Bioinformatic and experimental characterization of the RBM15 RNA binding protein.

Designing synthetic biomolecular condensates, or membraneless organelles, offers insights into the functions of their natural counterparts and is equally valuable for cellular and metabolic engineering. Choosing E. coli for its biotechnological relevance, we deploy RNA nanotechnology to design and express non-natural membraneless organelles in vivo. The designer condensates assemble co-transcriptionally from branched RNA motifs interacting via base-pairing. Exploiting binding selectivity, we express orthogonal, non-mixing condensates, and by embedding a protein-binding aptamer, we achieve selective protein recruitment. Condensates can be made to dissolve and reassemble upon thermal cycling, thereby reversibly releasing and re-capturing protein clients. The synthetic organelles are expressed robustly across the cell population and remain stable despite enzymatic RNA processing. Compared with existing solutions based on peptide building blocks or repetitive RNA sequences, these nanostructured RNA motifs enable algorithmic control over interactions, affinity for clients, and condensate microstructure, opening further directions in synthetic biology and biotechnology.

Riboswitches are non-coding RNA motifs that regulate gene expression in response to ligand binding. The glycine tandem riboswitch (GTR) contains two glycine aptamers that interact extensively, driving conformational changes in the downstream expression platform to control gene expression. Despite numerous studies, the role of glycine and RNA folding pathways in co-transcriptional regulation remains unclear. Here, we integrate single-molecule kinetic analysis, co-transcriptional RNA structure probing, and modeling to reveal that the GTR processes multiple molecular inputs sequentially, guided by polymerase pausing. Our findings elucidate its stepwise 5'-to-3' folding pathway and demonstrate how sequential glycine binding to each aptamer, K+ binding to a kink-turn, non-native folding intermediates, inter-aptamer docking driving binding site pre-organization, and modulation by transcription factor NusA collectively orchestrate co-transcriptional gene regulation. These results support a model wherein glycine binding cooperativity arises through non-equilibrium mechanisms, rather than a classical concerted model.

Hepatocellular carcinoma (HCC) is a highly lethal malignancy with obvious heterogeneity features. This study aims to identify circRNAs with key roles in promoting HCC malignancy and stemness properties. Through circRNA profiling comparison in HCC patients and functional screening in HCC cells, circRSU1 emerges as the top candidate. It exhibits a significant higher expression level in tumor tissues compared to adjacent non-tumor liver tissues from HCC patients. Functionally, circRSU1 promotes a spectrum of HCC malignant phenotypes both in vitro and in vivo, including an enrichment of CD24positive cancer stem cell population. Mechanistically, circRSU1 interacts with heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) via two RNA motifs on circRSU1 and two RNA-binding domains on hnRNPA1. This interaction increases hnRNPA1 protein level via reducing its proteasomal degradation. Furthermore, hnRNPA1 enhances HIF-1α protein translation via binding to its internal ribosome entry site (IRES), which subsequently increases the CD24positive cell population. Additionally, circRSU1 further enhances this process not only through increasing the hnRNPA1 protein level, but also through enhancing the interaction of hnRNPA1 with HIF1A IRES, consequently augmenting the CD24positive cell population and the associated malignancy/stemness features of HCC cells. Together, circRSU1 activates the hnRNPA1/HIF-1α/CD24 signaling axis, leading to the increased HCC malignancy and stemness features.

The emergence of a chemical system capable of self-replication and evolution is a critical event in the origin of life. RNA polymerase ribozymes can replicate RNA, but their large size and structural complexity impede self-replication and preclude their spontaneous emergence. Here we describe QT45: a 45-nucleotide polymerase ribozyme, discovered from random sequence pools, that catalyzes general RNA-templated RNA synthesis using trinucleotide triphosphate (triplet) substrates in mildly alkaline eutectic ice. QT45 can synthesize both its complementary strand using a random triplet pool at 94.1% per-nucleotide fidelity, and a copy of itself using defined substrates, both with yields of ~0.2% in 72 days. The discovery of polymerase activity in a small RNA motif suggests that polymerase ribozymes are more abundant in RNA sequence space than previously thought.

During mammalian aging, there are changes in abundance of noncoding RNAs including microRNAs, long noncoding RNAs, and circular RNAs. Although global profiles of the human transcriptome and epitranscriptome during the aging process are available, the existence and function of mitochondrial circular RNAs originating from the mitochondrial genome are poorly studied. Here, we report profiles of circular RNAs annotated to mitochondrial chromosome, chrM, in young and old cohorts. The most abundant circular RNA junctions are found in MT-RNR2, whose level is depleted in old cohorts and senescent fibroblast. The mitochondria-localized RNA-binding protein GRSF1 binds various mitochondrial transcripts, including linear and circular MT-RNR2, with a distinct RNA motif. Linear and circular MT-RNR2 bind a subset of TCA cycle enzymes, suggesting their possible function in regulating glucose metabolism in mitochondria to preserve proliferating status in young cohorts. In human fibroblasts, depletion of GRSF1 reduced levels of circMT-RNR2 and fumarate/succinate, concomitantly accelerating cellular senescence and mitochondrial dysfunction. Taken together, our findings demonstrate the existence and possible function of circular MT-RNR2 during human aging and senescence, implicating its role in promoting the TCA cycle.

Extracellular vesicles (EVs) deliver genetic material to recipient cells, thereby influencing immunity, development, and various diseases. However, the precise mechanism by which specific mRNAs are selected for packaging into EVs remains poorly understood. This study identifies Nucleophosmin 1 (NPM1) as a key protein responsible for sorting mRNAs into EVs. NPM1 achieves this by binding to specific RNA motifs, including those within mRNAs such as the epidermal growth factor receptor(EGFR), thereby initiating the formation of phase separation condensates containing intracellular RNA. These condensates are subsequently packaged into late endosomes and multivesicular bodies, facilitating their loading into EVs. Additionally, EVs derived from the serum of non-small cell lung cancer (NSCLC) patients exhibit elevated levels of NPM1 protein and EGFR mRNA, suggesting relevance for this transportation mechanism to NSCLC pathogenesis. This study, therefore, not only enhances the understanding of the molecular mechanism underlying mRNA sorting into EVs but also provides valuable insights for potential therapeutic strategies targeting NSCLC.

The RNA thermometer motif ROSE-G regulates ABC transporter gene expression in bacteria.

The long non-coding RNA MALAT1 encodes a micropeptide that promotes influenza A virus replication by suppressing innate immune responses.

Artificial biomolecular condensates have emerged as powerful tools to control cellular behaviors. Here we introduce a method to build artificial condensates within living mammalian cells through the design of modular RNA motifs formed by a single, short strand of RNA. These condensates emerge spontaneously, creating RNA-rich compartments that remain separated from their surrounding environment. The RNA sequences include stem-loop domains that fold as the RNA is transcribed, and then condense in the nucleus and cytoplasm through loop-loop interactions. These sequences can be optimized and diversified, enabling the generation of distinct, non-mixing condensate populations and the programmable control of their subcellular localization. The RNA motifs can also be modified to recruit small molecules, proteins, and RNA molecules in a sequence-specific manner to the RNA-rich phase. By introducing additional RNAs that link two distinct types of condensates, we can create droplets with multiple subcompartments, whose organization can be controlled by tuning the stoichiometry of different RNA sequences. These artificial condensates provide a versatile platform for studying and manipulating molecular functions inside living cells.

This study aimed to investigate the impact of the RNA-binding protein eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2) gene, also known as PKR, on the condition of islet beta cells. In this study, EIF2AK2 was overexpressed in INS1 cells, and transcriptome data following EIF2AK2 overexpression were obtained using RNA-seq technology. Additionally, potential target genes that bind to EIF2AK2 were identified through iRIP-seq technology. The proteins interacting with EIF2AK2 were characterized using co-immunoprecipitation (CO-IP) combined with mass spectrometry to elucidate the molecular regulatory mechanisms of EIF2AK2 in INS1 cells. RNA-seq results indicated that in INS1 cells overexpressing EIF2AK2, 1171 genes were differentially expressed, and 2161 alternative splicing events were significantly altered. iRIP-seq data demonstrated that reads from the immunoprecipitated samples were significantly enriched in the intronic and coding sequence (CDS) regions. EIF2AK2 preferentially binds to the GCGGCGG motif in RNA. Comprehensive analysis suggests that EIF2AK2 may directly bind to and regulate the expression of Dusp8, Btg1, and Prkce, thereby affecting pancreatic islet cell functions. Furthermore, EIF2AK2 may influence islet cell function by modulating the alternative splicing of Zfr and Pias2. Additionally, combined with Co-IP mass spectrometry data, it was discovered that EIF2AK2 can interact with 649 proteins, including various differentially expressed RNA-binding proteins, transcription factors, and histones, which may be associated with diabetes. Our results indicate that EIF2AK2 may regulate the expression or alternative splicing of mRNA related to type 2 diabetes through direct or indirect binding. Additionally, it may influence the progression of type 2 diabetes by interacting with other proteins. We propose that EIF2AK2 plays a significant role in diabetic islet beta cells, and its aberrant regulatory pattern is closely associated with the onset and progression of type 2 diabetes.

Repetitive and compositionally biased low-complexity (LC) motifs appear in biological sequences where they interact with the machinery controlling the abundance of their host molecules. They can have significant impacts on physiological function, and act as raw material for evolution of regulatory motifs. The extent to which LC motifs affect abundance is not known. Even definitions of LC sequences are not well established, let alone which motifs exists in LC sequences, and which of those are abundance associated. To fill these knowledge gaps for post-transcriptional impacts of LC motifs, we integrated data from the GTEx project, PaxDb, and the IGSR. We establish definitions for LC motifs in both RNA and protein sequences. We observed that the presence of LC motifs in the 5' untranslated regions (UTRs) were positively associated with transcript abundance. We present a method to de novo identify abundance associated motifs and identified trinucleotide repeats of (A/C)GG as most strongly abundance associated. We observed that m1A methylation sites were strongly associated with both LC motifs and abundance, an effect which is amplified as methylation signatures from unspecialized RNA-seq increased. Together, our results demonstrate that LC motifs play important roles in regulating gene expression.

RNA design aims to find a sequence that folds into a designated target structure under a specific RNA folding model, also known as the inverse problem of RNA folding. While numerous RNA design methods have been invented to search for sequences capable of folding into a target structure under the default (Turner) RNA folding model, little attention has been given to the identification of undesignable structures. This work bridges the gap between RNA design and undesignability by introducing a series of theorems and algorithms aimed at identifying both undesignable structures and their causative local structural components, which we define as minimal undesignable motifs. We first present theorems that provide sufficient conditions for recognizing undesignability structures and propose efficient, theorem-guided algorithms to verify whether an RNA structure is undesignable. While such global undesignability sheds light on the limits of RNA design, identifying the specific motifs responsible for undesignability is critical for understanding RNA folding models and advancing design methodologies. To this end, we develop a new theoretical framework for motif undesignability and propose scalable and interpretable algorithms to identify minimal undesignable motifs within a given RNA secondary structure. Our approach establishes motif undesignability by searching for rival motifs, rather than exhaustively enumerating all (partial) sequences that could potentially fold into the motif. Furthermore, we exploit rotational invariance in RNA structures to detect, group, and reuse equivalent motifs and to construct a database of unique minimal undesignable motifs. To achieve that, we propose a loop-pair graph representation for motifs and a recursive graph isomorphism algorithm for motif equivalence. Our algorithms successfully identified 24 unique minimal undesignable motifs among 18 undesignable puzzles from the Eterna100 benchmark. Surprisingly, we also find over 350 unique minimal undesignable motifs and 663 undesignable native structures in the ArchiveII dataset, drawn from a diverse set of RNA families. Last but not least, we demonstrate that our theory and algorithms can handle motifs with external loops-a critical advancement given the substantial impact of external loops on the quantity, diversity, and designability of RNA structure motifs.

Both Domains of APOBEC3F Recognize AA RNA Motifs to Support HIV-1 Virion Encapsidation and Antiviral Function.

RNA modification, which is evolutionarily conserved, is crucial for modulating various biological functions and disease pathogenesis. High resolution transcriptome-wide mapping of RNA modifications has facilitated both data resources and computational prediction of RNA modification. While these prediction algorithms are promising, they are limited in interpretability or generalizability, or the capacity for discovering novel post-transcriptional regulations. Here, we present NetRNApan, a deep learning framework for RNA modification site prediction, motif discovery and trans-regulatory factor identification. Using m5U profiles generated by FICC-seq and miCLIP-seq technologies and single-base resolution m6A sites from multiple experiments as cases, we demonstrated the accuracy of NetRNApan with more efficient and interpretive feature representations. For m5U modification, we uncovered five representative clusters with consensus motifs that may be essential by decoding the informative characteristics detected by NetRNApan. Furthermore, NetRNApan revealed interesting trans-regulatory factors and provided a protein-binding perspective for investigating the function of RNA modifications. Specifically, we discovered 21 potential functional RNA-binding proteins (RBPs) whose binding sites were significantly linked to the extracted top-scoring motifs for m5U modification. Two examples are ANKHD1 and RBM4 with potential regulatory function of m5U modifications. Meanwhile, the analysis of convolution layer parameters within the model offers valuable insights into the regulation of m6A in humans. Collectively, NetRNApan demonstrated high accuracy, interpretability and generalizability for study of RNA modification and mRNA regulation. NetRNApan is freely available at https://github.com/bsml320/NetRNApan.

The RNA-binding protein hnRNP E1 regulates p53 and p21 translation via KH1 and KH2 domain interactions with 3′ UTR C-rich motifs

Oral squamous cell carcinoma (OSCC) is a major global health concern with a 5-year survival rate of approximately 50%, driven by high recurrence and metastasis. N6-methyladenosine (m6A) RNA modification, regulated by METTL3, plays a critical role in cancer progression, yet its mechanisms in OSCC remain underexplored. This study investigates the METTL3/HNRNPA2B1/FOXQ1 axis in OSCC tumorigenesis. m6A RNA immunoprecipitation sequencing (MeRIP-Seq) and motif analysis were performed to identify m6A modification sites in OSCC cell lines. METTL3, HNRNPA2B1, and FOXQ1 expression levels were assessed in OSCC and normal tissues using immunohistochemistry as a retrospectively registered. METTL3 was modulated in CAL27 cells to evaluate its effects on m6A levels, HNRNPA2B1/FOXQ1 expression, and mRNA stability via RT-qPCR, Western blotting, and RNA immunoprecipitation (RIP)-PCR. Functional assays (EdU, wound healing, Transwell) assessed proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Xenograft models validated in vivo effects. MeRIP-Seq identified the “GGAC” motif as a predominant m6A site. METTL3, HNRNPA2B1, and FOXQ1 were significantly overexpressed in OSCC tissues (p < 0.05). METTL3 silencing reduced m6A levels, HNRNPA2B1/FOXQ1 expression, and mRNA stability, attenuating proliferation, migration, invasion, and EMT, while overexpression enhanced these phenotypes. RIP-PCR confirmed METTL3 binding to HNRNPA2B1/FOXQ1 mRNA. In vivo, METTL3 silencing decreased tumor growth and FOXQ1 expression. METTL3-mediated m6A modification promotes OSCC progression by stabilizing HNRNPA2B1 and FOXQ1 mRNA, driving EMT and malignancy. The METTL3/HNRNPA2B1/FOXQ1 axis is a potential diagnostic and therapeutic target for OSCC, offering novel insights into epigenetic regulation and treatment strategies.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-32025-7.

Guanidine is well known as a denaturing agent. However, recent studies have demonstrated both the widespread synthesis of guanidine, e.g., in plants and mammals, as well as the widespread occurrence of guanidine metabolism in bacteria, suggesting a broader biological role. Here, we provide insights into guanidine assimilation via guanidine hydrolases (GdmH) in cyanobacteria. The gdmH gene is widespread among cyanobacteria and enables growth on guanidine as the sole nitrogen source. Consistent with this, gdmH gene expression increased under nitrogen limitation, regulated by the transcription factor NtcA. However, guanidine is toxic above 5 mM, necessitating GdmH activity and adaptive mutations activating the multidrug efflux system PrqA. The gdmH gene is frequently colocalized with ABC transporter genes (named gimABC), which are driven by an additional NtcA-regulated promoter. The corresponding substrate-binding protein GimA showed high affinity to guanidine. Consistent with a high affinity import system, disruption of genes gimA or gimB impaired guanidine-dependent growth of Synechocystis sp. PCC 6803 at low concentrations. However, in presence of >1 mM guanidine, these mutants grew like wildtype, suggesting the existence of additional uptake mechanisms for guanidine. We also demonstrate the high-affinity binding of guanidine to a previously described, conserved RNA motif located within the gdmH 5'-untranslated region, validating it as a guanidine-I riboswitch. By combining it with various promoters, we achieved precise, titratable control of heterologous gene expression in cyanobacteria in vivo. Our findings establish guanidine assimilation as an integral element of cyanobacterial nitrogen metabolism and highlight guanidine riboswitches as valuable tools for synthetic biology.

Adenosine deaminases acting on RNA (ADAR) catalyze the deamination of adenosine to inosine in double-stranded RNA. Because inosine is read as guanosine during translation, this process enables programmable A-to-G recoding at the transcript level. ADARs can be harnessed for therapeutic correction of pathogenic mutations through site-directed RNA editing with guide RNAs. To expand the design space of editing-enabling guides, we applied EMERGe, a high-throughput screening platform, to identify motifs targeting a premature termination codon in the MeCP2 transcript associated with Rett syndrome. This uncovered a guide RNA motif that supported efficient ADAR2-mediated editing in vitro, featuring a 5'-GUG-3' sequence predicted to form an asymmetric loop. To enable therapeutic application, structure-activity relationship studies and chemical optimization were performed, yielding a fully modified guide RNA with 2'-O-methyl, 2'-fluoro, and phosphorothioate linkages. This stabilized guide retained the activity of unmodified RNA and showed enhanced nuclease resistance. The optimized guide induces dose-dependent editing at two MECP2 loci in reporter assays in HEK293T cells, demonstrating that EMERGe-selected motifs can be rendered viable in cells through targeted chemical modification. These findings highlight the utility of EMERGe as a discovery platform and establish a pipeline for identifying and optimizing editing-enabling guide RNA features beyond traditional design rules.

Abstract Viruses encode a limited number of proteins and often expropriate host factors for their successful infection and propagation. The molecular composition of the complex used for synthesis of the viral subgenomic (sg) RNAs remains largely unknown. In this study, an RNA motif (45 nt), equivalent to the sequence coding for the N-terminal portion of the tobamovirus coat protein (CP), known to be an enhancer-like element (tentatively designated as EL1) of the CP sg promoter, was used as a bait to capture bound proteins. Nicotiana benthamiana thioredoxin h-type 1 (NbTRXh1), a host defense factor with redox activity, was identified and its enzymatic active site was determined to be covered by EL1, thus blocking its reductase activity. In addition, the virus replicase Hel domain interacted with NbTRXh1, resulting in a decrease in NbTRXh1 protein accumulation in N. benthamiana . Thus, the viral RNA element and viral protein integrated their suppression of NbTRXh1 to facilitate viral CP sgRNA synthesis. Understanding this multiplexed regulatory mechanism between tobamovirus EL1 and associated proteins will assist in designing strategies for virus resistance and for optimization of the production of exogenous proteins under the control of the viral sg promoter.