RNA Motifs Research Articles

A KSHV RNA-binding protein promotes FOS to inhibit nuclease AEN and transactivate RGS2 for AKT phosphorylation.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an RNA-binding protein ORF57 in lytic infection. Using an optimized CLIP-seq in this report, we identified ORF57-bound transcripts from 544 host protein-coding genes. By comparing with the RNA-seq profiles from BCBL-1 cells with latent and lytic KSHV infection and from HEK293T cells with and without ORF57 expression, we identified FOS RNA as one of the major ORF57-specific RNA targets. FOS dimerizes with JUN as a transcription factor AP-1 involved in cell proliferation, differentiation, and transformation. Knockout of the ORF57 gene from the KSHV genome led BAC16-iSLK cells incapable of FOS expression in KSHV lytic infection. The dysfunctional KSHV genome in FOS expression could be rescued by Lenti-ORF57 virus infection. ORF57 protein does not regulate FOS translation but binds to the 13-nt RNA motif near the FOS RNA 5' end and prolongs FOS mRNA half-life 7.7 times longer than it is in the absence of ORF57. This binding of ORF57 to FOS RNA is likely competitive to the binding of host nuclease AEN (ISG20L1) of which physiological RNase activity remains unknown. KSHV infection inhibits the expression of AEN, but not exosomal RNA helicase MTR4. FOS expression mediated by ORF57 inhibits AEN transcription through FOS binding to AEN promoter but transactivates RGS2, a regulator of G-protein-coupled receptors. FOS binds a conserved AP-1 site in the RGS2 promoter and enhances RGS2 expression to phosphorylate AKT. Altogether, we have discovered that KSHV ORF57 specifically binds and stabilizes FOS RNA to increase FOS expression, thereby disturbing host gene expression and inducing pathogenesis during KSHV lytic infection.IMPORTANCEWe discovered that FOS, a heterodimer component of oncogenic transcription factor AP-1, is highly elevated in KSHV-infected cells by expression of a viral lytic RNA-binding protein, ORF57, which binds a 13-nt RNA motif near the FOS RNA 5' end to prolong FOS RNA half-life. This binding of ORF57 to FOS RNA is competitive to the binding of host RNA destabilizer(s). KSHV infection inhibits expression of host nuclease AEN, but not MTR4. FOS inhibits AEN transcription by binding to the AEN promoter but transactivates RGS2 by binding to a conserved AP-1 site in the RGS2 promoter, thereby enhancing RGS2 expression and phosphorylation of AKT. Thus, KSHV lytic infection controls the expression of a subset of genes for signaling, cell cycle progression, and proliferation to potentially contribute to viral oncogenesis.

An interpretable RNA foundation model for exploring functional RNA motifs in plants

AbstractThe complex ‘language’ of plant RNA encodes a vast array of biological regulatory elements that orchestrate crucial aspects of plant growth, development and adaptation to environmental stresses. Recent advancements in foundation models (FMs) have demonstrated their unprecedented potential to decipher complex ‘language’ in biology. In this study, we introduced PlantRNA-FM, a high-performance and interpretable RNA FM specifically designed for plants. PlantRNA-FM was pretrained on an extensive dataset, integrating RNA sequences and RNA structure information from 1,124 distinct plant species. PlantRNA-FM exhibits superior performance in plant-specific downstream tasks. PlantRNA-FM achieves an F1 score of 0.974 for genic region annotation, whereas the current best-performing model achieves 0.639. Our PlantRNA-FM is empowered by our interpretable framework that facilitates the identification of biologically functional RNA sequence and structure motifs, including both RNA secondary and tertiary structure motifs across transcriptomes. Through experimental validations, we revealed translation-associated RNA motifs in plants. Our PlantRNA-FM also highlighted the importance of the position information of these functional RNA motifs in genic regions. Taken together, our PlantRNA-FM facilitates the exploration of functional RNA motifs across the complexity of transcriptomes, empowering plant scientists with capabilities for programming RNA codes in plants.

Endo-bind-n-seq: identifying RNA motifs of RNA binding proteins isolated from endogenous sources.

RNA binding proteins (RBPs) are crucial regulators of gene expression and critically depend on the specific recognition of their target RNAs. Accordingly, a selection of methods to analyze RBP specificities has been developed, including protein-RNA crosslinking and sequencing (CLIP) and in vitro selection methods such as SELEX, RNA compete or RNA bind-n-seq. However, limitations like the availability for purified recombinant proteins and custom microarray platforms (RNAcompete) or extensive sequencing depth and sophisticated bioinformatic data processing (CLIP) may limit a broader implementation of these methods. Here, we present an RNA bind-n-seq method that uses short random RNA pools and enables multiple rounds of selection. This results in strong motif enrichment with low positional variance thus reducing sequencing depth requirements. Furthermore, we have coupled our protocol to immunoprecipitation of tagged or endogenous RBPs from cultured cells or tissue samples, eliminating the need for recombinant proteins. Our method also allows for the identification of indirect RNA motifs of proteins that are integral parts of multiprotein RNPs and result in physically more relevant RNA motifs.

R3DMCS: a web server for visualizing structural variation in RNA motifs across experimental 3D structures from the same organism or across species.

The recent progress in RNA structure determination methods has resulted in a surge of newly solved RNA 3D structures. However, there is an absence of a user-friendly browser-based tool that can facilitate the comparison and visualization of RNA motifs across multiple 3D structures. We introduce R3DMCS, a web server that allows users to compare selected RNA nucleotides across all 3D structures of a given molecule from a given species, or across all 3D structures mapped to a single Rfam family. Starting from one instance of the motif, R3DMCS retrieves, aligns, annotates, organizes, and displays 3D coordinates of corresponding sets of nucleotides from other 3D structures. With R3DMCS, one can explore conformational changes of motifs due to 3D structures being solved in different functional states or different experimental conditions. One can also investigate conservation of 3D structure across species, or changes in 3D structure due to changes in sequence. R3DMCS is open-source software and freely available at https://rna.bgsu.edu/correspondence/ and https://github.com/BGSU-RNA/RNA-3D-correspondence . Supplementary data are available at Bioinformatics online.

The perinucleolar compartment and the oncogenic super-enhancers are part of the same phase-separated structure filled with phosphatidylinositol 4,5 bisphosphate and long non-coding RNA HANR

The liquid-liquid phase separation in the cell nucleus regulates various processes such as gene regulation and transcription control, chromatin organization, and DNA repair. A plethora of proteins and RNAs contribute to the formation of biomolecular condensates and recently, several nuclear phosphoinositides were shown to be a part of these membrane-less complexes within the nucleus as well. Here we lipid-interacting RNA sequencing (LIPRNAseq) and confocal microscopy to uncover the RNA-binding capacity and localization of phosphatidylinositol 4,5 bisphosphate (PIP2). We discovered the consensus PIP2-binding AU-rich RNA motif and identified long non-coding RNA HANR (lncHANR) to colocalize with PIP2 in the proximity to the nucleolus in the perinucleolar compartment (PNC). Colocalization studies with different nuclear markers reveal that PIP2-HANR presence in the PNC correlates with oncogenic super-enhancers, and both PNC and oncogenic enhancers are part of the same structure. As lncHANR, PNC, and oncogenic super-enhancers are associated with cancer cell lines and tumors, we suggest that they can serve as interchangeable prognostic markers. Understanding of the interplay between lipid metabolism, and lncRNAs in subnuclear compartment phase separation can lead to future improvement in treatment strategies and personalized cancer management approaches.

Reconstructing the sequence specificities of RNA-binding proteins across eukaryotes.

RNA-binding proteins (RBPs) are key regulators of gene expression. Here, we introduce EuPRI (Eukaryotic Protein-RNA Interactions) - a freely available resource of RNA motifs for 34,736 RBPs from 690 eukaryotes. EuPRI includes in vitro binding data for 504 RBPs, including newly collected RNAcompete data for 174 RBPs, along with thousands of reconstructed motifs. We reconstruct these motifs with a new computational platform - Joint Protein-Ligand Embedding (JPLE) - which can detect distant homology relationships and map specificity-determining peptides. EuPRI quadruples the number of known RBP motifs, expanding the motif repertoire across all major eukaryotic clades, and assigning motifs to the majority of human RBPs. EuPRI drastically improves knowledge of RBP motifs in flowering plants. For example, it increases the number of Arabidopsis thaliana RBP motifs 7-fold, from 14 to 105. EuPRI also has broad utility for inferring post-transcriptional function and evolutionary relationships. We demonstrate this by predicting a role for 12 Arabidopsis thaliana RBPs in RNA stability and identifying rapid and recent evolution of post-transcriptional regulatory networks in worms and plants. In contrast, the vertebrate RNA motif set has remained relatively stable after its drastic expansion between the metazoan and vertebrate ancestors. EuPRI represents a powerful resource for the study of gene regulation across eukaryotes.

Determinants of selectivity in the dicing mechanism

Our research elucidates the cleavage processes of the RNase III enzyme, DICER, which plays a crucial role in the production of small RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Utilizing high-throughput dicing assays, we expose the bipartite pairing rule that dictates the cleavage sites of DICER. Furthermore, we decode the intricate recognition mechanism of the primary YCR motif and identify an analogous secondary YCR motif that influences DICER's cleavage choices. Collectively, our findings clarify the bipartite pairing rule and enhance our understanding of the role of RNA motifs in modulating DICER's cleavage activity, laying the groundwork for future research on their roles in miRNA biogenesis and gene regulation.

Interactive Structural Analysis of KH3-4 Didomains of IGF2BPs with Preferred RNA Motif Having m6A Through Dynamics Simulation Studies.

m6A modification is the most common internal modification of messenger RNA in eukaryotes, and the disorder of m6A can trigger cancer progression. The GGACU is considered the most frequent consensus sequence of target transcripts which have a GGAC m6A core motif. Newly identified m6A 'readers' insulin-like growth factor 2 mRNA-binding proteins modulate gene expression by binding to the m6A binding sites of target mRNAs, thereby affecting various cancer-related processes. The dynamic impact of the methylation at m6A within the GGAC motif on human IGF2BPs has not been investigated at the structural level. In this study, through in silico analysis, we mapped IGF2BPs binding sites for the GGm6AC RNA core motif of target mRNAs. Subsequent molecular dynamics simulation analysis at 400 ns revealed that only the KH4 domain of IGF2BP1, containing the 503GKGG506 motif and its periphery residues, was involved in the interaction with the GGm6AC backbone. Meanwhile, the methyl group of m6A is accommodated by a shallow hydrophobic cradle formed by hydrophobic residues. Interestingly, in IGF2BP2 and IGF2BP3 complexes, the RNA was observed to shift from the KH4 domain to the KH3 domain in the simulation at 400 ns, indicating a distinct dynamic behavior. This suggests a conformational stabilization upon binding, likely essential for the functional interactions involving the KH3-4 domains. These findings highlight the potential of targeting IGF2BPs' interactions with m6A modifications for the development of novel oncological therapies.

A compact stem-loop DNA aptamer targets a uracil-binding pocket in the SARS-CoV-2 nucleocapsid RNA-binding domain.

SARS-CoV-2 nucleocapsid (N) protein is a structural component of the virus with essential roles in the replication and packaging of the viral RNA genome. The N protein is also an important target of COVID-19 antigen tests and a promising vaccine candidate along with the spike protein. Here, we report a compact stem-loop DNA aptamer that binds tightly to the N-terminal RNA-binding domain of SARS-CoV-2 N protein. Crystallographic analysis shows that a hexanucleotide DNA motif (5'-TCGGAT-3') of the aptamer fits into a positively charged concave surface of N-NTD and engages essential RNA-binding residues including Tyr109, which mediates a sequence-specific interaction in a uracil-binding pocket. Avid binding of the DNA aptamer allows isolation and sensitive detection of full-length N protein from crude cell lysates, demonstrating its selectivity and utility in biochemical applications. We further designed a chemically modified DNA aptamer and used it as a probe to examine the interaction of N-NTD with various RNA motifs, which revealed a strong preference for uridine-rich sequences. Our studies provide a high-affinity chemical probe for the SARS-CoV-2 N protein RNA-binding domain, which may be useful for diagnostic applications and investigating novel antiviral agents.

Exploring the energetic and conformational properties of the sequence space connecting naturally occurring RNA tetraloop receptor motifs.

Folded RNAs contain tertiary contact motifs whose structures and energetics are conserved across different RNAs. The transferable properties of RNA motifs simplify the RNA folding problem, but measuring energetic and conformational properties of many motifs remains a challenge. Here, we use a high-throughput thermodynamic approach to investigate how sequence changes alter the binding properties of naturally-occurring motifs, the GAAA tetraloop•tetraloop receptor (TLR) interactions. We measured the binding energies and conformational preferences of TLR sequences that span mutational pathways from the canonical 11ntR to two other natural TLRs, the IC3R and Vc2R. While the IC3R and Vc2R share highly similar energetic and conformational properties, the landscapes that map the sequence changes for their conversion from the 11ntR to changes in these properties differ dramatically. Differences in the energetic landscapes stem from the mutations needed to convert the 11ntR to the IC3R and Vc2R rather than a difference in the intrinsic energetic architectures of these TLRs. The conformational landscapes feature several non-native TLR variants with conformational preferences that differ from both the initial and final TLRs; these species represent potential branching points along the multidimensional sequence space to sequences with greater fitness in other RNA contexts with alternative conformational preferences. Our high-throughput, quantitative approach reveals the complex nature of sequence-fitness landscapes and leads to models for their molecular origins. Systematic and quantitative molecular approaches provide critical insights into understanding the evolution of natural RNAs as they traverse complex landscapes in response to selective pressures.

The TRIM-NHL RNA-binding protein Brain Tumor coordinately regulates expression of the glycolytic pathway and vacuolar ATPase complex.

The essential Drosophila RNA-binding protein Brain Tumor (Brat) represses specific genes to control embryogenesis and differentiation of stem cells. In the brain, Brat functions as a tumor suppressor that diminishes neural stem cell proliferation while promoting differentiation. Though important Brat-regulated target mRNAs have been identified in these contexts, the full impact of Brat on gene expression remains to be discovered. Here, we identify the network of Brat-regulated mRNAs by performing RNAsequencing (RNA-seq) following depletion of Brat from cultured cells. We identify 158 mRNAs, with high confidence, that are repressed by Brat. De novo motif analysis identified a functionally enriched RNA motif in the 3' untranslated regions (UTRs) of Brat-repressed mRNAs that matches the biochemically defined Brat binding site. Integrative data analysis revealed a high-confidence list of Brat-repressed and Brat-bound mRNAs containing 3'UTR Brat binding motifs. Our RNA-seq and reporter assays show that multiple 3'UTR motifs promote the strength of Brat repression, whereas motifs in the 5'UTR are not functional. Strikingly, we find that Brat regulates expression of glycolytic enzymes and the vacuolar ATPase complex, providing new insight into its role as a tumor suppressor and the coordination of metabolism and intracellular pH.

Optimized periphery-core interface increases fitness of the Bacillus subtilis glmS ribozyme.

Like other functional RNAs, ribozymes encode a conserved catalytic center supported by peripheral domains that vary among ribozyme sub-families. To understand how core-periphery interactions contribute to ribozyme fitness, we compared the cleavage kinetics of all single base substitutions at 152 sites across the Bacillus subtilis glmS ribozyme by high-throughput sequencing (k-seq). The in vitro activity map mirrored phylogenetic sequence conservation in glmS ribozymes, indicating that biological fitness reports all biochemically important positions. The k-seq results and folding assays showed that most deleterious mutations lower activity by impairing ribozyme self-assembly. All-atom molecular dynamics simulations of the complete ribozyme revealed how individual mutations in the core or the IL4 peripheral loop introduce a non-native tertiary interface that rewires the catalytic center, eliminating activity. We conclude that the need to avoid non-native helix packing powerfully constrains the evolution of tertiary structure motifs in RNA.

Switchback RNA.

Intricately designed DNA and RNA motifs guide the assembly of robust and functional nucleic acid nanostructures. In this work, we present a globally left-handed RNA motif with two parallel strands called switchback RNA and report its assembly, biophysical, and biochemical characterization. Switchback RNA can be assembled in buffers without Mg2+, with improved thermal stability in buffers containing Mg2+, Na+, or K+. Differences in the binding of small molecules to switchback RNA and conventional RNA indicate design-based approaches for small molecule loading on RNA nanostructures. Further, the differential affinity of the two component strands in switchback or conventional duplex conformations allows for toehold-less strand displacement. Enzyme studies showed that the switchback and conventional RNA structures have similar levels of nuclease resistance. These results provide insights for employing switchback RNA as a structural motif in RNA nanotechnology. Our observation that RNA strands with switchback complementarity can form stable complexes at low magnesium concentrations encourages studies into the potential occurrence of switchback RNA in nature.

Machine learning-augmented molecular dynamics simulations (MD) reveal insights into the disconnect between affinity and activation of ZTP riboswitch ligands.

The challenge of targeting RNA with small molecules necessitates a better understanding of RNA-ligand interaction mechanisms. However, the dynamic nature of nucleic acids, their ligand-induced stabilization, and how conformational changes influence gene expression pose significant difficulties for experimental investigation. This work employs a combination of computational and experimental methods to address these challenges. By integrating structure-informed design, crystallography, and machine learning-augmented all-atom molecular dynamics simulations (MD) we synthesized, biophysically and biochemically characterized, and studied the dissociation of a library of small molecule activators of the ZTP riboswitch, a ligand-binding RNA motif that regulates bacterial gene expression. We uncovered key interaction mechanisms, revealing valuable insights into the role of ligand binding kinetics on riboswitch activation. Further, we established that ligand on-rates determine activation potency as opposed to binding affinity and elucidated RNA structural differences, which provide mechanistic insights into the interplay of RNA structure on riboswitch activation.

ARTEMIS: a method for topology-independent superposition of RNA 3D structures and structure-based sequence alignment.

Non-coding RNAs play a major role in diverse processes in living cells with their sequence and spatial structure serving as the principal determinants of their function. Superposition of RNA 3D structures is the most accurate method for comparative analysis of RNA molecules and for inferring structure-based sequence alignments. Topology-independent superposition is particularly relevant, as evidenced by structurally similar RNAs with sequence permutations such as tRNA and Y RNA. To date, state-of-the-art methods for RNA 3D structure superposition rely on intricate heuristics, and the potential for topology-independent superposition has not been exhausted. Recently, we introduced the ARTEM method for unrestrained pairwise superposition of RNA 3D modules and now we developed it further to solve the global RNA 3D structure alignment problem. Our new tool ARTEMIS significantly outperforms state-of-the-art tools in both sequentially-ordered and topology-independent RNA 3D structure superposition. Using ARTEMIS we discovered a helical packing motif to be preserved within different backbone topology contexts across various non-coding RNAs, including multiple ribozymes and riboswitches. We anticipate that ARTEMIS will be essential for elucidating the landscape of RNA 3D folds and motifs featuring sequence permutations that thus far remained unexplored due to limitations in previous computational approaches.

Therapeutic applications of RNA nanostructures.

RNA-based therapeutics have gained wide public interest in recent years. RNA is a versatile molecule that exists in many forms including mRNA, siRNA, miRNA, ribozymes, and other non-coding RNAs and is primarily applied for gene therapy. RNA is also used as a modular building block to construct RNA nanostructures. The programmable nature of RNA nanostructures enables the generation of simple, modulable, and multi-functional RNA-based therapeutics. Although the therapeutic application of RNA may be limited due to its structural instability, advances in RNA nanotechnology have improved the stability of RNA nanostructures for greater application. Various strategies have been developed to enhance the stability of RNA nanostructures enabling their application in vivo. In this review, we examine the therapeutic applications of RNA nanostructures. Non-immunogenic RNA nanostructures can be rationally designed with functional RNA molecules to modulate gene expression for gene therapy. On the other hand, nucleic acids can be sensed by cellular receptors to elicit an innate immune response, for which certain DNA and RNA motifs can function as adjuvants. Taking advantage of this adjuvant potential, RNA nanostructures can be used for immunotherapy and be designed for cancer vaccines. Thus, we examine the therapeutic application of immunogenic RNA nanostructures for cancer immunotherapy. RNA nanostructures represent promising platforms to design new nanodrugs, gene therapeutics, immunotherapeutic adjuvants, and cancer vaccines. Ongoing research in the field of RNA nanotechnology will continue to empower the development of RNA nanostructure-based therapeutics with high efficacy and limited toxicity.

Precise Insertion of AttB Sequences in Goat Genome Using Enhanced Prime Editor.

Prime editor, an editing tool based on the CRISPR/Cas9 system, allows for all 12 types of nucleotide exchanges and arbitrary indels in genomic sequences without the need for inducing DNA double-strand breaks. Despite its flexibility and precision, prime editing efficiency is still low and hindered by various factors such as target sites, editing types, and the length of the primer binding site. In this study, we developed a prime editing system by incorporating an RNA motif at the 3' terminal of the pegRNA and integrating all twin prime editor factors into a single plasmid. These two strategies enhanced prime editing efficiency at target sites by up to 3.58-fold and 2.19-fold, respectively. Subsequently, enhanced prime editor was employed in goat cells and embryos to efficiently insert a 38 bp attB sequence into the Gt(ROSA)26Sor (Rosa26) and C-C motif chemokine receptor 5 (CCR5) loci. The enhanced prime editor can mediate 11.9% and 6.8% editing efficiency in parthenogenetic activation of embryos through embryo microinjection. In summary, our study introduces a modified prime editing system with improved editing and transfection efficiency, making it more suitable for inserting foreign sequences into primary cells and embryos. These results broaden the potential applications of prime editing technologies in the production of transgenic animals.

A plant-specific clade of serine/arginine-rich proteins regulates RNA splicing homeostasis and thermotolerance in tomato.

Global warming poses a threat for crops, therefore, the identification of thermotolerance mechanisms is a priority. In plants, the core factors that regulate transcription under heat stress (HS) are well described and include several HS transcription factors (HSFs). Despite the relevance of alternative splicing in HS response and thermotolerance, the core regulators of HS-sensitive alternative splicing have not been identified. In tomato, alternative splicing of HSFA2 is important for acclimation to HS. Here, we show that several members of the serine/arginine-rich family of splicing factors (SRSFs) suppress HSFA2 intron splicing. Individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) combined with RNA-Seq revealed that RS2Z35 and RS2Z36, which make up a plant-specific clade of SR proteins, not only regulate HSFA2 but approximately 50% of RNAs that undergo HS-sensitive alternative splicing, with preferential binding to purine-rich RNA motifs. Single and double CRISPR rs2z mutant lines show a dysregulation of splicing and exhibit lower basal and acquired thermotolerance compared to wild type plants. Our results suggest that RS2Z35 and RS2Z36 have a central role in mitigation of the negative effects of HS on RNA splicing homeostasis, and their emergence might have contributed to the increased capacity of plants to acclimate to high temperatures.

Decoding the end of message: PASTA provides food for thought on mechanisms of alternative polyadenylation

In a recent publication in Cell, Kowalski etal.1 developed an interdisciplinary and multiplexed approach to uncover regulatory modules of alternative polyadenylation, involving single-cell-based gene perturbation, isoform abundance analysis, machine learning of RNA motifs, and massively parallel reporter assays.

Modular RNA motifs for orthogonal phase separated compartments

Recent discoveries in biology have highlighted the importance of protein and RNA-based condensates as an alternative to classical membrane-bound organelles. Here, we demonstrate the design of pure RNA condensates from nanostructured, star-shaped RNA motifs. We generate condensates using two different RNA nanostar architectures: multi-stranded nanostars whose binding interactions are programmed via linear overhangs, and single-stranded nanostars whose interactions are programmed via kissing loops. Through systematic sequence design, we demonstrate that both architectures can produce orthogonal (distinct and immiscible) condensates, which can be individually tracked via fluorogenic aptamers. We also show that aptamers make it possible to recruit peptides and proteins to the condensates with high specificity. Successful co-transcriptional formation of condensates from single-stranded nanostars suggests that they may be genetically encoded and produced in living cells. We provide a library of orthogonal RNA condensates that can be modularly customized and offer a route toward creating systems of functional artificial organelles for the task of compartmentalizing molecules and biochemical reactions.