An efficient C-to-U RNA recorder as a tool for profiling targets of RNA-binding proteins.

Physicochemical and RNA structural features of selective RNA binding by drug-like small molecules.

Human Staufen1 (hStau1, UniProt O95793.2) is a double-strand RNA (dsRNA) binding protein that modulates gene expression via mRNA-dependent mechanisms such as Staufen-mediated mRNA decay (SMD). This modular protein is dynamic and binds to both messanger RNA (mRNA) targets and proteins. The Staufen-swapping motif (SSM) domain is reported to play a key role in hStau1 dimerization. Our data confirm that SSM deletion decreases hStau1 dimerization. This protein shows higher protein disorder in the absence of SSM. Thus, SSM plays not only a key role in hStau1 dimerization but also modulates its tertiary structure. Surprisingly, increased disorder upon SSM deletion does not affect affinity for mRNA targets or protein/RNA stoichiometry but SMD efficiency since SMD targets are upregulated upon SSM deletion. In conclusion, hStau1 dimerization via SSM affects the overall structure and dynamics of the protein required for efficient regulation of gene expression via SMD.

The severe overlapping generations and rapid development of insecticide resistance in Panonychus citri pose significant challenges to chemical control. Due to that the cuticle serves as a critical defensive barrier for mite survival, elucidating its assembly mechanism may provide new targets for overcoming resistance. This study focused on the cuticular protein PcCPR12, which is highly expressed in the cuticle-enriched exoskeleton fraction. Functional analysis demonstrated that silencing PcCPR12 resulted in disordered cuticular wrinkling and high mortality, with the survival rate dropping to 45.68%. In vitro experiments confirmed that the CPR12 protein possesses chitin-binding ability, establishing its role in structural support. Transcriptome sequencing further revealed that PcCPR12 knockdown significantly down-regulated the chitin and lipid metabolism pathways. Subsequent validation indicated that silencing the key genes PcCHT1 (chitinase) and PcFABP (fatty acid binding protein) also led to lethal phenotypes, proving their essential roles in cuticle integrity. Based on this mechanism, a fusion double-stranded RNA (dsRNA) targeting PcCPR12-CHT1-FABP was constructed. Bioassays showed that this multi-target strategy resulted in a lower cumulative survival rate (37.23%) and significantly impaired female fecundity compared to single-gene interference. In summary, this study demonstrates the crucial function of PcCPR12 in upholding the structural integrity and stability of the P. citri cuticle. By delivering a multi-target strike against 'protein-chitin-lipid' components, the fusion RNA interference (RNAi) strategy developed here offers a theoretical foundation for P. citri management. © 2026 Society of Chemical Industry.

Clinical studies suggested that antibiotics (ABx) administration might increase esophagogastric junction adenocarcinoma risk, but the underlying mechanisms remain unclear. We previously demonstrated that the administration of a high-fat diet (HFD) and acid bile salts (ABS) to K19-Wnt1/C2mE mice might promote the metabolic-driven tumor growth at the squamocolumnar junction (SCJ) cooperatively with gut dysbiosis. To clarify whether ABx-induced dysbiosis promotes tumorigenesis, we evaluated the effects of HFD + ABS ± ABx treatment on tumor immune evasion in mice. In HFD + ABS + ABx-treated mice, SCJ tumor growth with increased tumor cell proliferation and infiltration of inflammatory cells positive for CD8, programmed cell death protein 1, and programmed cell death-ligand 1 (PD-L1) was observed, along with apoptosis suppression. Protein expressions of interferon-gamma (IFNγ) and phosphorylated signal transducer and activator of transcription (p-STAT) 3 were upregulated in the tumors of the HFD + ABS + ABx group, whose p-STAT1 expression was equivalent to that of the control group. The mice exhibited insulin resistance and metabolic endotoxemia, and metagenomic analysis of their ileal excrement revealed dysbiosis with a decrease in butyrate-producing bacteria and bacterial butanoate metabolism activity. Moreover, IFNγ stimulation of human-derived NUGC-4 cells increased the protein expression of PD-L1, p-STAT1, and p-STAT3, all of which decreased in response to STAT inhibitors. Transfection with small interfering RNA targeting STAT1 or STAT3 did not attenuate PD-L1 induction, which was inhibited by the combined knockdown. Therefore, oral HFD + ABS + ABx administration to K19-Wnt1/C2mE mice may promote SCJ tumors through tumor immune evasion via IFNγ-STAT1/STAT3-PD-L1 signaling, along with metabolic endotoxemia.NEW & NOTEWORTHY Coadministration of antibiotics with a high-fat diet and acid bile salts exacerbated dysbiosis, insulin resistance, and systemic inflammation, thereby promoting tumor progression via tumor immune evasion at the squamocolumnar junction (SCJ) in K19-Wnt1/C2mE mice. In the tumor, interferon-gamma-induced programmed death-ligand 1 through the activation of signal transducer and activator of transcription 1 (STAT1) and STAT3. Understanding the link between dysbiosis and tumor immunity might aid in the development of new immunotherapies for SCJ tumors.

Circ_0057105 promotes intrahepatic cholangiocarcinoma progression by sponging miR-1290 and regulating the MDM2/P53 pathway.

Reelin signaling regulates multiple pathways in neurodegenerative conditions, including neuronal migration, synaptic plasticity, tau phosphorylation, and amyloidogenic processing of amyloid precursor protein (APP). This study aimed to investigate the impact of reelin downregulation on the expression of topoisomerase IIβ (topo IIβ), given its crucial role in neuronal differentiation and its established association with neurodegenerative disorders such as Alzheimer's disease (AD). Furthermore, we sought to elucidate the potential relationship between reelin downregulation and proteins implicated in the pathophysiology of AD. Firstly, the optimum concentration of small interfering RNAs (siRNA) targeting reelin was transfected into SH-SY5Y cells using Lipofectamine RNAiMAX reagent. The downregulation of reelin was confirmed at the mRNA level by real-time quantitative polymerase chain reaction (qRT-PCR). Reelin-mediated molecular alterations at both the mRNA and protein levels were analyzed by qRT-PCR and Western blotting. Reelin downregulation led to a decrease in the number of viable cells as determined by the MTT assay. Consistent with the downregulation of reelin gene expression, topo IIβ, Psen1, and BACE1 expressions were also reduced, whereas tau and APP expressions were upregulated. Although siRNA treatment effectively decreased reelin mRNA levels and the proteolytic fragment of reelin protein, no significant change was observed in total full-length reelin protein levels, suggesting the involvement of post-transcriptional regulatory mechanisms. Moreover, pTAU and APP protein expressions were increased, while Nurr1 protein was decreased in reelin-silenced cells. These findings suggest that downregulation of reelin gene expression may contribute to neurodegeneration through alterations in topo IIβ and nurr1 expression, in addition to changes in proteins associated with AD pathology.

The type III-D2 CRISPR-Cas system comprises multiple Cas subunits and a CRISPR RNA, and is likely an evolutionary intermediate between the well-studied type III-A and III-E systems. Here we show that the type III-D2 complex synthesizes two distinct second messengers, SAM-AMP and SAM-AMP2, from S-adenosylmethionine (SAM) and ATP in response to target RNA recognition. We determined cryo-electron microscopy structures of the type III-D2 effector complex in different functional states, providing mechanistic insights into target RNA cleavage and second messenger synthesis. The structures reveal how SAM and ATP are recognized by the Cas10 subunit within the effector complex. Furthermore, our biological data suggest that both SAM-AMP and SAM-AMP2 act on the CorA ancillary effector, inducing growth arrest of infected bacterial cells and thereby conferring immunity. Thus, our study establishes the type III-D2 system as a unique anti-phage defense mechanism that employs both SAM-AMP and SAM-AMP2 as second messengers, expanding the repertoire of second messenger strategies in bacterial defense systems and highlighting the remarkable functional diversity of CRISPR-Cas systems.

In vitro fertilization-associated osteomodulin upregulation is linked to impaired placental angiogenesis.

Small interfering RNAs (siRNAs) are an expanding class of RNA therapeutics, with seven drugs approved by the US Food and Drug Administration and many more in development. Rational design, however, has been limited by incomplete understanding of how human Argonaute 2 (hAgo2) catalyzes target cleavage. Here we report high-resolution cryo-electron microscopy structures of hAgo2 bound to a target RNA in catalytic and noncatalytic conformations. The structures reveal that guide-target pairing alone is insufficient for slicing and catalysis requires deformation of the duplex through a coordinated network of RNA-protein interactions. Expansion of the central major groove positions the scissile phosphate, while compression toward the supplementary region docks the duplex into the hAgo2 cleft. A kink after guide nucleotide g6 disrupts seed-only pairing conformation and promotes the extended pairing required for catalysis. This rearrangement enables repositioning of K709 within the active site, while a pyrimidine at target position t10 optimally aligns R710 to accelerate cleavage. These findings provide a structural framework linking siRNA duplex geometry to catalytic efficiency and inform rational design of siRNAs with improved potency and specificity.

Traditional proximity biotinylation approaches require extensive genetic engineering or intricate purification steps. Here, we introduce FISH+, a ready-to-use RNA proximity labeling method that relies on the recruitment of peroxidase to RNA targets, and facilitates in situ proximity biotinylation in fixed cells. This method permits concurrent visualization of RNA molecules and identification of RNA-interacting proteins. Using this method, we visualized 45S and NEAT1 RNA, and captured their proximal proteins, demonstrating the capability to concurrently visualize RNA and identify proximal proteins. By targeting PNCTR (~36 copies per cell), we observed distinctly bright RNA dots, representing the combined biotinylation signals from both RNA and proximal proteins in situ. This indicates the potential of the FISH+ method for enhanced RNA visualization. We further generalized the FISH+ method to explore XIST-interacting proteins, a number of reported interactors were significantly enriched, such as SPEN, CIZ1 and RBM15. Using quantitative mass spectrometry, we show that FISH+ correctly identifies known RNA-protein interactions in the nucleus of human cells. Overall, we established a watch-and-catch punctate RNA method through the integration of RNA fluorescence in situ hybridization (FISH) with proximity biotinylation. This method provides additional spatial information for the characterization of RNA-centric interactions in fixed, genetically unperturbed samples.

The CRISPR/Cas12a system has revolutionized molecular diagnostics; however, conventional Cas12a-based methods for RNA detection typically require transcription and pre-amplification steps. Our group has recently developed a diagnostic technique known as the SCas12a assay, which combines Cas12a with a split crRNA, achieving amplification-free detection of miRNA. However, this method still encounters challenges in accurately quantifying long RNA molecules with complex secondary structures. Here, we report an enhanced version termed SCas12aV2 (split-crRNA Cas12a version 2 system), which enables direct detection of RNA molecules without sequence limitation while demonstrating high specificity in single-nucleotide polymorphism (SNP) applications. We describe the general procedure for preparing the SCas12a system and its application in detecting RNA targets from clinical samples. Key features • The SCas12aV2 assay enables efficient detection of long-chain RNA molecules with complex secondary structures. • PAM-distal sites can be effectively distinguished at the SNP detection level. • The entire experimental procedure can be completed in less than one hour.

Targeting RNA quality-control defects in tauopathies: Pharmacodynamic biomarkers and therapeutic development.

Recently, numerous nucleic acid therapeutics have been developed, and a substantial number of the approved agents are categorized as antisense oligonucleotides (ASOs). These therapeutics comprise phosphorothioate oligonucleotide (PS-DNA) or phosphorodiamidate morpholino oligonucleotide (PMO) featuring chemically modified nucleic acid backbones for nuclease resistance. Although chemical modifications to the backbone and sugar moieties of ASOs enhance nuclease resistance, increase binding affinity toward target RNAs, and optimize pharmacokinetic properties, information on the physicochemical properties of ASO therapeutics, which are mixtures of several stereoisomers, remains limited. In this study, the physicochemical properties of PS-DNA and PMO were analyzed using ultraviolet melting analysis, surface plasmon resonance, isothermal titration calorimetry, and nuclear magnetic resonance. Compared with natural deoxynucleotide, PS-DNA more rapidly dissociates from the complementary RNA, resulting in a lower RNA affinity, whereas PMO more quickly associates with the complementary RNA, leading to a higher RNA affinity. Furthermore, characteristic thermodynamic parameters governing the binding of PMO to RNA were determined. Although these physicochemical properties of ASOs are derived from a single model system, the accumulation of physicochemical data for ASOs featuring various sequences and lengths will contribute to the future development of nucleic acid-based therapeutics.

Sequence-specific RNA-binding proteins (RBPs) must efficiently locate their targets among a multitude of cellular RNAs. Cas13, an RNA-guided CRISPR protein, represents an ideal model system in which to study this search process. Cas13 combats bacteriophage infection by cleaving RNA nonspecifically upon binding of its crRNA to the target RNA sequence; thus, Cas13's search for its RNA target comes with a time constraint determined by the rate of phage multiplication. The mechanism by which Cas13 locates its target within this critical window remains unknown. Here, we investigate Cas13's mechanism of target search through integration of biophysical modeling, activity assays, and biochemical characterization. We show that Cas13 employs facilitated diffusion to accelerate its search, and find that Cas13's search time when targeting RNAs of different lengths cannot be explained by 1D sliding, the search mechanism used by many DNA-binding proteins. We propose that Cas13 primarily searches for its RNA target by intersegmental transfers (ITs), non-specifically binding the RNA at two locations and directly switching between them without fully dissociating from the RNA. We develop a biophysical model for ITs in an RNA context that we subsequently validate experimentally. Furthermore, we demonstrate that ITs can differentially accelerate the search process for a broad class of RNA-binding proteins, as opposed to their DNA-binding counterparts, due to RNA's short persistence length and the heterogeneity of RNA lengths in the cell. Our results illuminate how Cas13 achieves rapid target recognition in a complex RNA environment, and implicate ITs as a potentially widespread solution to the RNA search problem.

Spliceosome is a dynamic macromolecular complex responsible for splicing precursor mRNA. Single Nucleotide Polymorphisms (SNPs) in spliceosomal proteins have been implicated in various diseases. Mutational studies and high-resolution structures have provided important functional insights, and these data can be used to evaluate the impact of SNPs on splicing. Here, we applied integrated sequence and structure-based approaches to investigate the functional consequences of SNPs in spliceosomal proteins. Changes in binding free energy (ΔΔG) for SNPs were compared with those of mutations with known functional implications. When human crystal structures were unavailable, homologous protein complexes from other organisms were used, and variants were mapped onto these structures via multiple-sequence alignment before ΔΔG estimation. Protein-protein interactions annotated with FoldX ΔΔG values were visualized as interaction networks, and molecular dynamics (MD) simulations were performed to assess SNP and mutation-induced structural perturbations. Target RNAs affected by functionally characterized SNPs and mutations were compiled. Stage-specific effects were evaluated by mapping variants onto yeast spliceosome complexes representing different stages of the spliceosome cycle. For the U2AF35-U2AF65 complex, ClusPro docking produced ΔΔG values comparable to experimental and AlphaFold-predicted complexes, whereas ZDOCK showed a distinct trend. Following MD simulations, ΔΔG values across models were comparable. However, predicted and docked complexes showed ∼10–15% interface variation relative to the crystal structure, highlighting the need for cautious interpretation, while our analysis was limited to protein subcomplexes due to the spliceosome’s size and complexity. Overall, this study provides a structural systems biology framework for understanding how SNPs perturb spliceosome function and influence splicing.

The RNA-binding protein Pumilio-2 (PUM2), encoded by PUM2, is known as a translational repressor with important roles during embryonic development, cell differentiation and synapse function. Information of its roles in the ovary were sparse, yet data bank mining followed by immunohistochemistry revealed expression in nonhuman primate ovarian granulosa cells (GCs). Expression was also found in human IVF-derived GCs, in samples of human GC tumors (GCT), and in KGN cells, a well-established GCT-derived cell line. KGN cells were used to explore the roles of PUM2. A small interfering RNA (siRNA) targeting PUM2 reduced PUM2 levels in KGN cells, as seen in a proteomic analysis, which also revealed that a total of 51 proteins were lower, and the abundance of 66 proteins were increased. GO term analyses showed that the pathways mainly decreased were 'response to virus', 'defence response to virus', and 'innate immune response'. PUM2 may thus be a regulator of the innate immune abilities of GCs. The pathway increased most was 'cell division', a result in line with increased proliferation of siRNA transfected KGN cells. This underlies the increased ability to close the gap in scratch assays, as individual cell velocity or directionality of migration were comparable to controls. Down-regulation of PUM2 also impaired the steroidogenic potential of KGN cells (lower HSD3B2 levels, lower progesterone in supernatant). Taken together, the results indicate fundamental roles of PUM2 not only in in the regulation of proliferation and steroidogenesis of human granulosa cells, but also in regulation of their innate immune abilites.

Eukaryotic genomes generate a plethora of polyadenylated (pA+) RNAs1,2, which are packaged into ribonucleoprotein particles (RNPs). To ensure faithful gene expression, functional pA+ RNPs, including protein-coding RNPs, are exported to the cytoplasm, whereas transcripts within non-functional pA+ RNPs are degraded in the nucleus1-4. How cells distinguish these opposing fates remains unknown. The DExD-box ATPase UAP56 (also known as DDX39B) is a central component of functional pA+ RNPs, and promotes their docking to the nuclear pore complex-anchored TREX-25,6, which triggers transcript release from UAP56 to facilitate export7. Here we reveal that the poly(A) tail exosome targeting (PAXT) connection8 binds a TREX-2-like module, which releases pA+ RNAs from UAP56 for decay by the nuclear exosome. The core of this module consists of a LENG8-PCID2-SEM1 trimer, which we show is structurally and biochemically equivalent to the central GANP-PCID2-SEM1 trimer of TREX-2. Mutagenesis and transcriptomic data demonstrate that the nuclear fate of pA+ RNPs is governed by the contending actions of nucleoplasmic PAXT and nuclear pore complex-associated TREX-2, which interpret RNA-bound UAP56 as a signal for RNA decay or export, respectively. As RNA targets of PAXT are generally short and intron-poor, we propose an overall model for pA+ RNP fate determination whereby the distinct sub-nuclear localizations of PAXT and TREX-2 govern the degradation of short non-functional pA+ RNAs while allowing export of their longer and functional counterparts.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder caused by a dominant mutation in the ACVR1 gene (R206H, 97% of cases), leading to debilitating heterotopic ossification (HO) characterized by abnormal bone growth triggered by inflammatory flare-ups. Here, we report the development of disease-modifying, allele-selective small interfering RNA (siRNA) targeting ACVR1R206H. Allele selectivity is essential as wildtype ACVR1 is crucial for many functions including skeletal homeostasis and development. When conjugated to docosanoic acid (DCA), administration of the fully modified ACVR1 siRNA, either alone or in combination with an siRNA targeting IL1B (a key regulator of inflammation), results in profound reduction of HO using both responsive (post-trauma) and preventative (pre-trauma) intervention strategies in a murine FOP model. Notably, the combination therapy outperforms modulation of either target alone. We also describe the chemical engineering of a new class of lipophilic divalent siRNAs that target both pathways with a single compound, demonstrating superior muscle accumulation and therapeutic efficacy. siRNA treatment inhibits key signaling pathways (e.g. inflammatory, WNT, Notch, Hedgehog, and TGF-β), within muscle-resident fibroadipogenic progenitors (FAPs), leading to a significant reduction in cartilage, bone, and connective tissue formation. This work establishes a foundation for the development of disease-modifying treatments for FOP and offers a platform for targeting other musculoskeletal disorders involving multi-pathway dysregulation.

Decades of neuroscience research using the Drosophila model system has made enormous strides in uncovering genes that positively support the ability to form new memories or consolidate them for the long-term. However, recent research has revealed the existence of more rare but equally important memory suppressor genes whose normal function is to limit memory formation or even promote active forgetting. Here we report that RNAi targeting of Diacylglycerol kinase (Dgk), a major regulator of lipid signaling, within the mushroom body memory circuitry specifically enhances aversive olfactory memory retention while leaving learning and sensory-motor behavioural controls unaffected. Furthermore, memory retention is enhanced when targeting Dgk in these circuits with two additional RNAi lines. Finally, using in vivo functional imaging, we offer evidence that Dgk plays a role in regulating baseline synaptic transmission of memory circuits. While the exact mechanism for Dgk's synaptic effects and the implications for memory storage remain unclear, our findings implicate lipid signaling via Dgk as an important regulator of active-forgetting pathways. This work builds on our prior understanding of the importance of lipids in cognition by extended their role to memory suppression and active forgetting.