hnRNP A1 Protein Research Articles

P-coumaric acid regulates exon 12 splicing of the ATP7B gene by modulating hnRNP A1 protein expressions

P-coumaric acid regulates exon 12 splicing of the ATP7B gene by modulating hnRNP A1 protein expressions

Sequence-specific interactions determine viscoelasticity and aging dynamics of protein condensates.

Biomolecular condensates are viscoelastic materials. Here, we investigate the determinants of sequence-encoded and age-dependent viscoelasticity of condensates formed by the prion-like low-complexity domain of the protein hnRNP A1 and its designed variants. We find that the dominantly viscous forms of the condensates are metastable Maxwell fluids. A Rouse-Zimm model that accounts for the network-like organization of proteins within condensates reproduces the measured viscoelastic moduli. We show that the strengths of aromatic inter-sticker interactions determine sequence-specific amplitudes of elastic and viscous moduli, and the timescales over which elastic properties dominate. These condensates undergo physical ageing on sequence-specific timescales. This is driven by mutations to spacer residues that weaken the metastability of dominantly viscous phases. The ageing of condensates is accompanied by disorder-to-order transitions, leading to the formation of non-fibrillar, beta-sheet-containing, semi-crystalline, elastic, Kelvin-Voigt solids. Our results suggest that sequence grammars, which refer to amino acid identities of stickers versus spacers in prion-like low-complexity domains, have evolved to afford control over metastabilities of dominantly viscous fluid phases of condensates. This selection is likely to render barriers for conversion from metastable fluids to globally stable solids insurmountable on functionally relevant timescales.

ZMAT3 participated in benzene-caused disruption in self-renewal and differentiation of hematopoietic stem cells via TNF-α/NF-κB pathway

Benzene is a common environmental and occupational pollutant, benzene exposure causes damage to hematopoietic system. ZMAT3 is a zinc finger protein which has important biological functions. In this study, benzene-exposed mouse model and ZMAT3 overexpression and low expression hematopoietic stem cells (HSCs) models were constructed to explore the mechanism of ZMAT3 in benzene-induced hematopoietic toxicity. The results showed that benzene increased the expression of ZMAT3 in mouse bone marrow (BM) cells, HSCs and peripheral blood (PB) leukocyte, and the changes in HSCs were more sensitive than BM and PB cells. In addition, overexpression of ZMAT3 decreased the self-renewal ability of HSCs and reduced the HSCs differentiation into myeloid hematopoietic cells, while low expression has the opposite effect. Besides, over and low expression of ZMAT3 both increased the HSCs differentiation into lymphoid progenitor cells. Moreover, bioinformatics analysis suggested that ZMAT3 was associated with TNF-α signaling pathway, and the correlation was confirmed in mouse model. Meanwhile, the results indicated that ZMAT3 promoted TNF-α mRNA processing by binding to the ARE structural domain on TNF-α and interacting with hnRNP A2/B1 and hnRNP A1 proteins, ultimately activating the NF-κB signaling pathway. This study provides a new mechanism for the study of benzene toxicity.

Lactylome analyses suggest systematic lysine-lactylated substrates in oral squamous cell carcinoma under normoxia and hypoxia

Cancer cells tend to live in hypoxic environment characterized by enhanced glycolysis and accumulation of lactate. Intracellular lactate is shown to drive a novel type of post-translational modification (PTM), lysine lactylation (Kla). Kla has been confirmed to affect the malignant progression of tumors such as hepatocellular carcinoma (HCC) and colon cancer, whereas the global lactylomic profiling of oral squamous cell carcinoma (OSCC) is unclear. Here, the integrative lactylome and proteome analyses by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1011 Kla sites within 532 proteins and 1197 Kla sites within 608 proteins in SCC25 cells under normoxic and hypoxic environments, respectively. Among these lactylated proteins, histones accounted for only a small fraction, suggesting the presence of Kla modification of OSCC in a large number of non-histone proteins. Notably, Kla preferred to enrich in spliceosome, ribosome and glycolysis/gluconeogenesis pathway in both normoxic and hypoxic cultures. Compared with normoxia, 589 differential proteins with 898 differentially lactylated sites were detected under hypoxia, which were mainly associated with the glycolysis/gluconeogenesis pathway by KEGG analysis. Importantly, we verified the presence of lactylation modification in the spliceosomal proteins hnRNPA1, SF3A1, hnRNPU and SLU7, as well as in glycolytic enzyme PFKP. In addition, the differential alternative splicing analysis described the divergence of pre-mRNA splicing patterns in the presence or absence of sodium lactate and at different oxygen concentrations. Finally, a negative correlation between tissue Kla levels and the prognosis of OSCC patients was revealed by immunohistochemistry. Our study is the first report to elucidate the lactylome and its biological function in OSCC, which deepens our understanding of the mechanisms underlying OSCC progression and provides a novel strategy for targeted therapy for OSCC.

Pedigree Analysis and Molecular Mechanism Study of Hereditary Glanzmann Thrombasthenia Caused by Compound Heterozygous Mutation of the ITGA2B Gene

Objective: The phenotype and genotype of a pedigree with Glanzmann thrombasthenia caused by compound heterozygous mutation in the ITGA2B gene and its molecular pathogenesis were explored. Methods: The platelet aggregation rate of the proband and his family was detected by using a platelet aggregation test with adenosine diphosphate, collagen, epinephrine, arachidonic acid, and ristocetin. The expression levels of CD41 (αⅡb), CD61 (β3), and CD42b (GPⅠb) on the platelet surface was detected by flow cytometry. Gene sequencing technology was used for the genetic identification of the family. RT-PCR was used in the detection of mRNA splicing, and qRT-PCR was used in detecting the relative mRNA level of the ITGA2B gene. Bioinformatics analysis was used to evaluate the pathogenicity of mutation sites and their effects on protein structure and function. The expressions of total αⅡb and β3 in platelets were analyzed by Western blot. Results: Except ristocetin, the other four inducers could not induce platelet aggregation in the proband. Flow cytometry showed that the expression levels of αⅡb and β3 were only 0.25% and 9.76%, respectively, on the platelet surface of the proband, whereas GPⅠb expression was relatively normal. The expression levels of glycoproteins in the other family members were almost normal. c.480C>G and c.2929C>T mutations were detected in the proband through gene sequencing. The c.480C>G mutation was inherited from his mother, and the c.2929C>T mutation was inherited from his father. The RT-PCR and sequencing results showed that the c.480C>G mutation caused mRNA splicing in the proband and his mother, resulting in the deletion of 99 bases in c.476G-574A (p.S160-S192). qRT-PCR showed that the c.2929C>T variant reduced the mRNA level of the ITGA2B gene in the proband and his father. Bioinformatics analysis suggested that the c.480C>G mutation might form a binding sequence with hnRNP A1 protein and generate the 5'SS splice site. The three-dimensional structural model of the αⅡb subunit showed that the β-propeller domain of the p.S160-S192 deletion lost two β-strands and one α-helix in blade 2. The c.2929C>T nonsense mutation caused premature translation termination and produced a truncated protein with the deletion of p.R977-E1039, including the cytoplasmic domain, transmembrane domain, and a β chain of the extracellular Calf-2 domain. The total αⅡb expression of the proband was absent, and the relative expression of β3 was 11.36% of the normal level. Conclusion: The compound heterozygous mutation c.480C>G in exon 4 and c.2929C>T in exon 28 of the ITGA2B gene probably underlies Glanzmann thrombasthenia in this pedigree.

A mutation in the low-complexity domain of splicing factor hnRNPA1 linked to amyotrophic lateral sclerosis disrupts distinct neuronal RNA splicing networks.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by loss of motor neurons. Human genetic studies have linked mutations in RNA-binding proteins as causative for this disease. The hnRNPA1 protein, a known pre-mRNA splicing factor, is mutated in some ALS patients. Here, two human cell models were generated to investigate how a mutation in the C-terminal low-complexity domain (LCD) of hnRNPA1 can cause splicing changes of thousands of transcripts that collectively are linked to the DNA damage response, cilium organization, and translation. We show that the hnRNPA1 D262V mutant protein binds to new binding sites on differentially spliced transcripts from genes that are linked to ALS. We demonstrate that this ALS-linked hnRNPA1 mutation alters normal RNA-dependent protein-protein interactions. Furthermore, cells expressing this hnRNPA1 mutant exhibit a cell aggregation phenotype, markedly reduced growth rates, changes in stress granule kinetics, and aberrant growth of neuronal processes. This study provides insight into how a single amino acid mutation in a splicing factor can alter RNA splicing networks of genes linked to ALS.

Interference of gut-brain-gonad axis originating from triclocarban exposure to parent zebrafish induces offspring embryonic development abnormality by up-regulation of maternal circSGOL1

Triclocarban (TCC) is a widely used antibacterial ingredient possessing acute toxicity effects; however, its chronic toxicity and underlying molecular mechanisms remain uncertain. Herein, we demonstrated that chronic TCC exposure affects the growth and development of adult zebrafish through inducing an intestinal flora disorder in the gut. The imbalance of intestinal flora caused functional barriers within the intestinal-brain-gonadal axis. This resulted in a series of anomalous nerve and motor behaviors, and reproductive toxicity as reflected in pathological damage to parental gonads and F1-larval developmental malformations. Abnormal development of F1 larvae was attributed to apoptosis induced by the up-regulation of circSGOL1. This up-regulation affected the activity and localization of the hnRNP A1 protein, which then promoted overexpression of pro-apoptotic related genes that ultimately lead to apoptosis during early embryonic development. Overall, these novel findings systematically elucidated the TCC toxicity mechanism in parent-offspring dyads, and provide important theoretical guidance for early risk warning and control of chronic TCC toxicity.

N6-methyladenosine modification of OIP5-AS1 promotes glycolysis, tumorigenesis, and metastasis of gastric cancer by inhibiting Trim21-mediated hnRNPA1 ubiquitination and degradation

BackgroundOpa-interacting protein 5 antisense transcript 1 (OIP5-AS1) has been demonstrated to play vital roles in development and progression of tumors such as gastric cancer (GC). However, the detailed molecular mechanism of OIP5-AS1 has not been completely elucidated. Our study aimed to investigate the role and the epigenetic regulation mechanism of OIP5-AS1 in GC.MethodsOIP5-AS1 expression in GC tissues was detected by RT-qPCR. Loss- and gain-of-function experiments were conducted to assess the biological function of OIP5-AS1 in vitro and in vivo. The interaction of OIP5-AS1 with insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) or heterogeneous nuclear nucleoprotein A1 (hnRNPA1) was verified by bioinformatics analysis, RNA pull-down assays, and RNA immunoprecipitation assays.ResultsIn this study, we identified that OIP5-AS1 is specifically overexpressed in GC tumor tissues and cell lines and correlated with a poor prognosis. The loss of OIP5-AS1 suppressed the proliferation, migration, invasion, epithelial–mesenchymal transition (EMT), and glycolysis of GC cells, but the ectopic expression of OIP5-AS1 had the opposite impact. Meanwhile, knockdown of OIP5-AS1 inhibited tumor growth in patient-derived xenograft models, as well as repressed tumor metastasis. Mechanistically, IGF2BP3 could bind to OIP5-AS1 by N6-methyladenosine (m6A) modification sites on OIP5-AS1, thereby stabilizing OIP5-AS1. Moreover, OIP5-AS1 prevented Trim21-mediated ubiquitination and degradation of hnRNPA1, stabilizing hnRNPA1 protein and promoting the malignant progression of GC by regulating PKM2 signaling pathway.ConclusionsIn conclusion, this study highlighted that OIP5-AS1 is an oncogenic m6A-modified long non-coding RNA (lncRNA) in GC and that IGF2BP3/OIP5-AS1/hnRNPA1 axis may provide a potential diagnostic or prognostic target for GC.

Inhibition of the lncRNA 585189 prevents podocyte injury and mitochondria dysfunction by promoting hnRNP A1 and SIRT1 in diabetic nephropathy

Podocyte dysfunction has been identified as a crucial pathological characteristic of diabetic nephropathy (DN). However, the regulatory effects of long non-coding RNAs (lncRNAs) in this process have not been fully elucidated. Here, we performed an unbiased RNA-sequencing (RNA-seq) analysis of renal tissues and identified a significantly upregulated long non-coding RNA, ENST00000585189.1 (lncRNA 585189), in patients with DN. Furthermore, lncRNA 585189 was positively correlated with renal insufficiency and was upregulated in both DN patients and high-glucose-induced human podocytes. Gain- and loss-of-function experiments revealed that silencing lncRNA 585189 decreased the production of ROS, rescued aberrant mitochondrial morphology and membrane potential, and alleviated podocyte damage caused by high glucose. Mechanistically, bioinformatics analysis predicted an interaction between lncRNA 585189 and hnRNP A1, which was subsequently confirmed by RIP, pull-down, and EMSA assays. Further investigation revealed that lncRNA 585189 destabilizes the hnRNP A1 protein, leading to the downregulation of its expression. Conversely, hnRNP A1 promoted the expression of lncRNA 585189. Moreover, both RIP and pull-down assays demonstrated a direct interaction between hnRNP A1 and SIRT1, which enhanced SIRT1 mRNA stability. Our findings suggest that lncRNA 585189 suppresses SIRT1 through hnRNP A1, thereby hindering the recovery from mitochondrial abnormalities and podocyte damage. In summary, targeting lncRNA 585189 is a promising strategy for reversing mitochondrial dysfunction and treating DN.

Suppression of hnRNP A1 binding to HK1 RNA leads to glycolytic dysfunction in Alzheimer's disease models.

To investigate the mechanism of RNA-binding protein hnRNP A1 in mouse hippocampal neurons (HT22) on glycolysis. RIP and CLIP-qPCR were performed by HT22 in vitro to observe the mechanism of hnRNP A1 regulating the expression of key proteins in glycolysis. The RNA binding domain of hnRNP A1 protein in HT22 was inhibited by VPC-80051, and the effect of hnRNP A1 on glycolysis of HT22 was observed. Lentivirus overexpression of hnRNP A1 was used to observe the effect of overexpression of hnRNP A1 on glycolysis of Aβ25-35-injured HT22. The expression of hnRNP A1 in brain tissues of wild-type mice and triple-transgenic (APP/PS1/Tau) AD mice at different ages was studied by Western blot assay. The results of RIP experiment showed that hnRNP A1 and HK1 mRNA were significantly bound. The results of CLIP-qPCR showed that hnRNP A1 directly bound to the 2605-2821 region of HK1 mRNA. hnRNP A1 inhibitor can down-regulate the expression of HK1 mRNA and HK1 protein in HT22 cells. Overexpression of hnRNP A1 can significantly reduce the toxic effect of Aβ25-35 on neurons via the hnRNP A1/HK1/ pyruvate pathway. In addition, inhibition of hnRNP A1 binding to amyloid precursor protein (APP) RNA was found to increase Aβ expression, while Aβ25-35 also down-regulated hnRNP A1 expression by enhancing phosphorylation of p38 MAPK in HT22. They interact to form bidirectional regulation, further down-regulating the expression of hnRNP A1, and ultimately aggravating glycolytic dysfunction. Protein immunoblotting showed that hnRNP A1 decreased with age in mouse brain tissue, and the decrease was greater in AD mice, suggesting that the decrease of hnRNP A1 may be a predisposed factor in the pathogenesis of AD.

Cryo-EM Structure of the Full-length hnRNPA1 Amyloid Fibril

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a multifunctional RNA-binding protein that is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis and multisystem proteinopathy. In this study, we have used cryo-electron microscopy to investigate the three-dimensional structure of amyloid fibrils from full-length hnRNPA1 protein. We find that the fibril core is formed by a 45-residue segment of the prion-like low-complexity domain of the protein, whereas the remaining parts of the protein (275 residues) form a fuzzy coat around the fibril core. The fibril consists of two fibril protein stacks that are arranged into a pseudo-21 screw symmetry. The ordered core harbors several of the positions that are known to be affected by disease-associated mutations, but does not encompass the most aggregation-prone segments of the protein. These data indicate that the structures of amyloid fibrils from full-length proteins may be more complex than anticipated by current theories on protein misfolding.

Time-Dependent Material Properties of Aging Biomolecular Condensates from Different Viscoelasticity Measurements in Molecular Dynamics Simulations.

Biomolecular condensates are important contributors to the internal organization of the cell material. While initially described as liquid-like droplets, the term biomolecular condensates is now used to describe a diversity of condensed phase assemblies with material properties extending from low to high viscous liquids, gels, and even glasses. Because the material properties of condensates are determined by the intrinsic behavior of their molecules, characterizing such properties is integral to rationalizing the molecular mechanisms that dictate their functions and roles in health and disease. Here, we apply and compare three distinct computational methods to measure the viscoelasticity of biomolecular condensates in molecular simulations. These methods are the Green-Kubo (GK) relation, the oscillatory shear (OS) technique, and the bead tracking (BT) method. We find that, although all of these methods provide consistent results for the viscosity of the condensates, the GK and OS techniques outperform the BT method in terms of computational efficiency and statistical uncertainty. We thus apply the GK and OS techniques for a set of 12 different protein/RNA systems using a sequence-dependent coarse-grained model. Our results reveal a strong correlation between condensate viscosity and density, as well as with protein/RNA length and the number of stickers vs spacers in the amino acid protein sequence. Moreover, we couple the GK and the OS technique to nonequilibrium molecular dynamics simulations that mimic the progressive liquid-to-gel transition of protein condensates due to the accumulation of interprotein β-sheets. We compare the behavior of three different protein condensates, i.e., those formed by either hnRNPA1, FUS, or TDP-43 proteins, whose liquid-to-gel transitions are associated with the onset of amyotrophic lateral sclerosis and frontotemporal dementia. We find that both the GK and OS techniques successfully predict the transition from functional liquid-like behavior to kinetically arrested states once the network of interprotein β-sheets has percolated through the condensates. Overall, our work provides a comparison of different modeling rheological techniques to assess the viscosity of biomolecular condensates, a critical magnitude that provides information on the behavior of biomolecules inside condensates.

Serum/glucose starvation strikingly reduce heterogeneous nuclear ribonucleoprotein A1 protein and its target, cyclin D1.

Our investigation to explore cellular alterations related to undernutrition in cancer cells revealed that the protein level of heterogenous nuclear ribonucleoprotein A1 (hnRNP A1) is drastically decreased by serum/glucose starvation. Its loss was reversible, serum/glucose starvation-specific, and universal throughout cell types and species. The hnRNP A1 mRNA level and hnRNP A1 mRNA/protein stability were not altered under this condition. CCND1 mRNA, which we newly identified as the binding target of hnRNP A1, was decreased by serum/glucose starvation. Under similar conditions, CCND1 protein was reduced in vitro and in vivo, whereas hnRNP A1 mRNA level and CCND1 mRNA level revealed no correlation in most clinical samples. Functional analyzes revealed that CCND1 mRNA stability is certainly dependent on hnRNP A1 protein level and that RNA recognition motif-1 (RRM1) in hnRNP A1 plays a central role in maintaining CCND1 mRNA stability and subsequent protein expression. The injection of RRM1-deleted hnRNP A1-expressing cancer cells in the mouse xenograft model did not form any tumors, and that of hnRNP A1-expressing cancer cells retained CCND1 expression at the lesion adjacent to necrosis with a slight increase of tumor volume. Furthermore, RRM1 deletion caused growth suppression with induction of apoptosis and autophagy, whereas CCND1 restoration completely recovered it. Our results indicate that serum/glucose starvation triggers entire hnRNP A1 protein loss, and its loss may play a role in CCND1 mRNA destabilization and CCND1-mediated cellular event inhibition, i.e., growth promotion, apoptosis induction, and autophagosome formation.

N protein of PEDV plays chess game with host proteins by selective autophagy

ABSTRACT Macroautophagy/autophagy is a cellular degradation and recycling process that maintains the homeostasis of organisms. The protein degradation role of autophagy has been widely used to control viral infection at multiple levels. In the ongoing evolutionary arms race, viruses have developed various ways to hijack and subvert autophagy in favor of its replication. It is still unclear exactly how autophagy affects or inhibits viruses. In this study, we have found a novel host restriction factor, HNRNPA1, that could inhibit PEDV replication by degrading viral nucleocapsid (N) protein. The restriction factor activates the HNRNPA1-MARCHF8/MARCH8-CALCOCO2/NDP52-autophagosome pathway with the help of transcription factor EGR1 targeting the HNRNPA1 promoter. HNRNPA1 could also promote the expression of IFN to facilitate the host antiviral defense response for antagonizing PEDV infection through RIGI protein interaction. During viral replication, we found that PEDV can, in contrast, degrade the host antiviral proteins HNRNPA1 and others (FUBP3, HNRNPK, PTBP1, and TARDBP) through its N protein through the autophagy pathway. These results reveal the dual function of selective autophagy in PEDV N and host proteins, which could promote the ubiquitination of viral particles and host antiviral proteins and degradation both of the proteins to regulate the relationship between virus infection and host innate immunity. Abbreviations: 3-MA: 3-methyladenine; ATG: autophagy related; Baf A1: bafilomycin A1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4′,6-diamidino-2-phenylindole; GPI: glycosyl-phosphatidylinositol; hpi: hours post infection; MARCHF8/MARCH8: membrane-associated ring-CH-type finger 8; MOI: multiplicity of infection; N protein: nucleocapsid protein; PEDV: porcine epidemic diarrhea virus; siRNA: small interfering RNA; TCID50: 50% tissue culture infectious doses.

Complex coacervation contributes to the joint phase behaviors of mixtures of charge-deficient prion-like low-complexity domains.

Prion-like low-complexity domains (PLCDs) are involved in the formation and regulation of various cellular biomolecular condensates, including stress granules and transcriptional condensates, which form via phase separation coupled to percolation. Our recent work has uncovered how evolutionarily conserved sequence features drive PLCD phase behavior through homotypic interactions. Since many condensates encompass a diverse mixture of proteins with PLCDs, we asked if mixtures of PLCDs have emergent phase behaviors that go beyond the intrinsic phase behaviors that have been well-documented through recent, in-depth biophysical studies. Specifically, we combined coarse-grained, single-bead-per-residue stickers-and-spacers simulations with experimental phase separation assays to study mixtures of PLCDs from the proteins hnRNPA1 and FUS. We refer to these domains as A1-LCD and FUS-LCD, respectively. Surprisingly, we find that a 1:1 mixture of the two species phase separates more readily than either sequence on its own. We demonstrate that the enhanced driving forces for phase separation of mixtures of A1-LCD and FUS-LCD comes from complementary electrostatic interactions between the two proteins. This complex coacervation-like mechanism adds to complementary interactions among aromatic sticker residues. Our findings are striking because we find evidence for an enhancement mediated by complex coacervation even though only 12% and 2.8% of the residues in the A1- and the FUS-LCD are charged. Importantly, the condensates formed by mixtures of A1- and FUS-LCD show inhomogeneous spatial organization whereby molecules are spatially proximal to species with which they have the strongest interactions. This deviates from a random mixture model and instead reflects a fine interplay between homotypic and heterotypic associations. Our findings support the emerging view that multicomponent condensates involve complex networks of homotypic and heterotypic interactions that drive a coupling between associative and segregative phase transitions.

Multivalent interactions between RNA and RNA binding proteins drive differential intra-speckle positioning of RNA transcripts.

Nuclear speckles represent a type of membraneless organelles in higher eukaryotic cells which are rich in snRNP components, certain splicing factors, including SR proteins (a family of RNA binding proteins (RBPs) named for containing regions with repetitive serine and arginine residues), polyA+ RNAs, and certain long noncoding RNAs (lncRNA). Multivalent interactions between RNAs and residing protein components drive the formation, as well as layered organization of components in nuclear speckles. Furthermore, RBPs have differential localization with respect to nuclear speckles; while SRSF1 proteins is enriched in nuclear speckles, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is either uniformly distributed in the nucleoplasm or slightly depleted from nuclear speckles. Using super-resolution imaging we show that the pre-mRNA transcripts containing SRSF1 motifs in the exon and hnRNPA1 motifs in the intron, are orientated in a way such that the regions containing the SRSF1 binding motifs are positioned relatively towards the interior of nuclear speckles and hnRNPA1 binding motifs are placed relatively towards the periphery of speckles. Knocking down of SRSF1 protein leads to the migration of RNA transcripts further towards the periphery of nuclear speckles, whereas knocking down of hnRNPA1 protein leads to the inward migration of RNA transcripts with respect to the speckle center. A similar effect of protein knockdown is seen on the endogenous nuclear speckle associated lncRNA, MALAT1. Our results collectively demonstrate that RNAs can adopt non-random orientation and positioning within the membraneless organelles. These observations might help explain the need for nuclear RNAs for structural integrity of nuclear speckles and the context dependent effect of SR and hnRNP proteins on splicing outcomes.

Mapping inhomogeneous network structures within biomolecular condensate using single-molecule imaging and tracking of fluorogenic probes.

Biomolecular condensates form via phase separation coupled to percolation. These phase transitions are driven by multivalent macromolecules. Network structures within condensates and the interfaces of condensates are difficult to interrogate experimentally. Here, we leverage single-molecule (SM) techniques and three fluorogenic probes, Nile blue (NB), Nile red (NR), and merocyanine 540 (MC540), to study the network structures within biomolecule condensates of the low-complexity domain of the hnRNPA1 protein (A1-LCD). Importantly, the fluorescent probes are not covalently bound to A1-LCD but instead diffuse in solution and become fluorescent in response to inhomogeneities of the local chemical environments within condensates. Interestingly, epifluorescence microscopy shows that the three probes respond in unique ways within condensates: NB gives a uniform intensity distribution, MC540 shows a higher intensity at the interface, and NR highlights hot spots inside the condensates. We then use SM localization microscopy to correlate localizations of the three probes. Surprisingly, the high-resolution SM reconstructions all show inhomogeneous network structures but with different emphases: NR shows the largest number of hotspots, NB exhibits the most spatial uniformity, and MC540 shows the greatest spatial variation within each condensate. Further, NB and MC540 localizations are highly correlated within each condensate. SM tracking experiments show that slower-moving NB molecules form high-contrast inhomogeneous network structures with hot spots, in agreement with SMLM. On the other hand, highly diffusive NB molecules are uniformly distributed throughout the condensate. We therefore conclude that network structures within the condensate are heterogeneous; protein chains with strong connections to neighboring chains form a relatively stable inhomogeneous network with hot spots, while chains with weak interactions are diffusive throughout the condensates. The mesoscale and sub-mesoscale structural organizations of molecules within condensates inferred from SM measurements agree with recent computational studies.

Delivery of LINC00589 via mesoporous silica nanoparticles inhibits peritoneal metastasis in gastric cancer

Peritoneal metastasis is one of the common forms of metastasis in gastric cancer (GC). In this study, we identified the expression pattern of LINC00589 in GC patients and investigate the biological function in GC cells. RNA-pulldown assay was performed to explore the underlying molecular mechanism. Further, we utilize polyethyleneimine-modified mesoporous silica nanoparticles (PMSNs) as the nanocarriers for delivery of LINC00589 encoding plasmid and tested its therapeutic potential for GC with peritoneal dissemination. We revealed that LINC00589 was downregulated in GC tissues and suppressed the metastatic ability of GC cells. Mechanistically, LINC00589 exerted tumor suppressive function by promoting hnRNPA1 protein ubiquitination and proteasomal degradation, thus blocking alternative splicing of PKM to PKM2. Furthermore, LINC00589 delivered by PMSNs could suppress the peritoneal metastasis of GC in vivo and in vitro. This work may provide a new treatment option in GC peritoneal metastasis.

Optimization of Bifunctional Antisense Oligonucleotides for Regulation of Mutually Exclusive Alternative Splicing of PKM Gene.

Oligonucleotide tools, as modulators of alternative splicing, have been extensively studied, giving a rise to new therapeutic approaches. In this article, we report detailed research on the optimization of bifunctional antisense oligonucleotides (BASOs), which are targeted towards interactions with hnRNP A1 protein. We performed a binding screening assay, Kd determination, and UV melting experiments to select sequences that can be used as a high potency binding platform for hnRNP A1. Newly designed BASOs were applied to regulate the mutually exclusive alternative splicing of the PKM gene. Our studies demonstrate that at least three repetitions of regulatory sequence are necessary to increase expression of the PKM1 isoform. On the other hand, PKM2 expression can be inhibited by a lower number of regulatory sequences. Importantly, a novel branched type of BASOs was developed, which significantly increased the efficiency of splicing modulation. Herein, we provide new insights into BASOs design and show, for the first time, the possibility to regulate mutually exclusive alternative splicing via BASOs.

AMPK-related protein kinase ARK5 regulates subcellular localization of RNA-binding protein hnRNP A1 during hypertonic stress

The heterogeneous nuclear ribonucleoprotein hnRNP A1 is a nucleocytoplasmic-shuttling RNA-binding protein that plays an important role in nucleic acid metabolism and gene expression regulation. The function of hnRNP A1 is determined in part by its specific location within the cell. Although some work has been done to elucidate the signaling pathways that regulate the cellular localization of hnRNP A1, the precise mechanism(s), including physiological and pathophysiological conditions that alter hnRNP A1 localization, are not known. We previously conducted an unbiased RNAi-based kinome-wide screen to identify kinases that regulate hnRNP A1 localization during hypertonic stress. One of the hits from this screen is AMPK-related protein kinase 5 (ARK5). Here, we validate ARK5 as the kinase responsible for controlling hnRNP A1 subcellular localization in response to hypertonic stress. We find using immunoprecipitation and in vitro kinase assay methods that ARK5 directly interacts with and phosphorylates hnRNP A1 on serine residues within the F-peptide region. We further show that the M9 motif of hnRNP A1 is essential for the ARK5-hnRNP A1 interaction and subsequent phosphorylation. In addition, the silencing of ARK5 increases the expression of antiapoptotic protein Bcl-xL and consequently delays caspase activation during hypertonic stress. Our results indicate that ARK5 phosphorylates hnRNP A1 and regulates its subcellular localization during hypertonic stress.