The RNA-binding protein La is found in most eukaryotes, and despite being essential in many organisms, its function is not completely clear. Trypanosoma brucei, the causative agent of human African trypanosomiasis, encodes a 'classical' La protein (TbLa) composed of a La-motif, two RNA recognition motifs (RRM1 and RRM2α), a C-terminal short basic motif (SBM), and a nuclear localization signal (NLS). In T. brucei, like in most eukaryotes, position 34 of tRNATyr, -Asp, -Asn and -His is modified with queuosine (Q34). The steady-state levels of queuosine-modified tRNA in the insect form (procyclic) of T. brucei can fluctuate dynamically depending on growth conditions, but the mechanism(s) controlling Q34 levels are not well understood. A well-established function of La is in precursor-tRNA 3'-end metabolism, but in this work, we demonstrate that La also controls Q34-tRNA levels. Individual domain deletions showed that while deletion of La motif or RRM1 causes dysregulation of Q34-tRNA levels, no other domain plays a similar role. We also show that La is important for the normal balance of several additional tRNA modifications. These findings are discussed in the context of substrate competition between La and modification enzymes, also highlighting subcellular localization as a key determinant of tRNA function.

• MeSH
• nukleosid Q metabolismus   analogy a deriváty   MeSH
• posttranskripční úpravy RNA * MeSH
• proteinové domény MeSH
• proteiny vázající RNA * metabolismus   chemie   genetika   MeSH
• protozoální proteiny * metabolismus   chemie   genetika   MeSH
• RNA transferová * metabolismus   genetika   MeSH
• Trypanosoma brucei brucei * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• nukleosid Q MeSH
• proteiny vázající RNA * MeSH
• protozoální proteiny * MeSH
• RNA transferová * MeSH

RNA recognition motifs (RRMs) are a key class of proteins that primarily bind single-stranded RNAs. In this study, we applied standard atomistic molecular dynamics simulations to obtain insights into the intricate binding dynamics between uridine-rich RNAs and TbRGG2 RRM using the recently developed OL3-Stafix AMBER force field, which improves the description of single-stranded RNA molecules. Complementing structural experiments that unveil a primary binding mode with a single uridine bound, our simulations uncover two supplementary binding modes in which adjacent nucleotides encroach upon the binding pocket. This leads to a unique molecular mechanism through which the TbRGG2 RRM is capable of rapidly transitioning the U-rich sequence. In contrast, the presence of non-native cytidines induces stalling and destabilization of the complex. By leveraging extensive equilibrium dynamics and a large variety of binding states, TbRGG2 RRM effectively expedites diffusion along the RNA substrate while ensuring robust selectivity for U-rich sequences despite featuring a solitary binding pocket. We further substantiate our description of the complex dynamics by simulating the fully spontaneous association process of U-rich sequences to the TbRGG2 RRM. Our study highlights the critical role of dynamics and auxiliary binding states in interface dynamics employed by RNA-binding proteins, which is not readily apparent in traditional structural studies but could represent a general type of binding strategy employed by many RNA-binding proteins.

• MeSH
• konformace nukleové kyseliny MeSH
• motiv rozpoznávající RNA * MeSH
• proteiny vázající RNA * chemie   metabolismus   MeSH
• RNA * metabolismus   chemie   MeSH
• simulace molekulární dynamiky * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny vázající RNA * MeSH
• RNA * MeSH

Repression of msl-2 mRNA translation is essential for viability of Drosophila melanogaster females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3' untranslated region (UTR) of the msl-2 transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with msl-2 are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of msl-2 3' UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to msl-2.

• Klíčová slova
• Dosage compensation, Hrp48, RNA binding protein, RNA recognition motif, Translation regulation,
• MeSH
• 3' nepřekládaná oblast * MeSH
• DNA vazebné proteiny MeSH
• Drosophila melanogaster * metabolismus   genetika   MeSH
• heterogenní jaderné ribonukleoproteiny MeSH
• messenger RNA metabolismus   genetika   chemie   MeSH
• proteiny Drosophily * metabolismus   chemie   genetika   MeSH
• proteiny vázající RNA * metabolismus   chemie   genetika   MeSH
• proteosyntéza MeSH
• simulace molekulární dynamiky MeSH
• transkripční faktory metabolismus   chemie   genetika   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3' nepřekládaná oblast * MeSH
• DNA vazebné proteiny MeSH
• heterogenní jaderné ribonukleoproteiny MeSH
• Hrb27C protein, Drosophila MeSH Prohlížeč
• messenger RNA MeSH
• msl-2 protein, Drosophila MeSH Prohlížeč
• proteiny Drosophily * MeSH
• proteiny vázající RNA * MeSH
• transkripční faktory MeSH

Atomic force microscopy (AFM) has recently received increasing interest in molecular biology. This technique allows quick and reliable detection of biomolecules. However, studying RNA-protein complexes using AFM poses significant challenges. Here, we describe a simple and reliable method to visualize positively charged proteins bound to RNA that does not require metallic cations. This method allowed us to effectively detect and visualize Staufen-RNA complexes by height or logarithmic stiffness. The study of the mechanical properties is particularly important in the case of protein-coated RNA complexes, where RNA cannot be detected by height channel. In any case, it is necessary to compare AFM data with the data derived from other techniques like nuclear magnetic resonance, X-ray crystallography, cryogenic electron microscopy, and small-angle X-ray scattering. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Preparation and visualization of RNA-protein complex.

• Klíčová slova
• AFM, RNA–protein complex formation, Staufen,
• MeSH
• mikroskopie atomárních sil * metody   MeSH
• proteiny vázající RNA * chemie   metabolismus   MeSH
• RNA * chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny vázající RNA * MeSH
• RNA * MeSH

N6-Methyladenosine (m6A) is a prevalent RNA post-transcriptional modification that plays crucial roles in RNA stability, structural dynamics, and interactions with proteins. The YT521-B (YTH) family of proteins, which are notable m6A readers, functions through its highly conserved YTH domain. Recent structural investigations and molecular dynamics (MD) simulations have shed light on the mechanism of recognition of m6A by the YTHDC1 protein. Despite advancements, using MD to predict the stabilization induced by m6A on the free energy of binding between RNA and YTH proteins remains challenging due to inaccuracy of the employed force field and limited sampling. For instance, simulations often fail to sufficiently capture the hydration dynamics of the binding pocket. This study addresses these challenges through an innovative methodology that integrates metadynamics, alchemical simulations, and force-field refinement. Importantly, our research identifies hydration of the binding pocket as giving only a minor contribution to the binding free energy and emphasizes the critical importance of precisely tuning force-field parameters to experimental data. By employing a fitting strategy built on alchemical calculations, we refine the m6A partial charge parameters, thereby enabling the simultaneous reproduction of N6 methylation on both the protein binding free energy and the thermodynamic stability of nine RNA duplexes. Our findings underscore the sensitivity of binding free energies to partial charges, highlighting the necessity for thorough parametrization and validation against experimental observations across a range of structural contexts.

• MeSH
• adenosin * analogy a deriváty   chemie   metabolismus   MeSH
• metylace MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• RNA * chemie   metabolismus   MeSH
• simulace molekulární dynamiky * MeSH
• termodynamika * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosin * MeSH
• N-methyladenosine MeSH Prohlížeč
• proteiny vázající RNA MeSH
• RNA * MeSH

Z-DNA and Z-RNA are functionally important left-handed structures of nucleic acids, which play a significant role in several molecular and biological processes including DNA replication, gene expression regulation and viral nucleic acid sensing. Most proteins that have been proven to interact with Z-DNA/Z-RNA contain the so-called Zα domain, which is structurally well conserved. To date, only eight proteins with Zα domain have been described within a few organisms (including human, mouse, Danio rerio, Trypanosoma brucei and some viruses). Therefore, this paper aimed to search for new Z-DNA/Z-RNA binding proteins in the complete PDB structures database and from the AlphaFold2 protein models. A structure-based similarity search found 14 proteins with highly similar Zα domain structure in experimentally-defined proteins and 185 proteins with a putative Zα domain using the AlphaFold2 models. Structure-based alignment and molecular docking confirmed high functional conservation of amino acids involved in Z-DNA/Z-RNA, suggesting that Z-DNA/Z-RNA recognition may play an important role in a variety of cellular processes.

• Klíčová slova
• Z-DNA, Z-RNA, Zα domain, bioinformatics, protein binding,
• MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• interakční proteinové domény a motivy * MeSH
• konformace nukleové kyseliny MeSH
• konformace proteinů MeSH
• molekulární modely * MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• RNA chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Z-DNA chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• proteiny vázající RNA MeSH
• RNA MeSH
• Z-DNA MeSH

During eukaryotic transcription elongation, RNA polymerase II (RNAP2) is regulated by a chorus of factors. Here, we identified a common binary interaction module consisting of TFIIS N-terminal domains (TNDs) and natively unstructured TND-interacting motifs (TIMs). This module was conserved among the elongation machinery and linked complexes including transcription factor TFIIS, Mediator, super elongation complex, elongin, IWS1, SPT6, PP1-PNUTS phosphatase, H3K36me3 readers, and other factors. Using nuclear magnetic resonance, live-cell microscopy, and mass spectrometry, we revealed the structural basis for these interactions and found that TND-TIM sequences were necessary and sufficient to induce strong and specific colocalization in the crowded nuclear environment. Disruption of a single TIM in IWS1 induced robust changes in gene expression and RNAP2 elongation dynamics, which underscores the functional importance of TND-TIM surfaces for transcription elongation.

• 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
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• DNA vazebné proteiny MeSH
• Iws1 protein, human MeSH Prohlížeč
• PPP1R10 protein, human MeSH Prohlížeč
• proteiny vázající RNA MeSH
• PSIP1 protein, human MeSH Prohlížeč
• RNA-polymerasa II MeSH
• transcription factor S-II MeSH Prohlížeč
• transkripční elongační faktory MeSH
• transkripční faktory MeSH
• vnitřně neuspořádané proteiny MeSH

Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.

• Klíčová slova
• axonal transport, cell size regulation, local translation, protein-membrane interaction, subcellular localization,
• MeSH
• axonální transport genetika   MeSH
• buněčné jadérko metabolismus   ultrastruktura   MeSH
• exprese genu MeSH
• fosfoproteiny chemie   genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• kineziny genetika   metabolismus   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• mutace MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nervus ischiadicus cytologie   metabolismus   MeSH
• neurony cytologie   metabolismus   MeSH
• nukleolin MeSH
• primární buněčná kultura MeSH
• proteinové domény MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• spinální ganglia cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fosfoproteiny MeSH
• kineziny MeSH
• Kns2 protein, mouse MeSH Prohlížeč
• messenger RNA MeSH
• proteiny vázající RNA MeSH

BACKGROUND: Eukaryotic gene expression is controlled by a number of RNA-binding proteins (RBP), such as the proteins from the Puf (Pumilio and FBF) superfamily (PufSF). These proteins bind to RNA via multiple Puf repeat domains, each of which specifically recognizes a single RNA base. Recently, three diversified PufSF proteins have been described in model organisms, each of which is responsible for the maturation of ribosomal RNA or the translational regulation of mRNAs; however, less is known about the role of these proteins across eukaryotic diversity. RESULTS: Here, we investigated the distribution and function of PufSF RBPs in the tree of eukaryotes. We determined that the following PufSF proteins are universally conserved across eukaryotes and can be broadly classified into three groups: (i) Nop9 orthologues, which participate in the nucleolar processing of immature 18S rRNA; (ii) 'classical' Pufs, which control the translation of mRNA; and (iii) PUM3 orthologues, which are involved in the maturation of 7S rRNA. In nearly all eukaryotes, the rRNA maturation proteins, Nop9 and PUM3, are retained as a single copy, while mRNA effectors ('classical' Pufs) underwent multiple lineage-specific expansions. We propose that the variation in number of 'classical' Pufs relates to the size of the transcriptome and thus the potential mRNA targets. We further distinguished full set of PufSF proteins in divergent metamonad Giardia intestinalis and initiated their cellular and biochemical characterization. CONCLUSIONS: Our data suggest that the last eukaryotic common ancestor (LECA) already contained all three types of PufSF proteins and that 'classical' Pufs then underwent lineage-specific expansions.

• Klíčová slova
• Giardia intestinalis, LECA, Puf superfamily proteins, RNA processing, RNA-binding protein,
• MeSH
• Eukaryota genetika   metabolismus   MeSH
• fylogeneze MeSH
• messenger RNA metabolismus   MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• proteosyntéza * MeSH
• RNA ribozomální 18S metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• proteiny vázající RNA MeSH
• RNA ribozomální 18S MeSH

Precise guided pollen tube growth by the female gametophyte is a prerequisite for successful sexual reproduction in flowering plants. Cysteine-rich proteins (CRPs) secreted from the embryo sac are known pollen tube attractants perceived by pollen tube receptor-like kinases. How pre-mRNA splicing facilitates this cell-to-cell communication is not understood. Here, we report a novel function of Pre-mRNA PROCESSING factor 8 paralogs, PRP8A and PRP8B, as regulators of pollen tube attraction. Double mutant prp8a prp8b ovules cannot attract pollen tubes, and prp8a prp8b pollen tubes fail to sense the ovule's attraction signals. Only 3% of ovule-expressed genes were misregulated in prp8a prp8b Combination of RNA sequencing and the MYB98/LURE1.2-YFP reporter revealed that the expression of MYB98, LUREs and 49 other CRPs were downregulated, suggesting loss of synergid cell fate. Differential exon usage and intron retention analysis revealed autoregulation of PPR8A/PRP8B splicing. In vivo, PRP8A co-immunoprecipitates with splicing enhancer AtSF3A1, suggesting involvement of PRP8A in 3'-splice site selection. Our data hint that the PRP8A/PRP8B module exhibits spliceosome autoregulation to facilitate pollen tube attraction via transcriptional regulation of MYB98, CRPs and LURE pollen tube attractants.

• Klíčová slova
• Cell-to-cell signaling, Cysteine-rich proteins, Pollen tube attraction and reception, Post-transcriptional regulation, Pre-mRNA PROCESSING factor 8, Sexual reproduction, Splicing,
• MeSH
• Arabidopsis metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• geneticky modifikované rostliny metabolismus   MeSH
• místa sestřihu RNA MeSH
• mutageneze MeSH
• podjednotky proteinů genetika   metabolismus   MeSH
• proteiny huseníčku chemie   genetika   metabolismus   MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• pylová láčka růst a vývoj   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• sestřihové faktory genetika   metabolismus   MeSH
• spliceozomy metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• místa sestřihu RNA MeSH
• MYB98 protein, Arabidopsis MeSH Prohlížeč
• podjednotky proteinů MeSH
• proteiny huseníčku MeSH
• proteiny vázající RNA MeSH
• sestřihové faktory MeSH
• transkripční faktory MeSH