liver and hepatocellular carcinoma. We identify 24 primary microRNAs with significant changes in processing efficiency from normal liver to hepatocellular carcinoma, among those the highly expressed miRNA-122 and miRNA-21, demonstrating that differential processing of primary microRNAs is occurring and could be involved in disease. With our method presented here we provide means to study pri-miRNA processing in disease from clinical samples.

molecular biology↗

bioRxiv2018

Transient N-6-methyladensosine Transcriptome sequencing reveals a regulatory role of m6A in splicing efficiency

Splicing efficiency varies among transcripts, and tight control of splicing kinetics is crucial for coordinated gene expression. N-6-methyladenosine (m6A) is the most abundant RNA modification and is involved in regulation of RNA biogenesis and function. The impact of m6A on the regulation of RNA splicing kinetics has not been investigated. Here, we provide the first time-resolved high-resolution assessment of m6A on nascent RNA transcripts and unveil its importance for the control of RNA splicing kinetics. We identify that early co-transcriptional m6A deposition near splice junctions promotes fast splicing, while m6A modification of introns is associated with long, slowly processed introns and alternative splicing events. In conclusion, by directly comparing the processing dynamics of individual transcripts in the methylated versus unmethylated state on a transcriptome-wide scale we show that early m6A deposition marks transcripts for a fast-track processing.

molecular biology↗

bioRxiv2017

Chromatin-release of the long ncRNA A-ROD is required for transcriptional activation of its target gene DKK1

Long non-coding RNAs (ncRNAs) are involved in both positive and negative regulation of transcription. Long ncRNAs are often enriched in the nucleus and at chromatin but whether chromatin-release plays a functional role is unknown. Here, we used epigenetic marks, expression level and strength of chromatin interactions to group long ncRNAs and find that those engaged in strong chromatin interactions are less enriched at chromatin in MCF-7 cells, suggesting a functional involvement of chromatin-release of long ncRNAs in transcriptional regulation. To study this further, we identify the long ncRNA A-ROD, an activating regulator of the Wnt signaling inhibitor DKK1. We show that A-ROD enhances transcription elongation of DKK1 in an RNA-dependent manner and that A-ROD recruits EBP1 to the DKK1 promoter. Our data suggest that the activating function depends on the release of A-ROD from chromatin, and further identify a functional regulatory interaction mediated by A-ROD in the transcription activation of DKK1. We propose that the release of a subset of long ncRNAs is important for their function, adding a new mechanistic perspective to the subcellular localization of long ncRNAs.

molecular biology↗

bioRxiv2016

Microprocessor dynamics shows co- and post-transcriptional processingof pri-miRNAs

miRNAs are small regulatory RNAs involved in the regulation of translation of target transcripts. miRNA biogenesis is a multi-step process starting with the cleavage of the primary miRNA transcript in the nucleus by the Microprocessor complex. Endogenous processing of pri-miRNAs is challenging to study and the in vivo kinetics of this process is not known. Here, we present a method for determining the processing kinetics of pri-miRNAs within intact cells over time using a pulse-chase approach to obtain nascent RNA within a 1-hour window after labeling with bromouridine. We show, that pri-miRNAs exhibit different processing kinetics ranging from fast over intermediate to slow processing and provide evidence that pri-miRNA processing can occur both co-transcriptionally and post-transcriptionally.

molecular biology↗