Influenza A viruses, causing seasonal epidemics and occasional pandemics, rely on interactions with host proteins for their RNA genome transcription and replication. The viral RNA polymerase utilizes host RNA polymerase II (Pol II) and interacts with the serine 5 phosphorylated (pS5) C-terminal domain (CTD) of Pol II to initiate transcription. Our study, using single-particle electron cryomicroscopy (cryo-EM), reveals the structure of the 1918 pandemic influenza A virus polymerase bound to a synthetic pS5 CTD peptide composed of four heptad repeats mimicking the 52 heptad repeat mammalian Pol II CTD. The structure shows that the CTD peptide binds at the C-terminal domain of the PA viral polymerase subunit (PA-C) and reveals a previously unobserved position of the 627 domain of the PB2 subunit near the CTD. We identify crucial residues of the CTD peptide that mediate interactions with positively charged cavities on PA-C, explaining the preference of the viral polymerase for pS5 CTD. Functional analysis of mutants targeting the CTD-binding site within PA-C reveals reduced transcriptional function or defects in replication, highlighting the multifunctional role of PA-C in viral RNA synthesis. Our study provides insights into the structural and functional aspects of the influenza virus polymerase-host Pol II interaction and identifies a target for antiviral development.IMPORTANCEUnderstanding the intricate interactions between influenza A viruses and host proteins is crucial for developing targeted antiviral strategies. This study employs advanced imaging techniques to uncover the structural nuances of the 1918 pandemic influenza A virus polymerase bound to a specific host protein, shedding light on the vital process of viral RNA synthesis. The study identifies key amino acid residues in the influenza polymerase involved in binding host polymerase II (Pol II) and highlights their role in both viral transcription and genome replication. These findings not only deepen our understanding of the influenza virus life cycle but also pinpoint a potential target for antiviral development. By elucidating the structural and functional aspects of the influenza virus polymerase-host Pol II interaction, this research provides a foundation for designing interventions to disrupt viral replication and transcription, offering promising avenues for future antiviral therapies.

• MeSH
• chřipka lidská virologie   MeSH
• elektronová kryomikroskopie * MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• lidé MeSH
• molekulární modely MeSH
• proteinové domény MeSH
• replikace viru MeSH
• RNA virová metabolismus   genetika   MeSH
• RNA-dependentní RNA-polymerasa * metabolismus   chemie   MeSH
• RNA-polymerasa II * metabolismus   chemie   MeSH
• vazba proteinů MeSH
• virové proteiny * metabolismus   chemie   genetika   MeSH
• virus chřipky A * metabolismus   genetika   enzymologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Double-strand breaks (DSBs) are the most severe type of DNA damage. Previously, we demonstrated that RNA polymerase II (RNAPII) phosphorylated at the tyrosine 1 (Y1P) residue of its C-terminal domain (CTD) generates RNAs at DSBs. However, the regulation of transcription at DSBs remains enigmatic. Here, we show that the damage-activated tyrosine kinase c-Abl phosphorylates hSSB1, enabling its interaction with Y1P RNAPII at DSBs. Furthermore, the trimeric SOSS1 complex, consisting of hSSB1, INTS3, and c9orf80, binds to Y1P RNAPII in response to DNA damage in an R-loop-dependent manner. Specifically, hSSB1, as a part of the trimeric SOSS1 complex, exhibits a strong affinity for R-loops, even in the presence of replication protein A (RPA). Our in vitro and in vivo data reveal that the SOSS1 complex and RNAPII form dynamic liquid-like repair compartments at DSBs. Depletion of the SOSS1 complex impairs DNA repair, underscoring its biological role in the R-loop-dependent DNA damage response.

• MeSH
• DNA vazebné proteiny * metabolismus   MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• RNA-polymerasa II * metabolismus   MeSH
• separace fází MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In contrast to the catalytic subunit of telomerase, its RNA subunit (TR) is highly divergent in size, sequence and biogenesis pathways across eukaryotes. Current views on TR evolution assume a common origin of TRs transcribed with RNA polymerase II in Opisthokonta (the supergroup including Animalia and Fungi) and Trypanosomida on one hand, and TRs transcribed with RNA polymerase III under the control of type 3 promoter, found in TSAR and Archaeplastida supergroups (including e.g. ciliates and Viridiplantae taxa, respectively). Here, we focus on unknown TRs in one of the largest Animalia order - Hymenoptera (Arthropoda) with more than 300 available representative genomes. Using a combination of bioinformatic and experimental approaches, we identify their TRs. In contrast to the presumed type of TRs (H/ACA box snoRNAs transcribed with RNA Polymerase II) corresponding to their phylogenetic position, we find here short TRs of the snRNA type, likely transcribed with RNA polymerase III under the control of the type 3 promoter. The newly described insect TRs thus question the hitherto assumed monophyletic origin of TRs across Animalia and point to an evolutionary switch in TR type and biogenesis that was associated with the divergence of Arthropods.

• MeSH
• Eukaryota genetika   MeSH
• fylogeneze MeSH
• Hymenoptera * genetika   MeSH
• konformace nukleové kyseliny MeSH
• RNA-polymerasa II genetika   metabolismus   MeSH
• RNA-polymerasa III genetika   metabolismus   MeSH
• RNA genetika   MeSH
• rostliny genetika   MeSH
• telomerasa * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.

• MeSH
• aktivace lymfocytů MeSH
• Burkittův lymfom genetika   imunologie   MeSH
• cytidindeaminasa genetika   MeSH
• genetická transkripce MeSH
• imunoglobuliny genetika   metabolismus   MeSH
• kur domácí MeSH
• lidé MeSH
• mutace genetika   MeSH
• mutageneze cílená MeSH
• podskupiny B-lymfocytů imunologie   MeSH
• ptačí proteiny genetika   metabolismus   MeSH
• RNA-polymerasa II genetika   metabolismus   MeSH
• rozmanitost protilátek MeSH
• somatická hypermutace imunoglobulinových genů MeSH
• zesilovače transkripce genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

MIR139 is a tumor suppressor and is commonly silenced in acute myeloid leukemia (AML). However, the tumor-suppressing activities of miR-139 and molecular mechanisms of MIR139-silencing remain largely unknown. Here, we studied the poorly prognostic MLL-AF9 fusion protein-expressing AML. We show that MLL-AF9 expression in hematopoietic precursors caused epigenetic silencing of MIR139, whereas overexpression of MIR139 inhibited in vitro and in vivo AML outgrowth. We identified novel miR-139 targets that mediate the tumor-suppressing activities of miR-139 in MLL-AF9 AML. We revealed that two enhancer regions control MIR139 expression and found that the polycomb repressive complex 2 (PRC2) downstream of MLL-AF9 epigenetically silenced MIR139 in AML. Finally, a genome-wide CRISPR-Cas9 knockout screen revealed RNA Polymerase 2 Subunit M (POLR2M) as a novel MIR139-regulatory factor. Our findings elucidate the molecular control of tumor suppressor MIR139 and reveal a role for POLR2M in the MIR139-silencing mechanism, downstream of MLL-AF9 and PRC2 in AML. In addition, we confirmed these findings in human AML cell lines with different oncogenic aberrations, suggesting that this is a more common oncogenic mechanism in AML. Our results may pave the way for new targeted therapy in AML.

• MeSH
• akutní myeloidní leukemie genetika   MeSH
• epigeneze genetická MeSH
• fúzní onkogenní proteiny genetika   MeSH
• karcinogeneze genetika   MeSH
• lidé MeSH
• mikro RNA genetika   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• nádorové buněčné linie MeSH
• protoonkogenní protein MLL genetika   MeSH
• regulace genové exprese u leukemie MeSH
• RNA-polymerasa II genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

[Figure: see text].

• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• elongace genetické transkripce * MeSH
• exprese genu MeSH
• interakční proteinové domény a motivy genetika   MeSH
• lidé MeSH
• mapy interakcí proteinů MeSH
• molekulární modely MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• proteinové domény MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• RNA-polymerasa II chemie   metabolismus   MeSH
• transkripční elongační faktory chemie   metabolismus   MeSH
• transkripční faktory chemie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• vnitřně neuspořádané proteiny chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.

• MeSH
• buněčné linie MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• genový knockdown MeSH
• lidé MeSH
• myši knockoutované MeSH
• neurony chemie   metabolismus   MeSH
• posttranskripční úpravy RNA MeSH
• proteinové domény MeSH
• regulace genové exprese MeSH
• RNA-polymerasa II chemie   genetika   metabolismus   MeSH
• RNA * chemie   genetika   metabolismus   MeSH
• stabilita RNA MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats.

• MeSH
• genetická transkripce MeSH
• konformace nukleové kyseliny MeSH
• molekulární evoluce * MeSH
• mutace MeSH
• RNA rostlin biosyntéza   chemie   genetika   MeSH
• RNA-polymerasa II metabolismus   MeSH
• RNA-polymerasa III metabolismus   MeSH
• RNA biosyntéza   chemie   genetika   MeSH
• sekvenční seřazení MeSH
• telomerasa biosyntéza   chemie   genetika   MeSH
• telomery chemie   MeSH
• transkriptom MeSH
• Viridiplantae genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery have fundamental functional consequences for gene expression, its detailed studies have been abrogated by the limits of classical light microscopy for a long time. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.

• MeSH
• fluorescenční mikroskopie * metody   MeSH
• genetická transkripce * MeSH
• lidé MeSH
• regulace genové exprese * MeSH
• RNA-polymerasa II metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• vazba proteinů MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Here, we provide evidence for the presence of Myosin phosphatase rho-interacting protein (MPRIP), an F-actin-binding protein, in the cell nucleus. The MPRIP protein binds to Phosphatidylinositol 4,5-bisphosphate (PIP2) and localizes to the nuclear speckles and nuclear lipid islets which are known to be involved in transcription. We identified MPRIP as a component of RNA Polymerase II/Nuclear Myosin 1 complex and showed that MPRIP forms phase-separated condensates which are able to bind nuclear F-actin fibers. Notably, the fibrous MPRIP preserves its liquid-like properties and reforms the spherical shaped condensates when F-actin is disassembled. Moreover, we show that the phase separation of MPRIP is driven by its long intrinsically disordered region at the C-terminus. We propose that the PIP2/MPRIP association might contribute to the regulation of RNAPII transcription via phase separation and nuclear actin polymerization.

• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• aktiny metabolismus   MeSH
• buněčné jádro účinky léků   metabolismus   MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• glykoly farmakologie   MeSH
• lidé MeSH
• myosin typu I metabolismus   MeSH
• nádorové buněčné linie MeSH
• proteinové domény MeSH
• RNA-polymerasa II metabolismus   MeSH
• subcelulární frakce metabolismus   MeSH
• vazba proteinů účinky léků   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH