Biology subjects

Stanek, D.

Publications and source records attributed to Stanek, D..

8 recordsLinked to original sources

bioRxiv2026

Molecular mechanism of coilin interaction with core snRNPs

Cajal bodies (CBs) are nuclear membrane-less organelles that accumulate various short non-coding RNAs and facilitate their biogenesis. They also function in quality control during the assembly of small nuclear ribonucleoprotein particles (snRNPs), sequestering immature or defective complexes. In this paper, we show that coilin, the key scaffolding protein of CBs, is the factor that discriminates between mature and immature snRNPs. We provide evidence that the C-terminus of coilin contains a bipartite snRNP-interaction module composed of a nonspecific RNA-binding region formed by RG repeats and a Tudor-like domain that interacts specifically with Sm proteins. The Tudor-like domain contains two conserved loops that protrude from the core barrel and bind the Sm proteins E, F, and G. Both the RNA-binding and Sm-binding regions are essential for productive interactions between coilin and core snRNPs, providing a molecular explanation for the specificity of coilin-mediated sequestration of immature snRNPs in Cajal bodies.

molecular biology↗

bioRxiv2025

PRPF8-associated retinitis pigmentosa variant induces human neural retina-autonomous photoreceptor defects

Retinitis pigmentosa (RP) is an inherited retinal disorder characterized by the progressive loss of photoreceptors that currently lacks effective treatment. Here, we investigated the impact of the pathogenic PRPF8-Y2334N variant on neural retina cells in hiPSC-derived retinal organoids. Expression of this variant resulted in photoreceptor defects, including thinning of the outer segment layer. At the molecular level, we observed relatively minor changes in mRNA expression in multiple retinal cells, indicating that neural retina cells are impacted independently of retinal pigment epithelium (RPE). We found splicing alterations in genes associated with neural and retinal diseases, including those involved in intraflagellar transport, suggesting that these genes may represent common targets of splicing factor mutations. We further detected the misexpression of several circular RNAs (circRNAs), which could serve as early biomarkers of splicing defects caused by RP mutations. Together, we present a model of RP that recapitulates photoreceptor degeneration and demonstrates that these defects are independent of RPE erosion.

cell biology↗

bioRxiv2024

Rapid depletion and super-resolution microscopy reveal an unexpected role of the nuclear-speckle protein SRSF5 in paraspeckle assembly and dynamics during cellular stress

Nuclear speckles (NS) and paraspeckles (PS) are adjacent condensates with distinct protein composition, with serine-arginine-rich splicing factors (SRSFs) concentrated in NS. Surprisingly, we find that SRSF5 is present in both. Combining super-resolution imaging, proximity proteomics and iCLIP, we show that SRSF5 binds with PS core proteins to the PS-scaffold RNA NEAT1 and locates between PS spheres. Acute SRSF5 depletion results in reduced PS with differently packaged NEAT1. Under stress, SRSF5s association with PS increases, and without SRSF5, PS cluster assembly is impaired. Interfering with binding to purine-rich RNAs even causes PS-NS fusion. In an intriguing over-compensation, longer SRSF5 depletion reduces TDP-43 levels via premature polyadenylation, leading to NEAT1 isoform switching and more PS. We propose that SRSF5 forms a stress-specific PS shell and acts as a glue for PS clusters. Additionally, we uncover SRSF5 as a novel regulator of TDP-43 and demonstrate how acute depletion distinguishes direct from compensatory effects. HighlightsO_LINS protein SRSF5 associates with PS shells and enriches between PS spheres C_LIO_LISRSF5 binds the PS-scaffold RNA NEAT1 and ensures proper NEAT1 packaging C_LIO_LISRSF5 association with PS increases under stress and promotes cluster formation C_LIO_LIInterfering with binding to purine-rich RNAs causes the fusion of PS and NS C_LIO_LIAcute SRSF5 depletion reveals compensatory effects on PS assembly and dynamics C_LIO_LISRSF5 regulates TDP-43 levels via premature polyadenylation C_LI

cell biology↗

bioRxiv2022

HIV-1 infection reduces NAD capping of host cell snRNA and snoRNA

Nicotinamide adenine dinucleotide (NAD) is a critical component of the cellular metabolism and also serves as an alternative 5' cap on various RNAs. However, the function of the NAD RNA cap is still under investigation. We studied NAD capping of RNAs in HIV-1-infected cells because HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causing intracellular pellagra. By applying the NAD captureSeq protocol to HIV-1-infected and uninfected cells, we revealed that four snRNAs (e.g. U1) and four snoRNAs lost their NAD cap when infected with HIV-1. Here, we provide evidence that the presence of the NAD cap decreases the stability of the U1/HIV-1 pre-mRNA duplex. Additionally, we demonstrate that reducing the quantity of NAD-capped RNA by overexpressing the NAD RNA decapping enzyme DXO results in an increase in HIV-1 infectivity. This suggests that NAD capping is unfavorable for HIV-1 and plays a role in its infectivity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/515957v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@16d5cf0org.highwire.dtl.DTLVardef@f0bc60org.highwire.dtl.DTLVardef@df83bborg.highwire.dtl.DTLVardef@41a2db_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Retinitis pigmentosa associated mutations in mouse Prpf8 cause misexpression of circRNAs and degeneration of cerebellar granule neurons

A subset of patients suffering from a familial retinitis pigmentosa (RP) carry mutations in several spliceosomal components including PRPF8 protein. Here, we established two novel alleles of murine Prpf8 that genocopy or mimic aberrant PRPF8 found in RP patients - the substitution p.Tyr2334Asn and an extended protein variant p.Glu2331ValfsX15. Homozygous mice expressing either of the aberrant Prpf8 variants developed within first 2 months progressive atrophy of the cerebellum due to extensive granule neuron loss. Comparison of transcriptome from pre-degenerative and degenerative tissues revealed a subset of circRNAs that were deregulated in all tissues and both Prpf8-RP mouse strains. To identify potential risk factors that sensitize cerebellum for Prpf8 mutations we monitored expression of several splicing proteins during first eight weeks. We observed downregulation of all selected splicing proteins in wild-type cerebellum, which coincided with neurodegeneration onset. The decrease in splicing protein expression was further pronounced in mouse strains expressing mutated Prpf8. Collectively, we propose a model where physiological reduction of spliceosomal components during postnatal tissue maturation sensitizes cells to expression of aberrant Prpf8 and the subsequent deregulation of circRNAs triggers neuron death.

neuroscience↗

bioRxiv2022

Genomic deletions and rearrangements in monkeypox virus from the 2022 outbreak, USA

Genomic surveillance of monkeypox virus (MPXV) during the 2022 outbreak has been mainly focused on single nucleotide polymorphism (SNP) changes. DNA viruses, including MPXV, have a lower SNP mutation rate than RNA viruses due to higher fidelity replication machinery. We identified a large genomic rearrangement in a MPXV sequence from a 2022 case in the state of Minnesota (MN), USA, from an abnormal, uneven MPXV read mapping coverage profile in whole-genome sequencing (WGS) data. We further screened WGS data of 206 U.S. MPXV samples and found seven (3.4 percent) sequenced genomes contained similar abnormal read coverage profiles that suggested putative large deletions or genomic rearrangements. Here, we present three MPXV genomes containing deletions ranging from 2.3 to 15 kb and four genomes containing more complex rearrangements. Five genomic changes were each only seen in one sample, but two sequences from linked cases shared an identical 2.3 kb deletion in the 3 terminal region. All samples were positive using VAC1 and Clade II (formerly West African)-specific MPXV diagnostic tests; however, large deletions and genomic rearrangements like the ones reported here have the potential to result in viruses in which the target of a PCR diagnostic test is deleted. The emergence of genomic rearrangements during the outbreak may have public health implications and highlight the importance of continued genomic surveillance.

genomics↗

bioRxiv2021

SATB1 undergoes isoform-specific phase transitions in T cells

Intra-thymic T cell development is coordinated by the regulatory actions of SATB1 genome organizer. In this report, we show that SATB1 is involved in the regulation of transcription and splicing, both of which displayed deregulation in Satb1 knockout murine thymocytes. More importantly, we characterized a novel SATB1 protein isoform and described its distinct biophysical behavior, implicating potential functional differences compared to the commonly studied isoform. SATB1 utilized its prion-like domains to transition through liquid-like states to aggregated structures. This behavior was dependent on protein concentration as well as phosphorylation and interaction with nuclear RNA. Notably, the long SATB1 isoform was more prone to aggregate following phase separation. Thus, the tight regulation of SATB1 isoforms expression levels alongside with protein post-translational modifications, are imperative for SATB1s mode of action in T cell development. Our data indicate that deregulation of these processes may also be linked to disorders such as cancer.

molecular biology↗

bioRxiv2018

International travelers and genomics uncover a ‘hidden’ Zika outbreak

The ongoing Zika epidemic in the Americas has challenged public health surveillance, response, and control systems. Even as the epidemic appears to be near its end in the Americas, it is unclear whether substantial Zika virus transmission may still be ongoing. This issue is exacerbated by large discrepancies in local case reporting and significant delays in detecting outbreaks due to surveillance gaps. To uncover locations with lingering outbreaks in the Americas, we investigated travel-associated Zika cases diagnosed in the United States and Europe to identify signatures of transmission dynamics that were not captured by local reporting. We found that a large and unreported Zika outbreak occurred in Cuba during 2017, a year after peak transmission in neighboring countries, with cases still appearing in 2018. By sequencing Zika virus from infected travelers, we show that the 2017 outbreak in Cuba was sparked by long-lived lineages of Zika virus introduced from multiple places in the Americas a year prior. Our data suggest that while aggressive mosquito control in Cuba may initially have been effective at mitigating Zika virus transmission, in the absence of vaccines, herd immunity, or strong international coordination, such control measures may need to be maintained to be effective. Our study highlights how Zika virus may still be silently spreading in the Americas and provides a framework for more accurately understanding outbreak dynamics.

epidemiology↗