RNA Dimerization Research Articles

An architectural role of specific RNA-RNA interactions in oskar granules.

Ribonucleoprotein (RNP) granules are membraneless condensates that organize the intracellular space by compartmentalization of specific RNAs and proteins. Studies have shown that RNA tunes the phase behaviour of RNA-binding proteins, but the role of intermolecular RNA-RNA interactions in RNP granules in vivo remains less explored. Here we determine the role of a sequence-specific RNA-RNA kissing-loop interaction in assembly of mesoscale oskar RNP granules in the female Drosophila germline. We show that a two-nucleotide mutation that disrupts kissing-loop-mediated oskar messenger RNA dimerization impairs condensate formation in vitro and oskar granule assembly in the developing oocyte, leading to defective posterior localization of the RNA and abrogation of oskar-associated processing bodies upon nutritional stress. This specific trans RNA-RNA interaction acts synergistically with the scaffold RNA-binding protein, Bruno, in driving condensate assembly. Our study highlights the architectural contribution of an mRNA and its specific secondary structure and tertiary interactions to the formation of an RNP granule that is essential for embryonic development.

Role of RNA structural plasticity in modulating HIV-1 genome packaging and translation

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5' cap exposure were both poorly packaged and more efficiently translated than those that favored 5' cap sequestration. We propose that structural plasticity of 5' polyA and other conserved RNA elements place the 5' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

A specific phosphorylation-dependent conformational switch in SARS-CoV-2 nucleocapsid protein inhibits RNA binding.

The nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 encapsidates the viral genome and is essential for viral function. The central disordered domain comprises a serine-arginine-rich (SR) region that is hyperphosphorylated in infected cells. This modification regulates function, although mechanistic details remain unknown. We use nuclear magnetic resonance to follow structural changes occurring during hyperphosphorylation by serine arginine protein kinase 1, glycogen synthase kinase 3, and casein kinase 1, that abolishes interaction with RNA. When eight approximately uniformly distributed sites have been phosphorylated, the SR domain binds the same interface as single-stranded RNA, resulting in complete inhibition of RNA binding. Phosphorylation by protein kinase A does not prevent RNA binding, indicating that the pattern resulting from physiologically relevant kinases is specific for inhibition. Long-range contacts between the RNA binding, linker, and dimerization domains are abrogated, phenomena possibly related to genome packaging and unpackaging. This study provides insight into the recruitment of specific host kinases to regulate viral function.

Unraveling the assembly mechanism of SADS-CoV virus nucleocapsid protein: insights from RNA binding, dimerization, and epitope diversity profiling.

SADS-CoV is a porcine coronavirus that originated from a bat HKU2-related coronavirus. It causes devastating swine diseases and poses a high risk of spillover to humans. The coronavirus N protein, as the most abundant viral protein in infected cells, likely plays a key role in viral assembly and replication. However, the structure and function of this protein remain unclear. Therefore, this study employed a combination of biochemistry and X-ray crystallography to uncover distinct structural domains in the N protein, including RNA-binding domains, two disordered domains, and dimerization domains. Additionally, we made the novel discovery that the disordered domain elicited a significant antibody response. These findings provide new insights into the structure and functions of the SADS-CoV N protein, which have important implications for future studies on SADS-CoV diagnosis, as well as the development of vaccines and anti-viral drugs.

A platform to deliver single and bi-specific Cas9/guide RNA to perturb genes in vitro and in vivo

Although CRISPR/Cas9 technology is poised to revolutionize the treatment of diseases with underlying genetic mutations, it faces some significant issues limiting clinical entry. They include low-efficiency in vivo systemic delivery and undesired off-target effects. Here, we demonstrate, by modifying Cas9 with phosphorothioate DNA oligos (PS), one can efficiently deliver single and bi-specific CRISPR/Cas9/guide RNA (gRNA) dimers in vitro and in vivo with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion in vitro and in vivo. PS-Cas9/gRNA mediates gene perturbation in patient-derived tumor organoids and mouse xenograft tumors, leading to potent tumor antitumor effects. Further, HER2 antibody-PS-Cas9/gRNA conjugate selectively perturbs targeted genes in HER2+ ovarian cancer xenografts in vivo. Moreover, we created bi-specific PS-Cas9 with two gRNAs to target two adjacent sequences of the same gene, leading to efficient targeted gene disruption ex vivo and in vivo with markedly reduced unintended gene perturbation. Thus, the cell-penetrating PS-Cas9/gRNA can achieve efficient systemic delivery and precision in gene disruption.

SERPINE1AS2 regulates intramuscular adipogenesis by inhibiting PAI1 protein expression

Antisense long non-coding RNAs (lncRNAs) played a crucial role in the precise regulation of essential biological processes and were abundantly present in animals. Many of these antisense lncRNAs have been identified as key roles in adipose tissue accumulation in livestock, underscoring their vital role in the regulation of animal physiology. Nonetheless, the functional roles of these antisense lncRNAs in regulating adipogenesis and the specific molecular mechanisms these processes were still unclear, which was a significant gap in current scientific research.In this study, we identified and characterized SERPINE1AS2, a novel natural antisense lncRNA, was highly expressed in the fat tissues of adult cattle and calves. Its expression gradually increased during the differentiation of intramuscular adipocytes. Through functional studies, we observed that knockdown of SERPINE1AS2 inhibited the proliferation and adipogenesis of intramuscular adipocytes, while overexpression of SERPINE1AS2 produced the opposite effect. RNA sequencing (RNA-seq) analysis following SERPINE1AS2 knockdown revealed that differential expression genes (DEGs) were significantly enriched in key signaling pathways, notably the MAPK, Wnt, and mTOR signaling pathways. Furthermore, SERPINE1AS2 interacted with Plasminogen Activator Inhibitor-1 (PAI1), forming RNA dimers through complementary base pairing and consequently influencing PAI1 expression. Interestingly, studies on PAI1 suggested that reduced expression facilitated adipogenesis and the downregulation of PAI1 alleviated the inhibitory effect of reduced SERPINE1AS2 on adipogenesis. In summary, this study suggested that SERPINE1AS2 played a crucial role in the adipogenesis of bovine intramuscular adipocytes by modulating the expression of PAI1. SERPINE1AS2 also regulated adipogenesis by engaging in the MAPK, Wnt, and mTOR signaling pathways. Our results suggested that SERPINE1AS2 had a complex regulatory mechanism on adipogenesis in intramuscular adipocytes.

Prediction of 3D RNA Structures from Sequence Using Energy Landscapes of RNA Dimers: Application to RNA Tetraloops.

Access to the three-dimensional structure of RNA enables an ability to gain a more profound understanding of its biological mechanisms, as well as the ability to design RNA-targeting drugs, which can take advantage of the unique chemical environment imposed by a folded RNA structure. Due to the dynamic and structurally complex properties of RNA, both experimental and traditional computational methods have difficulty in determining RNA's 3D structure. Herein, we introduce TAPERSS (Theoretical Analyses, Prediction, and Evaluation of RNA Structures from Sequence), a physics-based fragment assembly method for predicting 3D RNA structures from sequence. Using a fragment library created using discrete path sampling calculations of RNA dinucleoside monophosphates, TAPERSS can sample the physics-based energy landscapes of any RNA sequence with relatively low computational complexity. We have benchmarked TAPERSS on 21 RNA tetraloops, using a combinatorial algorithm as a proof-of-concept. We show that TAPERSS was successfully able to predict the apo-state structures of all 21 RNA hairpins, with 16 of those structures also having low predicted energies as well. We demonstrate that TAPERSS performs most accurately on GNRA-like tetraloops with mostly stacked loop-nucleotides, while having limited success with more dynamic UNCG and CUYG tetraloops, most likely due to the influence of the RNA force field used to create the fragment library. Moreover, we show that TAPERSS can successfully predict the majority of the experimental non-apo states, highlighting its potential in anticipating biologically significant yet unobserved states. This holds great promise for future applications in drug design and related studies. With discussed improvements and implementation of more efficient sampling algorithms, we believe TAPERSS may serve as a useful tool for a physics-based conformational sampling of large RNA structures.

Therapeutic efficacy of a novel self-assembled immunostimulatory siRNA combining apoptosis promotion with RIG-I activation in gliomas

BackgroundCurrent cancer therapies often fall short in addressing the complexities of malignancies, underscoring the urgent need for innovative treatment strategies. RNA interference technology, which specifically suppresses gene expression, offers a promising new approach in the fight against tumors. Recent studies have identified a novel immunostimulatory small-interfering RNA (siRNA) with a unique sequence (sense strand, 5’-C; antisense strand, 3’-GGG) capable of activating the RIG-I/IRF3 signaling pathway. This activation induces the release of type I and III interferons, leading to an effective antiviral immune response. However, this class of immunostimulatory siRNA has not yet been explored in cancer therapy.MethodsIsiBCL-2, an innovative immunostimulatory siRNA designed to suppress the levels of B-cell lymphoma 2 (BCL-2), contains a distinctive motif (sense strand, 5’-C; antisense strand, 3’-GGG). Glioblastoma cells were subjected to 100 nM isiBCL-2 treatment in vitro for 48 h. Morphological changes, cell viability (CCK-8 assay), proliferation (colony formation assay), migration/invasion (scratch test and Transwell assay), apoptosis rate, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were evaluated. Western blotting and immunofluorescence analyses were performed to assess RIG-I and MHC-I molecule levels, and ELISA was utilized to measure the levels of cytokines (IFN-β and CXCL10). In vivo heterogeneous tumor models were established, and the anti-tumor effect of isiBCL-2 was confirmed through intratumoral injection.ResultsIsiBCL-2 exhibited significant inhibitory effects on glioblastoma cell growth and induced apoptosis. BCL-2 mRNA levels were significantly decreased by 67.52%. IsiBCL-2 treatment resulted in an apoptotic rate of approximately 51.96%, accompanied by a 71.76% reduction in MMP and a 41.87% increase in ROS accumulation. Western blotting and immunofluorescence analyses demonstrated increased levels of RIG-I, MAVS, and MHC-I following isiBCL-2 treatment. ELISA tests indicated a significant increase in IFN-β and CXCL10 levels. In vivo studies using nude mice confirmed that isiBCL-2 effectively impeded the growth and progression of glioblastoma tumors.ConclusionsThis study introduces an innovative method to induce innate signaling by incorporating an immunostimulatory sequence (sense strand, 5’-C; antisense strand, 3’-GGG) into siRNA, resulting in the formation of RNA dimers through Hoogsteen base-pairing. This activation triggers the RIG-I signaling pathway in tumor cells, causing further damage and inducing a potent immune response. This inventive design and application of immunostimulatory siRNA offer a novel perspective on tumor immunotherapy, holding significant implications for the field.

HIV-1 capsid stability and reverse transcription are finely balanced to minimize sensing of reverse transcription products via the cGAS-STING pathway.

In HIV-1 particles, the dimeric RNA genome and associated viral proteins and enzymes are encased in a proteinaceous lattice composed of the viral capsid protein. Herein, we assessed how altering the stability of this capsid lattice through orthogonal genetic and chemical approaches impacts the induction of innate immune responses. Specifically, we found that decreasing capsid lattice stability results in more potent sensing of viral reverse transcription products, but not the genomic RNA, in a cGAS-STING-dependent manner. The recently developed capsid inhibitors lenacapavir and GS-CA1 enhanced the innate immune sensing of HIV-1. Unexpectedly, due to increased levels of reverse transcription and cytosolic accumulation of the resulting viral cDNA, capsid mutants with hyperstable cores also resulted in the potent induction of type I interferon-mediated innate immunity. Our findings suggest that HIV-1 capsid lattice stability and reverse transcription are finely balanced to minimize exposure of reverse transcription products in the cytosol of host cells.

HIV-1 RNA genome packaging: it's G-rated.

A member of the Retroviridae, human immunodeficiency virus type 1 (HIV-1), uses the RNA genome packaged into nascent virions to transfer genetic information to its progeny. The genome packaging step is a highly regulated and extremely efficient process as a vast majority of virus particles contain two copies of full-length unspliced HIV-1 RNA that form a dimer. Thus, during virus assembly HIV-1 can identify and selectively encapsidate HIV-1 unspliced RNA from an abundant pool of cellular RNAs and various spliced HIV-1 RNAs. Several "G" features facilitate the packaging of a dimeric RNA genome. The viral polyprotein Gag orchestrates virus assembly and mediates RNA genome packaging. During this process, Gag preferentially binds unpaired guanosines within the highly structured 5' untranslated region (UTR) of HIV-1 RNA. In addition, the HIV-1 unspliced RNA provides a scaffold that promotes Gag:Gag interactions and virus assembly, thereby ensuring its packaging. Intriguingly, recent studies have shown that the use of different guanosines at the junction of U3 and R as transcription start sites results in HIV-1 unspliced RNA species with 99.9% identical sequences but dramatically distinct 5' UTR conformations. Consequently, one species of unspliced RNA is preferentially packaged over other nearly identical RNAs. These studies reveal how conformations affect the functions of HIV-1 RNA elements and the complex regulation of HIV-1 replication. In this review, we summarize cis- and trans-acting elements critical for HIV-1 RNA packaging, locations of Gag:RNA interactions that mediate genome encapsidation, and the effects of transcription start sites on the structure and packaging of HIV-1 RNA.

Cryo-EMstructure of SRP68/72 reveals an extended dimerization domain with RNA-binding activity.

The signal recognition particle (SRP) is a critical component in protein sorting pathways in all domains of life. Human SRP contains six proteins bound to the 7S RNA and their structures and functions have been mostly elucidated. The SRP68/72 dimer is the largest SRP component and is essential for SRP function. Although the structures of the SRP68/72 RNA binding and dimerization domains have been previously reported, the structure and function of large portions of the SRP68/72 dimer remain unknown. Here, we analyse full-length SRP68/72 using cryo-EM and report that SRP68/72 depend on each other for stability and form an extended dimerization domain. This newly observed dimerization domain is both a protein- and RNA-binding domain. Comparative analysis with current structural models suggests that this dimerization domain undergoes dramatic translocation upon SRP docking onto SRP receptor and eventually comes close to the Alu domain. We propose that the SRP68/72 dimerization domain functions by binding and detaching the Alu domain and SRP9/14 from the ribosomal surface, thus releasing elongation arrest upon docking onto the ER membrane.

Bipartite viral RNA genome heterodimerization influences genome packaging and virion thermostability.

Multi-segmented viruses often multimerize their genomic segments to ensure efficient and stoichiometric packaging of the correct genetic cargo. In the bipartite Nodaviridae family, genome heterodimerization is also observed and conserved among different species. However, the nucleotide composition and biological function for this heterodimer remain unclear. Using Flock House virus as a model system, we developed a next-generation sequencing approach ("XL-ClickSeq") to probe heterodimer site sequences. We identified an intermolecular base-pairing site which contributed to heterodimerization in both wild-type and defective virus particles. Mutagenic disruption of this heterodimer site exhibited significant deficiencies in genome packaging and encapsidation specificity to viral genomic RNAs. Furthermore, the disruption of this intermolecular interaction directly impacts the thermostability of the mature virions. These results demonstrate that the intermolecular RNA-RNA interactions within the encapsidated genome of an RNA virus have an important role on virus particle integrity and thus may impact its transmission to a new host.IMPORTANCEFlock House virus is a member of Nodaviridae family of viruses, which provides a well-studied model virus for non-enveloped RNA virus assembly, cell entry, and replication. The Flock House virus genome consists of two separate RNA molecules, which can form a heterodimer upon heating of virus particles. Although similar RNA dimerization is utilized by other viruses (such as retroviruses) as a packaging mechanism and is conserved among Nodaviruses, the role of heterodimerization in the Nodavirus replication cycle is unclear. In this research, we identified the RNA sequences contributing to Flock House virus genome heterodimerization and discovered that such RNA-RNA interaction plays an essential role in virus packaging efficiency and particle integrity. This provides significant insight into how the interaction of packaged viral RNA may have a broader impact on the structural and functional properties of virus particles.

CRISPR delivery with extracellular vesicles: Promises and challenges

AbstractThe CRISPR gene editing tool holds great potential for curing genetic disorders. However, the safe, efficient, and specific delivery of the CRISPR/Cas9 components into cells and tissues remains a challenge. While many currently available delivery methods achieve high levels of gene editing effects in vivo, they often result in genotoxicity and immunogenicity. Extracellular vesicles (EVs), which are cell‐derived lipid nanoparticles, are capable of transferring protein and nucleic acid cargoes between cells, making them a promising endogenous alternative to synthetic delivery methods. This review provides a comprehensive analysis of the currently available strategies for EV‐mediated delivery of CRISPR/Cas9. These strategies include cell‐based, passive loading obtained by overexpression of CRISPR/Cas9, active loading involving protein or RNA dimerization, and loading into already purified EVs. All these approaches suggest that EV‐based CRISPR/Cas9 delivery is useful for achieving both in vitro and in vivo gene editing. Despite that, substantial variations in cellular uptake and gene editing efficiencies indicate that further improvement and standardization are required for the therapeutic use of EVs as a CRISPR/Cas9 delivery vehicle. These improvements include, but is not limited to, the high‐yield purification of EVs, increased loading and release efficiencies, as well as improved tissue‐ or cell‐specific targeting specificities.

Co-crystal structures of the fluorogenic aptamer Beetroot show that close homology may not predict similar RNA architecture

Beetroot is a homodimeric in vitro selected RNA that binds and activates DFAME, a conditional fluorophore derived from GFP. It is 70% sequence-identical to the previously characterized homodimeric aptamer Corn, which binds one molecule of its cognate fluorophore DFHO at its interprotomer interface. We have now determined the Beetroot-DFAME co-crystal structure at 1.95 Å resolution, discovering that this RNA homodimer binds two molecules of the fluorophore, at sites separated by ~30 Å. In addition to this overall architectural difference, the local structures of the non-canonical, complex quadruplex cores of Beetroot and Corn are distinctly different, underscoring how subtle RNA sequence differences can give rise to unexpected structural divergence. Through structure-guided engineering, we generated a variant that has a 12-fold fluorescence activation selectivity switch toward DFHO. Beetroot and this variant form heterodimers and constitute the starting point for engineered tags whose through-space inter-fluorophore interaction could be used to monitor RNA dimerization.

Solution structure and behaviour of the Arabidopsis thaliana HYL1 protein

In plants, microRNA biogenesis involves the complex assembly of molecular processes that are mostly governed by three proteins: RNase III protein DCL1 and two RNA binding proteins, SERRATE and HYL1. HYL1 protein is a double stranded RNA binding protein that is needed for the precise excision of miRNA/miRNA* duplex from the stem-loop containing primary miRNA gene transcripts. Moreover, HYL1 protein partners with HSP90 and CARP9 proteins to load the miRNA molecules onto the AGO1 endonuclease. HYL1 protein as a crucial player in the biogenesis pathway is regulated by its phosphorylation status to fine tune the levels of miRNA in various physiological conditions. HYL1 protein consists of two dsRNA binding domains (dsRBD) that are involved in RNA binding and dimerization and a C-terminal disordered tail of unknown function. Although the spatial structures of the individual dsRBDs have been determined there is a lack of information about the behaviour and structure of the full length protein. Using small the angle X-ray scattering (SAXS) technique we investigated the structure and dynamic of the HYL1 protein from Arabidopsis thaliana in solution. We show that the C-terminal domain is disordered and dynamic in solution and that HYL1 protein dimerization is dependent on the concentration. HYL1 protein lacking a C-terminal tail and a nuclear localisation signal (NLS) fragment is almost exclusively monomeric and similarly to full-length protein has a dynamic nature in solution. Our results point for the first time to the role of the C-terminal fragment in stabilisation of HYL1 dimer formation.

Nonsuppressible viremia during HIV-1 therapy meets molecular virology.

HIV-1 replication can be suppressed with antiretroviral therapy (ART), but individuals who stop taking ART soon become viremic again. Some people experience extended times of detectable viremia despite optimal adherence to ART. In this issue of the JCI, White, Wu, and coauthors elucidate a source of nonsuppressible viremia (NSV) in treatment-adherent patients - clonally expanded T cells harboring HIV-1 proviruses with small deletions or mutations in the 5'-leader, the UTR that includes the major splice donor site of viral RNA. These mutations altered viral RNA-splicing efficiency and RNA dimerization and packaging, yet still allowed production of detectable levels of noninfectious virus particles. These particles lacked the HIV-1 Env surface protein required for cell entry and failed to form the mature capsid cone required for infectivity. These studies improve our understanding of NSV and the regulation of viral functions in the 5'-leader with implications for rationalized care in individuals with NSV.

Engineer RNA-Protein Nanowires as Light-Responsive Biomaterials.

RNA molecules have emerged as increasingly attractive biomaterials with important applications such as RNA interference (RNAi) for cancer treatment and mRNA vaccines against infectious diseases. However, it remains challenging to engineer RNA biomaterials with sophisticated functions such as non-covalent light-switching ability. Herein, light-responsive RNA-protein nanowires are engineered to have such functions. It first demonstrates that the high affinity of RNA aptamer enables the formation of long RNA-protein nanowires through designing a dimeric RNA aptamer and an engineered green fluorescence protein (GFP) that contains two TAT-derived peptides at N- and C- termini. GFP is then replaced with an optogenetic protein pair system, LOV2 (light-oxygen-voltage) protein and its binding partner ZDK (Z subunit of protein A), to confer blue light-controlled photo-switching ability. The light-responsive nanowires are long (>500 nm) in the dark, but small (20-30 nm) when exposed to light. Importantly, the co-assembly of this RNA-protein hybrid biomaterial does not rely on the photochemistry commonly used for light-responsive biomaterials, such as bond formation, cleavage, and isomerization, and is thus reversible. These RNA-protein structures can serve as a new class of light-controlled biocompatible frameworks for incorporating versatile elements such as RNA, DNA, and enzymes.

Unusual topological RNA G-quadruplex formed by an RNA duplex: implications for the dimerization of SARS-CoV-2 RNA.

The infectious disease coronavirus 2019 (SARS-CoV-2) is caused by a virus that has RNA as its genetic material. To understand the detailed structural features of SARS-COV-2 RNA, we probed the RNA structure by NMR. Two RNA sequences form a duplex and self-associate to form a dimeric G-quadruplex. The FrG nucleoside was employed as a 19F sensor to confirm the RNA structure in cells by 19F NMR. A FRET assay further demonstrated that the dimeric G-quadruplex resulted in RNA dimerization in cells. These results provide the basis for the elucidation of SARS-COV-2 RNA function, which provides new insights into developing novel antiviral drugs against SARS-COV-2.

Genome sequence analysis suggests coevolution of the DIS, SD, and Psi hairpins in HIV-1 genomes

HIV-1 RNA dimerization is a critical step in viral life cycle. It is a prerequisite for genome packaging and plays an important role in reverse transcription and recombination. Dimerization is promoted by the DIS (dimerization initiation site) hairpin located in the 5′ leader of HIV-1 genome. Despite the high genetic diversity in HIV-1 group M, only five apical loops (AAGCGCGCA, AAGUGCGCA, AAGUGCACA, AGGUGCACA and AGUGCAC) are commonly found in DIS hairpins. We refer to the parent DISes with these apical loops as DISLai, DISTrans, DISF, DISMal, and DISC, respectively. Based on identity or similarity of DIS hairpins to parent DISes, we distributed HIV-1 M genomes into five dimerization groups. Comparison of the primary and secondary structures of DIS, SD and Psi hairpins in about 3000 HIV-1 M genomes showed that the mutation frequencies at particular nucleotide positions of these hairpins differ among the dimerization groups, and DISF may be an origin of other parent DISes. We found that DIS, SD and Psi hairpins have hundreds of variants, only some of them occurring rather frequently. The lower part of DIS hairpin with G x AGG internal loop is highly conserved in both HIV-1 and SIV genomes. We supposed that the G-quadruplex, located 56 nts downstream of the Gag start codon, may participate in switching of HIV-1 leader RNA from BMH (branched multiple hairpins) to LDI (long distance interaction) conformation.

Molecular Modelling of NONO and SFPQ Dimerization Process and RNA Recognition Mechanism

NONO and SFPQ are involved in multiple nuclear processes (e.g., pre-mRNA splicing, DNA repair, and transcriptional regulation). These proteins, along with NEAT1, enable paraspeckle formation, thus promoting multiple myeloma cell survival. In this paper, we investigate NONO and SFPQ dimer stability, highlighting the hetero- and homodimer structural differences, and model their interactions with RNA, simulating their binding to a polyG probe mimicking NEAT1guanine-rich regions. We demonstrated in silico that NONO::SFPQ heterodimerization is a more favorable process than homodimer formation. We also show that NONO and SFPQ RRM2 subunits are primarily required for protein–protein interactions with the other DBHS protomer. Simulation of RNA binding to NONO and SFPQ, beside validating RRM1 RNP signature importance, highlighted the role of β2 and β4 strand residues for RNA specific recognition. Moreover, we demonstrated the role of the NOPS region and other protomer’s RRM2 β2/β3 loop in strengthening the interaction with RNA. Our results, having deepened RNA and DBHS dimer interactions, could contribute to the design of small molecules to modulate the activity of these proteins. RNA-mimetics, able to selectively bind to NONO and/or SFPQ RNA-recognition site, could impair paraspeckle formation, thus representing a first step towards the discovery of drugs for multiple myeloma treatment.