Biology subjects

Sajek, M. P.

Publications and source records attributed to Sajek, M. P..

9 recordsLinked to original sources

bioRxiv2026

Fatty Acid Synthase Binds Cellular mRNAs and its Depletion Reduces Translational Efficiency

Metabolic enzymes with non-canonical RNA binding activity have drawn a growing interest, due to their implications for basic biology and disease. Here we present the first endogenous RNA interactome of fatty acid synthase (FASN), an emerging oncological and virological target. Utilizing crosslinking-based approaches, we characterize the direct RNA association of FASN in two human cell lines, identifying over 6000 binding sites across over 3500 protein-coding genes, with a striking preference for the 5' UTR. We provide a functional cross-screen on the effects of FASN depletion on cellular translation. This integration with ribosome profiling indicated a preferential reduction in the translational efficiencies of FASN-associated mRNAs - an effect shown to be dependent on binding location and site number. Associated transcripts were enriched for morphogenic and cell division-related functions, as well as palmitoylated proteins. This correlative work primes further investigation of FASN as a regulator of translation of its target mRNAs.

molecular biology↗

bioRxiv2026

Isoform-Specific Localization Diversifies Human MSI2 Function

Musashi-2 (MSI2) is an RNA-binding protein implicated in stem cell regulation and cancer, yet reports of its regulatory activity span translational activation, repression, and effects on mRNA stability, sometimes within the same cellular context. The basis for this diversity has remained unclear. Here, we identify alternative splicing as a determinant of MSI2 regulatory activity, acting through isoform-specific subcellular localization. Using a tethered reporter assay, we show that the canonical MSI2-328 isoform promotes translation without changing mRNA abundance. Truncation and mutation analysis identify a 40-amino-acid region (residues 194-234) as necessary and sufficient for cytoplasmic localization and necessary for translational activation. In contrast, the alternatively spliced MSI2-324 isoform localizes predominantly to the nucleus and fails to promote translation. We map this difference to an 18-amino-acid sequence introduced by an alternative 3 splice acceptor in exon 12; this sequence is sufficient to direct nuclear localization, and its removal restores MSI2-328-like cytoplasmic localization and translational activation. Consistent with these compartmental differences, the two isoforms associate with distinct protein networks, MSI2-328 with translation factors and ribosomal proteins, and MSI2-324 with chromatin and pre-mRNA processing factors. These isoform-specific activities are conserved across cell types, and the relative abundance of MSI2 isoforms shifts toward MSI2-324 in several cancers. Altogether, alternative splicing controls MSI2 subcellular localization, interaction networks, and regulatory output, providing a mechanistic framework for the context-dependent roles of MSI2 in gene regulation.

molecular biology↗

bioRxiv2024

Circular RNA - circCLIP2 is predominantly expressed in GSC niche and enhances glioblastoma aggressiveness via EMT and ECM signaling

Glioblastoma (GBM) is the most common and malignant type of brain tumor in adults and no effective therapies exist to combat this disease. Surgical resection, radio- and temozolomide-based-chemotherapy are insufficient mainly due to inter- and intra-tumoral heterogeneity of GBM tumors, therefore personalized approaches are required. Additionally, diverse tumor microenvironment with a leading role of glioblastoma stem cells (GSCs) in hypoxic core underlie the aggressiveness and recurrence of GBM. However, the molecules and pathways which directly promote tumor aggressiveness together with the driving process of epithelial-to-mesenchymal transition (EMT) and the components of extracellular matrix (ECM), remain only partially covered with prior studies focused mainly on coding oncogenes and tumor suppressor genes. Over the past years, a special attention has been paid to non-coding, circular RNAs (circRNAs) differentially expressed in various cancers, including GBM. Despite high abundance and stability, the exact biological roles of most individual circRNAs have not been fully revealed, so far. Besides anti-cancer targets, circRNAs have been considered as essential biomarkers and stratifying criterion of malignancy. Thus, studies on functions of differentially expressed circRNAs in GBM are an emerging field to be explored. In herein study, the biological role of circRNA CLIP2 (circCLIP2) was found highly overexpressed in primary tumor tissues. Upon the knock-down of circCLIP2 in 2D and 3D in vitro models, functional tests performed clearly indicate a significant contribution of circCLIP2 to aggressive potential of GBM as showed by decreased rates of proliferation, migration and invasion. Additional studies indicated predominant expression of circCLIP2 in GSC fractions additionally elevated by hypoxic conditions. The increased rates of proliferation and migration as well as molecular analyses were also confirmed in in vivo settings where xenograft model was applied. The expression of EMT marker Snail2 and ECM-degrading enzyme metalloproteinase 9 (MMP9) were significantly downregulated in the tumors formed from circCLIP2 silenced cells. Taken together, these results indicate a strong contribution of circCLIP2 in the aggressive phenotype of GBM which is due to a modulation of GSC proliferative potential as well as migration and invasion via EMT together with the modulator of ECM composition.

cancer biology↗

bioRxiv2024

Nanopore sequencing of intact aminoacylated tRNAs

Transfer RNAs (tRNA) are decorated during biogenesis with a variety of modifications that modulate their stability, aminoacylation, and decoding potential during translation. The complex landscape of tRNA modification presents significant analysis challenges and to date no single approach enables the simultaneous measurement of important but disparate chemical properties of individual, mature tRNA molecules. We developed a new, integrated approach to analyze the sequence, modification, and aminoacylation state of tRNA molecules in a high throughput nanopore sequencing experiment, leveraging a chemical ligation that embeds the charged amino acid in an adapted tRNA molecule. During nanopore sequencing, the embedded amino acid generates unique distortions in ionic current and translocation speed, enabling application of machine learning approaches to classify charging status and amino acid identity. Specific applications of the method indicate it will be broadly useful for examining relationships and dependencies between tRNA sequence, modification, and aminoacylation.

molecular biology↗

bioRxiv2024

PDGFRα signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking

Signaling through the platelet-derived growth factor receptor alpha (PDGFR) plays a critical role in craniofacial development, as mutations in PDGFRA are associated with cleft lip/palate in humans and Pdgfra mutant mouse models display varying degrees of facial clefting. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFR signaling during skeletal development in the mouse. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFR signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in severe midline facial clefting, due to defects in proliferation and survival of cranial neural crest cells, and widespread alternative RNA splicing (AS) changes. Here, we sought to determine the molecular mechanisms by which Srsf3 activity is regulated downstream of PDGFR signaling to control AS of transcripts necessary for craniofacial development. We demonstrated via enhanced UV-crosslinking and immunoprecipitation (eCLIP) of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons and loss of binding to canonical Srsf3 CA-rich motifs. Through the analysis of complementary RNA-seq data, we showed that Srsf3 activity results in the preferential inclusion of exons with increased GC content and lower intron to exon length ratio. Moreover, we found that the subset of transcripts that are bound by Srsf3 and undergo AS upon PDGFR signaling commonly encode regulators of PI3K signaling and early endosomal trafficking. Functional validation studies further confirmed that Srsf3 activity downstream of PDGFR signaling leads to retention of the receptor in early endosomes and increases in downstream PI3K-mediated Akt signaling. Taken together, our findings reveal that growth factor-mediated phosphorylation of an RNA-binding protein underlies gene expression regulation necessary for mammalian craniofacial development.

molecular biology↗

bioRxiv2024

Peptidyl-tRNA hydrolysis rate influences the efficiency of nonsense-mediated mRNA decay

Nonsense variants underlie many genetic diseases. The phenotypic impact of nonsense variants is determined by nonsense-mediated mRNA decay (NMD), which degrades transcripts with premature termination codons (PTCs). Despite its clinical importance, the factors controlling transcript-specific and context-dependent variation in NMD activity remain poorly understood. Through analysis of human genetic datasets, we discovered that the amino acid preceding the PTC strongly influences NMD activity. Notably, glycine codons promote robust NMD efficiency and show striking enrichment before PTCs but depletion before normal termination codons (NTCs). This glycine-PTC enrichment is particularly pronounced in genes tolerant to loss-of-function variants, suggesting evolutionary selection or neutrality conferred by efficient elimination of truncated proteins from non-essential genes. Using biochemical assays and massively parallel reporter analysis, we demonstrated that the peptide release rate during translation termination varies substantially with the identity of the preceding amino acid and serves as the primary determinant of NMD activity. We propose a "window of opportunity" model where translation termination kinetics modulate NMD efficiency. By revealing how sequence context shapes NMD activity through translation termination dynamics, our findings provide a mechanistic framework for improved clinical interpretation of nonsense variants.

molecular biology↗

bioRxiv2022

Small molecule inhibition of multiple RNA binding proteins underlies Musashi-2 independent phenotypes

RNA binding proteins (RBPs) are key regulators of gene expression. Small molecules targeting these RBP-RNA interactions are a rapidly emerging class of therapeutics for treating a variety of diseases. Ro-08-2750 (Ro) is a small molecule inhibitor identified as a competitive inhibitor of Musashi(MSI)-RNA interactions. Here we show Ro potently inhibits adrenocortical steroidogenesis and viability independent of MSI2 in multiple cell lines. We identified Ro-interacting proteins using an unbiased proteome-wide approach and discovered it is broadly targeting RBPs. To confirm this finding, we leveraged the large-scale ENCODE data and found a subset of RBPs whose depletion phenocopies Ro inhibition. We conclude that Ro is a promiscuous inhibitor of multiple RBPs, many containing RRM1 domains. Moreover, we provide a general framework for validating the specificity and identifying targets of RBP inhibitors in a cellular context.

molecular biology↗

bioRxiv2020

RNAlign2D, a novel RNA structural alignment tool based on pseudo-amino acid substitution matrix

BackgroundThe functions of RNA molecules are mainly determined by their secondary structures. These functions can also be predicted using bioinformatic tools that enable the alignment of multiple RNAs to determine functional domains and/or classify RNA molecules into RNA families. However, the existing multiple RNA alignment tools, which use structural information, are slow in aligning long molecules and/or a large number of molecules. Therefore, a more rapid tool for multiple RNA alignment may improve the classification of known RNAs and help to reveal the functions of newly discovered RNAs. ResultsHere, we introduce an extremely fast Python-based tool called RNAlign2D. It converts RNA sequences to pseudo-amino acid sequences, which incorporate structural information, and uses a customizable scoring matrix to align these RNA molecules via the multiple protein sequence alignment tool MUSCLE. ConclusionsRNAlign2D produces accurate RNA alignments in a very short time. The pseudo-amino acid substitution matrix approach utilized in RNAlign2D is applicable for virtually all protein aligners.

bioinformatics↗

bioRxiv2019

Kinesin KIF18A is a novel PUM regulated target promoting mitotic progression and survival of human male germ cell line - TCam-2

Regulation of proliferation, apoptosis and cell cycle is crucial for the physiology of germ cells. Their malfunction contributes to infertility and germ cell tumours. Kinesin KIF18A is an important germ cell specific regulator which downregulates apoptosis while promoting cell proliferation in animal models. Whereas regulation of KIF18A expression was studied at the transcriptional level, its posttranscriptional regulation has not been extensively explored. Due to the presence of two PUM Binding Elements (PBEs) within 3UTR, KIF18A mRNA is a potential target of PUMs, well known RNA-binding proteins involved in posttranscriptional gene regulation (PTGR). We investigated that possibility in TCam-2 cells originating from seminoma, representing human male germ cells. We conducted RNA co-immunoprecipitation combined with RT-qPCR, as well as luciferase reporter assay by applying appropriate luciferase construct encoding the wild type KIF18A 3UTR, upon PUM1 and PUM2 overexpression or knockdown. We found that KIF18A is repressed by PUM1 and PUM2. To study how this regulation influences KIF18A function in TCam-2 cells, MTS proliferation assay, apoptosis and cell cycle, analysis using flow cytometry was performed upon KIF18A siRNA knockdown. We uncovered that KIF18A significantly influences proliferation, apoptosis and cell cycle, these effects being opposite to PUM effects in TCam-2 cells. We propose that repression by PUM proteins may represent one of mechanisms influencing KIF18A level in controlling proliferation, cell cycle and apoptosis in TCam-2 cells. To the best of our knowledge, this paper identifies the first mammalian functionally germ cell specific gene that is regulated by Pum proteins via 3UTR.

developmental biology↗