Search Results (26)

Serine/arginine splicing factors (SRSFs) are critical regulators of gene expression that influence alternative splicing through RNA binding via the RNA recognition motif (RRM). Circular RNAs (circRNAs) are a subset of non-coding RNAs that exhibit differential expression in WSSV-infected Litopenaeus vannamei. This study investigates the role of LvSRSF2 in regulating circRNA expression in response to WSSV infection. LvSRSF2 was highly expressed in hemocytes and upregulated during WSSV infection. Silencing LvSRSF2 using dsRNA significantly upregulated the expression of circRNAs (circ-Alpha2, circ-Anillin, circ-Hemocytin, circ-Nephrin, and circ-Toll) in both WSSV-infected and uninfected shrimps at 72 h post-injection with dsRNAs. Knockdown of LvSRSF2 also significantly reduced WSSV copy numbers at 24 h post-infection and extended shrimp survival, with knockdown shrimp surviving up to 9 d compared to the control group. In addition, circ-Hemocytin, an SRSF2-related circRNA, was predicted to interact with six miRNAs targeting immune-related genes such as Toll, STAT, NF-κB, and Vago4. Following WSSV infection, circ-Hemocytin expression increased at 24 and 48 hpi, and the immune genes STAT and Vago4 were also upregulated, suggesting a potential circRNA–miRNA–mRNA regulatory axis in shrimp antiviral defense. Furthermore, targeted suppression of circ-Hemocytin expression using siRNAs significantly reduced its expression without affecting the corresponding linear transcript and resulted in a notable decrease in WSSV load in shrimp gills, highlighting its potential role in antiviral defense. Full article

The cap-binding protein complex (CBC), comprising Cbp20 and Cbp80, is crucial for gene expression, yet its role in the notorious crop pathogen Botrytis cinerea remains unclear. Immunoprecipitation coupled with LC-MS/MS demonstrated that BcCbp20 interacts with BcCbp80. Yeast two-hybrid, GST pull-down, and Split-luciferase complementation assays confirmed that the conserved RNA recognition motif (RRM, 54–127 aa) of BcCbp20 and the N-terminal MIF4G domain (1–370 aa, 1–577 aa) of BcCbp80 constitute the core interaction regions. Genetic transformation experiments revealed that BcCBP80 exerts a more dominant role than BcCBP20 in regulating hyphal morphology, growth rate, conidiophore development, and conidial yield. Furthermore, BcCBP20 and BcCBP80 differentially regulate sclerotium formation to maintain sclerotial quantity. Based on pathogenicity assays, BcCBP80 associated with infection cushion development, with this phenotypic alteration possibly being among the factors correlated with altered pathogenicity. However, the increased sensitivity of ΔBccbp20 to various stress factors may be the primary reason for the diminished pathogenicity. Taken together, these results indicate that BcCBP20 and BcCBP80 play important roles in multiple aspects of B. cinerea growth, development, stress response, and pathogenicity. Full article

Poly(A) Binding Protein Nuclear 1 (PABPN1) is a nuclear poly(A)-binding protein that is highly conserved in eukaryotes. It plays multifaceted roles in RNA processing and metabolism, with its dysregulation closely linked to various diseases. PABPN1 contains an alanine-rich N-terminus, a central coiled-coil domain (CC), a conserved RNA recognition motif (RRM) and a C-terminal extension. PABPN1 influences mRNA splicing and stability through its RNA-binding capabilities, thereby modulating gene expression. While PABPN1 is known to interact with RNA, the molecular mechanism underlying this interaction with RNA awaits further investigation. Here, we designed and purified a PABPN1 fragment encompassing the RNA-binding domain (CCRRM fragment, amino acids 114–254). Using a combination of 3D modeling, small-angle X-ray scattering (SAXS) and selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) assay, our result indicated that CCRRM exhibits a high affinity for poly(A) RNA, a moderate affinity for GU-rich and CU-rich sequences, and negligible binding to AU-rich and CA-rich sequences. RNA binding induces conformation change in the CC. These results suggest that PABPN1 could potentially be involved in cytoplasmic polyadenylation and may influence the regulation of mRNA translation and degradation, although further investigation is required to confirm this role. Full article

During the life cycle of the influenza virus, viral RNPs (vRNPs) are transported to the nucleus for replication. Given that a large number of progeny viral RNA occupies the nucleus, whether there is any host protein located in the nucleus that recognizes the viral RNA and inhibits the viral replication remains largely unknown. In this study, to explore the role of hnRNPH1 in influenza virus infection, we knocked down and over-expressed the hnRNPH1 proteins in 293T cells, then infected the cells with the influenza virus. The results showed that the host hnRNPH1 inhibits the replication of H1N1 and H9N2 influenza viruses by restraining the polymerase activity of viruses. hnRNPH1 contains two RNA recognition motifs (RRM1) and RRM2. Further studies indicated that hnRNPH1 specifically binds to the viral RNA of the PB1, PA, and NP genes. Mutation of the key residues tryptophan and tyrosine in RRM1 and RRM2 abolished the binding affinity to viral RNA and the suppression of polymerase activity of the influenza virus. All the results suggested that hnRNPH1 suppresses polymerase activity and replication of the influenza virus by binding viral RNA. Full article

T-cell intracellular antigen 1 (TIA1) is an RNA-binding protein (RBP) that plays a multifunctional role in RNA metabolism. TIA1 has three RNA-Recognition Motifs (RRMs) and a prion-like carboxyl C-terminal domain (LCD) with intrinsically disordered regions (IDR) implicated in the dynamics (i.e., formation, assembly, and disassembly) of transient RNA-protein aggregates known as stress granules (SGs). A protein related to TIA1 is its paralog TIA1-related/like protein (TIAR/TIAL1), whose amino acid sequence, structural organisation, and molecular and cellular functions are highly conserved with TIA1. Both proteins are the main components of SGs, which are non-membranous RNA-protein condensates formed under stress to promote cell survival. Welander distal myopathy (WDM) is a late-onset muscular dystrophy that has been linked to a single-nucleotide substitution (c.1362G>A; p.E384K) in the gene encoding the TIA1 protein, which impacts TIA1-dependent SGs dynamics. Herein, we have analysed cellular and molecular aspects by targeting mutagenesis to position 384 to understand its molecular grammar in an amino acid/proteinogenic-dependent or -independent manner under oxidative stress. The observations suggest differential, even opposing, behaviours between TIA1 and TIAR in the presence of specific amino acids with negative and positive charges, and also uncharged acids, at equivalent positions of TIA1 and TIAR, respectively. Collectively, these findings illustrate a characteristic molecular grammar of TIAR- and TIA1-dependent SGs under oxidative conditions, suggesting a gain of versatility between two structurally and functionally highly conserved/related proteins. Full article

Dengue virus (DENV) is an enveloped, positive sense, single-stranded RNA virus belonging to the Flaviviridae. Translation initiation of the DENV mRNA (vRNA) can occur following a cap-dependent, 5′-3’end-dependent internal ribosome entry site (IRES)-independent or IRES-dependent mechanism. This study evaluated the activity of DENV IRES in BHK-21 cells and the role of the polypyrimidine-tract binding protein (PTB) isoforms PTB1, PTB2, and PTB4 as IRES-transacting factors (ITAFs) for the DENV IRES. The results show that DENV-IRES activity is stimulated in DENV-replicating BHK-21 cells and cells expressing the Foot-and-mouth disease virus leader or Human rhinovirus 2A proteases. Protease activity was necessary, although a complete shutdown of cap-dependent translation initiation was not a requirement to stimulate DENV IRES activity. Regarding PTB, the results show that PTB1 > PTB2 > PTB4 stimulates DENV-IRES activity in BHK-21 cells. Mutations in the PTB RNA recognition motifs (RRMs), RRM1/RRM2 or RRM3/RRM4, differentially impact PTB1, PTB2, and PTB4’s ability to promote DENV IRES-mediated translation initiation in BHK-21 cells. PTB1-induced DENV-IRES stimulation is rescinded when RRM1/RRM2 or RRM3/RRM4 are disrupted. Mutations in RRM1/RRM2 or RRM3/RRM4 do not affect the ITAF activity of PTB2. Mutating RRM3/RRM4, but not RRM1/RRM2, abolishes the ability of PTB4 to stimulate the DENV IRES. Thus, PTB1, PTB2, and PTB4 are ITAFs for the DENV IRES. Full article

In plants, nucleotide-binding site and leucine-rich repeat proteins (NLRs) play pivotal roles in effector-triggered immunity (ETI). However, the precise mechanisms underlying NLR-mediated disease resistance remain elusive. Previous studies have demonstrated that the NLR gene pair Pik-H4 confers resistance to rice blast disease by interacting with the transcription factor OsBIHD1, consequently leading to the upregulation of hormone pathways. In the present study, we identified an RNA recognition motif (RRM) protein, OsRRM2, which interacted with Pik₁-H4 and Pik₂-H4 in vesicles and chloroplasts. OsRRM2 exhibited a modest influence on Pik-H4-mediated rice blast resistance by upregulating resistance genes and genes associated with chloroplast immunity. Moreover, the RNA-binding sequence of OsRRM2 was elucidated using systematic evolution of ligands by exponential enrichment. Transcriptome analysis further indicated that OsRRM2 promoted RNA editing of the chloroplastic gene ndhB. Collectively, our findings uncovered a chloroplastic RRM protein that facilitated the translocation of the NLR gene pair and modulated chloroplast immunity, thereby bridging the gap between ETI and chloroplast immunity. Full article

Apobec-1 complementation factor (A1CF) functions as an RNA-binding cofactor for APO-BEC1-mediated C-to-U conversion during RNA editing and as a hepatocyte-specific regulator in the alternative pre-mRNA splicing of metabolic enzymes. Its role in RNA editing has not been clearly established. Western blot, co-immunoprecipitation (Co-IP), immunofluorescence (IF), methyl thiazolyl tetrazolium (MTT), and 5-ethynyl-2′-deoxyuridine (EdU) assays were used to examine the role of A1CF beyond RNA editing in renal carcinoma cells. We demonstrated that A1CF interacts with NKRF, independent of RNA and DNA, without affecting its expression or nuclear translocation; however, it modulates p65(Ser536) phosphorylation and IFN-β levels. Truncation of A1CF or deletion on NKRF revealed that the RRM1 domain of A1CF and the p65 binding motif of NKRF are required for their interaction. Deletion of RRM1 on A1CF abrogates NKRF binding, and the decrease in IFN-β expression and p65(Ser536) phosphorylation was induced by A1CF. Moreover, full-length A1CF, but not an RRM1 deletion mutant, promoted cell proliferation in renal carcinoma cells. Perturbation of A1CF levels in renal carcinoma cells altered anchorage-independent growth and tumor progression in nude mice. Moreover, p65(Ser536) phosphorylation and IFN-β expression were lower, but ki67 was higher in A1CF-overexpressing tumor tissues of a xenograft mouse model. Notably, primary and metastatic samples from renal cancer patients exhibited high A1CF expression, low p65(Ser536) phosphorylation, and decreased IFN-β levels in renal carcinoma tissues compared with the corresponding paracancerous tissues. Our results indicate that A1CF-decreased p65(Ser536) phosphorylation and IFN-β levels may be caused by A1CF competitive binding to the p65-combined site on NKRF and demonstrate the direct binding of A1CF independent of RNA or DNA in signal pathway regulation and tumor promotion in renal carcinoma cells. Full article

Polypyrimidine tract binding protein 1 (PTBP1) is one of the most well-described RNA binding proteins, known initially for its role as a splicing repressor before later studies revealed its numerous roles in RNA maturation, stability, and translation. While PTBP1’s various biological roles have been well-described, it remains unclear how its four RNA recognition motif (RRM) domains coordinate these functions. The early PTBP1 literature saw extensive effort placed in detailing structures of each of PTBP1’s RRMs, as well as their individual RNA sequence and structure preferences. However, limitations in high-throughput and high-resolution genomic approaches (i.e., next-generation sequencing had not yet been developed) precluded the functional translation of these findings into a mechanistic understanding of each RRM’s contribution to overall PTBP1 function. With the emergence of new technologies, it is now feasible to begin elucidating the individual contributions of each RRM to PTBP1 biological functions. Here, we review all the known literature describing the apo and RNA bound structures of each of PTBP1’s RRMs, as well as the emerging literature describing the dependence of specific RNA processing events on individual RRM domains. Our goal is to provide a framework of the structure–function context upon which to facilitate the interpretation of future studies interrogating the dynamics of PTBP1 function. Full article

Translation initiation of the hepatitis C virus (HCV) mRNA depends on an internal ribosome entry site (IRES) that encompasses most of the 5′UTR and includes nucleotides of the core coding region. This study shows that the polypyrimidine-tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the HCV 5′UTR, stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Our results show that PTB1 and PTB4, but not PTB2, stimulate HCV IRES activity in HuH-7 and HEK293T cells. In HuH-7 cells, PTB1 promotes HCV IRES-mediated initiation more strongly than PTB4. Mutations in PTB1, PTB4, RRM1/RRM2, or RRM3/RRM4, which disrupt the RRM’s ability to bind RNA, abrogated the protein’s capacity to stimulate HCV IRES activity in HuH-7 cells. In HEK293T cells, PTB1 and PTB4 stimulate HCV IRES activity to similar levels. In HEK293T cells, mutations in RRM1/RRM2 did not impact PTB1′s ability to promote HCV IRES activity; and mutations in PTB1 RRM3/RRM4 domains reduced, but did not abolish, the protein’s capacity to stimulate HCV IRES activity. In HEK293T cells, mutations in PTB4 RRM1/RRM2 abrogated the protein’s ability to promote HCV IRES activity, and mutations in RRM3/RRM4 have no impact on PTB4 ability to enhance HCV IRES activity. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity in a cell type-specific manner. We conclude that PTB1 and PTB4, but not PTB2, act as IRES transacting factors of the HCV IRES. Full article

The poly(A)-binding protein cytoplasmic 1 (PAB1 or PABPC1) protein is associated with the long poly(A) mRNA tails, inducing stability. Herein, we investigated the dynamics of the PABPC1 protein, along with tracing its mRNA binding specificity. During molecular dynamics simulations (MDS), the R176-Y408 amino acids (RRM3–4 domains; RNA recognition motifs) initiated a folded structure that resulted in the formation of different conformations. The RRM4 domain formed high-frequency intramolecular interactions, despite such induced flexibility. Residues D45, Y54, Y56, N58, Q88, and N100 formed long-lasting interactions, and specifically, aromatic residues (Y14, Y54, Y56, W86, and Y140) gained a unique binding pattern with the poly(A) mRNA. In addition, the poly(A) mRNA motif assembled a PABPC1-specific conformation, by inducing movement of the center three nucleotides to face towards RRM1–2 domains. The majority of the high-frequency cancer mutations in PAB1 reside within the RRM4 domain and amino acids engaging in high-frequency interactions with poly(A) mRNA were found to be preserved in different cancer types. Except for the G123C variant, other studied cancer-derived mutants hindered the stability of the protein. Molecular details from this study will provide a detailed understanding of the PABPC1 structure, which can be used to modulate the activity of this gene, resulting in production of mutant peptide or neoantigens in cancer. Full article

Many viruses from the realm Riboviria infecting eukaryotic hosts encode protein domains with sequence similarity to S-adenosylmethionine-dependent methyltransferases. These protein domains are thought to be involved in methylation of the 5′-terminal cap structures in virus mRNAs. Some methyltransferase-like domains of Riboviria are homologous to the widespread cellular FtsJ/RrmJ-like methyltransferases involved in modification of cellular RNAs; other methyltransferases, found in a subset of positive-strand RNA viruses, have been assigned to a separate “Sindbis-like” family; and coronavirus-specific Nsp13/14-like methyltransferases appeared to be different from both those classes. The representative structures of proteins from all three groups belong to a specific variety of the Rossmann fold with a seven-stranded β-sheet, but it was unclear whether this structural similarity extends to the level of conserved sequence signatures. Here I survey methyltransferases in Riboviria and derive a joint sequence alignment model that covers all groups of virus methyltransferases and subsumes the previously defined conserved sequence motifs. Analysis of the spatial structures indicates that two highly conserved residues, a lysine and an aspartate, frequently contact a water molecule, which is located in the enzyme active center next to the methyl group of S-adenosylmethionine cofactor and could play a key role in the catalytic mechanism of the enzyme. Phylogenetic evidence indicates a likely origin of all methyltransferases of Riboviria from cellular RrmJ-like enzymes and their rapid divergence with infrequent horizontal transfer between distantly related viruses. Full article

The spliceosome protein U1A is a prototype case of the RNA recognition motif (RRM) ubiquitous in biological systems. The in vitro kinetics of the chemical denaturation of U1A indicate that the unfolding of U1A is a two-state process but takes place via high energy channeling and a malleable transition state, an interesting variation of typical two-state behavior. Molecular dynamics (MD) simulations have been applied extensively to the study of two-state unfolding and folding of proteins and provide an opportunity to obtain a theoretical account of the experimental results and a molecular model for the transition state ensemble. We describe herein all-atom MD studies including explicit solvent of up to 100 ns on the thermal unfolding (UF) of U1A and 13 mutants. Multiple MD UF trajectories are carried out to ensure accuracy and reproducibility. A vector representation of the MD unfolding process in RMSD space is obtained and used to calculate a free energy landscape for U1A unfolding. A corresponding MD simulation and free energy landscape for the protein CI2, well known to follow a simple two state folding/unfolding model, is provided as a control. The results indicate that the unfolding pathway on the MD calculated free energy landscape of U1A shows a markedly extended transition state compared with that of CI2. The MD results support the interpretation of the observed chevron plots for U1A in terms of a high energy, channel-like transition state. Analysis of the MDUF structures shows that the transition state ensemble involves microstates with most of the RRM secondary structure intact but expanded by ~14% with respect to the radius of gyration. Comparison with results on a prototype system indicates that the transition state involves an ensemble of molten globule structures and extends over the region of ~1–35 ns in the trajectories. Additional MDUF simulations were carried out for 13 U1A mutants, and the calculated φ-values show close accord with observed results and serve to validate our methodology. Full article

Acinetobacter baumannii is a Gram-negative pathogen, known to acquire resistance to antibiotics used in the clinic. The RNA-binding proteome of this bacterium is poorly characterized, in particular for what concerns the proteins containing RNA Recognition Motif (RRM). Here, we browsed the A. baumannii proteome for homologous proteins to the human HuR(ELAVL1), an RNA binding protein containing three RRMs. We identified a unique locus that we called AB-Elavl, coding for a protein with a single RRM with an average of 34% identity to the first HuR RRM. We also widen the research to the genomes of all the bacteria, finding 227 entries in 12 bacterial phyla. Notably we observed a partial evolutionary divergence between the RNP1 and RNP2 conserved regions present in the prokaryotes in comparison to the metazoan consensus sequence. We checked the expression at the transcript and protein level, cloned the gene and expressed the recombinant protein. The X-ray and NMR structural characterization of the recombinant AB-Elavl revealed that the protein maintained the typical β₁α₁β₂β₃α₂β₄ and three-dimensional organization of eukaryotic RRMs. The biochemical analyses showed that, although the RNP1 and RNP2 show differences, it can bind to AU-rich regions like the human HuR, but with less specificity and lower affinity. Therefore, we identified an RRM-containing RNA-binding protein actually expressed in A. baumannii. Full article

RNA-Binding Proteins (RBP) are very diverse and cover a large number of functions in the cells. This review focuses on RBM22, a gene encoding an RBP and belonging to the RNA-Binding Motif (RBM) family of genes. RBM22 presents a Zinc Finger like and a Zinc Finger domain, an RNA-Recognition Motif (RRM), and a Proline-Rich domain with a general structure suggesting a fusion of two yeast genes during evolution: Cwc2 and Ecm2. RBM22 is mainly involved in pre-mRNA splicing, playing the essential role of maintaining the conformation of the catalytic core of the spliceosome and acting as a bridge between the catalytic core and other essential protein components of the spliceosome. RBM22 is also involved in gene regulation, and is able to bind DNA, acting as a bona fide transcription factor on a large number of target genes. Undoubtedly due to its wide scope in the regulation of gene expression, RBM22 has been associated with several pathologies and, notably, with the aggressiveness of cancer cells and with the phenotype of a myelodysplastic syndrome. Mutations, enforced expression level, and haploinsufficiency of RBM22 gene are observed in those diseases. RBM22 could represent a potential therapeutic target in specific diseases, and, notably, in cancer. Full article