Biology subjects

Makeyev, E. V.

Publications and source records attributed to Makeyev, E. V..

7 recordsLinked to original sources

bioRxiv2025

FUS post-transcriptional splicing is autoregulated via RNA condensation with therapeutic potential for ALS-FUS

Mutations in the FUS gene cause aggressive and often juvenile forms of amyotrophic lateral sclerosis (ALS-FUS). In addition to mRNA, the FUS gene gives rise to a partially processed RNA with retained introns 6 and 7. We demonstrate that these FUSint6&7-RNAs form nuclear condensates scaffolded by the highly structured intron 7 and associated with nuclear speckles. Using hybridization-proximity labelling proteomics, we show that the FUSint6&7-RNA condensates are enriched in splicing factors and the m6A reader YTHDC1. These ribonucleoprotein structures facilitate post-transcriptional FUS splicing and depend on m6A/YTHDC1 for their maintenance. FUSint6&7-RNAs become hypermethylated in cells expressing mutant FUS, leading to their enhanced condensation and consequently, splicing. We further demonstrate that FUS protein is repelled by m6A. Thus, ALS-FUS mutations may cause an abnormal activation of FUS post-transcriptional splicing via altered RNA methylation. Strikingly, ectopic expression of FUS intron 6&7 sequences dissolves the endogenous FUSint6&7-RNA condensates, downregulating FUS mRNA and protein. Overall, we describe an RNA condensation-dependent mechanism regulating FUS splicing that can be harnessed for developing new therapies.

cell biology↗

bioRxiv2024

Recurrent disruption of tumour suppressor genes in cancer by somatic mutations in cleavage and polyadenylation signals

The expression of eukaryotic genes relies on the precise 3-terminal cleavage and polyadenylation of newly synthesized pre-mRNA transcripts. Defects in these processes have been associated with various diseases, including cancer. While cancer-focused sequencing studies have identified numerous driver mutations in protein-coding sequences, noncoding drivers - particularly those affecting the cis-elements required for pre-mRNA cleavage and polyadenylation - have received less attention. Here, we systematically analysed cancer somatic mutations affecting 3UTR polyadenylation signals using the Pan-Cancer Analysis of Whole Genomes (PCAWG) dataset. We found a striking enrichment of cancer-specific somatic mutations that disrupt strong and evolutionarily conserved cleavage and polyadenylation signals within tumour suppressor genes. Further bioinformatics and experimental analyses conducted as a part of our study suggest that these mutations have a profound capacity to downregulate the expression of tumour suppressor genes. Thus, this work uncovers a novel class of noncoding somatic mutations with significant potential to drive cancer progression.

cancer biology↗

bioRxiv2023

Regulation potential of transcribed simple repeated sequences in developing neurons

Simple repeated sequences (SRSs), defined as tandem iterations of microsatellite- to satellite-sized DNA units, occupy a substantial part of the human genome. Some of these elements are known to be transcribed in the context of repeat expansion disorders. Mounting evidence suggests that the transcription of SRSs may also contribute to normal cellular functions. Here, we used genome-wide bioinformatics approaches to systematically examine SRS transcriptional activity in cells undergoing neuronal differentiation. We identified thousands of long noncoding RNAs containing >200-nucleotide-long SRSs (SRS-lncRNAs), with hundreds of these transcripts significantly upregulated in the neural lineage. We show that SRS-lncRNAs often originate from telomere-proximal regions and that they have a strong potential to form multivalent contacts with a wide range of RNA-binding proteins. Our analyses also uncovered a cluster of neurally upregulated SRS-lncRNAs encoded in a centromere-proximal part of chromosome 9, which underwent an evolutionarily recent segmental duplication. Using a newly established in vitro system for rapid neuronal differentiation of induced pluripotent stem cells, we demonstrate that at least some of the bioinformatically predicted SRS-lncRNAs, including those encoded in the segmentally duplicated part of chromosome 9, indeed increase their expression in developing neurons to readily detectable levels. These data suggest that many SRSs may be expressed in a cell type and developmental stage-specific manner, providing a valuable resource for further studies focused on the functional consequences of SRS-lncRNAs in the normal development of the human brain.

genomics↗

bioRxiv2023

Alternative splicing coupled to nonsense-mediated decay coordinates downregulation of non-neuronal genes in developing neurons

The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript abundance. Previous studies have identified several examples of such a regulation in developing neurons. However, the systems-level effects of AS-NMD in this context are poorly understood. We developed an R package, factR2, which offers a comprehensive suite of AS-NMD analysis functions. Using this tool, we conducted a longitudinal analysis of gene expression in pluripotent stem cells undergoing induced neuronal differentiation. Our analysis uncovered hundreds of AS-NMD events with significant potential to regulate gene expression. Notably, this regulation was significantly overrepresented in specific functional groups of developmentally downregulated genes. Particularly strong association with gene downregulation was detected for alternative cassette exons stimulating NMD (NS-CEs) upon their inclusion into mature mRNA. By combining bioinformatics analyses with CRISPR/Cas9 genome editing and other experimental approaches we show that NS-CEs regulated by the RNA-binding protein PTBP1 dampen the expression of their genes in developing neurons. We also provide evidence that the NS-CE activity is temporally coordinated with NMD-independent gene repression mechanisms. Our study provides an accessible workflow for the discovery and prioritization of AS-NMD targets. It further argues that the AS-NMD pathway plays a widespread role in developing neurons by facilitating the downregulation of functionally related non-neuronal genes.

molecular biology↗

bioRxiv2021

Neural stem cells alter nucleocytoplasmic partitioning and accumulate nuclear polyadenylated transcripts during quiescence

Quiescence is a cellular state characterised by reversible cell-cycle arrest and diminished biosynthetic activity that protects against environmental insults, replicative exhaustion and proliferation-induced mutations1. Entry into and exit from this state controls development, maintenance and repair of tissues plus, in the adult central nervous system, generation of new neurons and thus cognition and mood2-4. Cancer stem cells too can undergo quiescence, which confers them resistance to current therapies5, 6. Despite clinical relevance, quiescence is poorly understood and is defined functionally given lack of molecular markers. Decrease of the most resource-intensive cellular process of protein synthesis is a feature of quiescence, controlled across species and cell types by inhibition of the Target of Rapamycin (TOR) pathway1, 7. Here, we combine Drosophila genetics and a mammalian model to show that altered nucleocytoplasmic partitioning and nuclear accumulation of polyadenylated RNAs are novel evolutionarily conserved hallmarks of quiescence regulation. Furthermore, nuclear accumulation of messenger RNA (mRNA) in quiescent NSCs (qNSCs) largely predicts protein downregulation, accounting for uncoupling between transcriptome and proteome in quiescence. These mechanisms provide a previously unappreciated regulatory layer to reducing protein synthesis in quiescent cells, whilst priming them for reactivation in response to appropriate cues.

developmental biology↗

bioRxiv2020

Intronic enhancer region governs transcript-specific BDNF expression in neurons

Brain-derived neurotrophic factor (BDNF) controls the survival, growth, and function of neurons both during the development and in the adult nervous system. BDNF gene is transcribed from several distinct promoters generating transcripts with alternative 5 exons. BDNF transcripts initiated at the first cluster of exons have been associated with the regulation of body weight and various aspects of social behavior, but the mechanisms driving the expression of these transcripts have remained poorly understood. Here, we identify an evolutionarily conserved intronic enhancer region inside the BDNF gene that regulates both basal and stimulus-dependent expression of the BDNF transcripts starting from the first cluster of 5 exons in neurons. We further uncover a functional E-box element in the enhancer region, linking the expression of BDNF and various pro-neural basic helix-loop-helix transcription factors. Collectively, our results shed new light on the cell type- and stimulus-specific regulation of the important neurotrophic factor BDNF.

neuroscience↗

bioRxiv2020

A conserved role for SFPQ in repression of pathogenic cryptic last exons

The RNA-binding protein SFPQ plays an important role in neuronal development and has been associated with several neurodegenerative disorders, including ALS, FTLD, and Alzheimers Disease. Here, we report that loss of sfpq leads to premature termination of multiple transcripts due to widespread activation of previously unannotated cryptic last exons (CLEs). These CLEs appear preferentially in long introns of genes with neuronal functions and dampen gene expression outputs and/or give rise to short peptides interfering with the normal gene functions. We show that one such peptide encoded by the CLE-containing epha4b mRNA isoform is responsible for neurodevelopmental defects in the sfpq mutant. The uncovered CLE-repressive activity of SFPQ is conserved in mouse and human, and SFPQ-inhibited CLEs are found across ALS iPSC-derived neurons. These results greatly expand our understanding of SFPQ function and uncover a new gene regulation mechanism with wide relevance to human pathologies.

neuroscience↗