Search Results (18)

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA-binding protein containing low-complexity domains (LCDs), which are known to regulate protein behavior under stress conditions. This study demonstrates the ability to control hnRNPK’s transitions into four distinct material states—monomer, soluble aggregate, liquid droplet, and fibrillar hydrogel—by modulating environmental factors such as temperature and protein concentration. Importantly, the phase-separated and hydrogel states are newly identified for eGFP-hnRNPK, marking a significant advancement in understanding its material properties. A combination of biophysical techniques, including DLS and SEC-LS, were used to further characterize hnRNPK in monomeric and soluble aggregate states. Structural methods, such as SANS, SAXS, and TEM, revealed the elongated morphology of the hnRNPK monomer. Environmental perturbations, such as decreased temperature or crowding agents, drove hnRNPK into phase-separated or gel-like states, each with distinct biophysical characteristics. These novel states were further analyzed using SEM, X-ray diffraction, and fluorescence microscopy. Collectively, these results demonstrate the complex behaviors of hnRNPK under different conditions and illustrate the properties of the protein in each material state. Transitions of hnRNPK upon condition changes could potentially affect functions of hnRNPK, playing a significant role in regulation of hnRNPK-involved processes in the cell. Full article

Chikungunya virus (CHIKV) is an emerging, mosquito-borne arthritic alphavirus increasingly associated with severe neurological sequelae and long-term morbidity. However, there is limited understanding of the crucial host components involved in CHIKV replicase assembly complex formation, and thus virus replication and virulence-determining factors, within the central nervous system (CNS). Furthermore, the majority of CHIKV CNS studies focus on neuronal infection, even though astrocytes represent the main cerebral target. Heterogeneous ribonucleoprotein K (hnRNP K), an RNA-binding protein involved in RNA splicing, trafficking, and translation, is a regulatory component of alphavirus replicase assembly complexes, but has yet to be thoroughly studied in the context of CHIKV infection. We identified the hnRNP K CHIKV viral RNA (vRNA) binding site via sequence alignment and performed site-directed mutagenesis to generate a mutant, ΔhnRNPK-BS1, with disrupted hnRNPK–vRNA binding, as verified through RNA coimmunoprecipitation and RT-qPCR. CHIKV ΔhnRNPK-BS1 demonstrated hampered replication in both NSC-34 neuronal and C8-D1A astrocytic cultures. In astrocytes, disruption of the hnRNPK–vRNA interaction curtailed viral RNA transcription and shut down subgenomic RNA translation. Our study demonstrates that hnRNP K serves as a crucial RNA-binding host factor that regulates CHIKV replication through the modulation of subgenomic RNA translation. Full article

LTR-retrotransposons are transposable elements characterized by the presence of long terminal repeats (LTRs) directly flanking an internal coding region. They share genome organization and replication strategies with retroviruses. Steamer-like Element-1 (MchSLE-1) is an LTR-retrotransposon identified in the genome of the Chilean blue mussel Mytilus chilensis. MchSLE-1 is transcribed; however, whether its RNA is also translated and the mechanism underlying such translation remain to be elucidated. Here, we characterize the MchSLE-1 translation mechanism. We found that the MchSLE-1 5′ and 3′LTRs command transcription of sense and antisense RNAs, respectively. Using luciferase reporters commanded by the untranslated regions (UTRs) of MchSLE-1, we found that in vitro 5′UTR sense is unable to initiate translation, whereas the antisense 5′UTR initiates translation even when the eIF4E-eIF4G interaction was disrupted, suggesting the presence of an internal ribosomal entry site (IRES). The antisense 5′UTR IRES activity was tested using bicistronic reporters. The antisense 5′UTR has IRES activity only when the mRNA is transcribed in the nucleus, suggesting that nuclear RNA-binding proteins are required to modulate its activity. Indeed, heterogeneous nuclear ribonucleoprotein K (hnRNPK) was identified as an IRES trans-acting factor (ITAF) of the MchSLE-1 IRES. To our knowledge, this is the first report describing an IRES in an antisense mRNA derived from a mussel LTR-retrotransposon. Full article

Chinese soft-shelled turtles display obvious sex dimorphism. The exogenous application of hormones (estradiol and methyltestosterone) can change the direction of gonadal differentiation of P. sinensis to produce sex reversed individuals. However, the molecular mechanism remains unclear. In this study, TMT-based quantitative proteomics analysis of four types of P. sinensis (female, male, pseudo-female, and pseudo-male) gonads were compared. Quantitative analysis of 6107 labeled proteins in the four types of P. sinensis gonads was performed. We identified 440 downregulated and 423 upregulated proteins between pseudo-females and males, as well as 394 downregulated and 959 upregulated proteins between pseudo-males and females. In the two comparisons, the differentially expressed proteins, including K7FKG1, K7GIQ2, COL4A6, K7F2U2, and K7FF80, were enriched in some important pathways, such as focal adhesion, endocytosis, apoptosis, extracellular matrix-receptor interaction, and the regulation of actin cytoskeleton, which were upregulated in pseudo-female vs. male and downregulated in pseudo-male vs. female. In pathways such as ribosome and spliceosome, the levels of RPL28, SRSF3, SNRNP40, and HNRNPK were increased from male to pseudo-female, while they decreased from female to pseudo-male. All differentially expressed proteins after sexual reversal were divided into six clusters, according to their altered levels in the four types of P. sinensis, and associated with cellular processes, such as embryonic development and catabolic process, that were closely related to sexual reversal. These data will provide clues for the sexual reversal mechanism in P. sinensis. Full article

HnRNPK is a heterogeneous nuclear ribonucleoprotein (hnRNP) that has been firmly implicated in transcriptional and post-transcriptional regulation. However, the molecular mechanisms by which hnRNPK orchestrates transcriptional or post-transcriptional regulation are not well understood due to early embryonic lethality in homozygous knockout mice, especially in a tissue-specific context. Strikingly, in this study, we demonstrated that hnRNPK is strongly expressed in the mouse testis and mainly localizes to the nucleus in spermatogonia, spermatocytes, and round spermatids, suggesting an important role for hnRNPK in spermatogenesis. Using a male germ cell-specific hnRNPK-depleted mouse model, we found that it is critical for testicular development and male fertility. The initiation of meiosis of following spermatogenesis was not affected in Hnrnpk cKO mice, while most germ cells were arrested at the pachytene stage of the meiosis and no mature sperm were detected in epididymides. The further RNA-seq analysis of Hnrnpk cKO mice testis revealed that the deletion of hnRNPK disturbed the expression of genes involved in male reproductive development, among which the meiosis genes were significantly affected, and Hnrnpk cKO spermatocytes failed to complete the meiotic prophase. Together, these results identify hnRNPK as an essential regulator of spermatogenesis and male fertility. Full article

RNA-binding proteins (RBPs) regulate cell physiology via the formation of ribonucleic-protein complexes with coding and non-coding RNAs. RBPs have multiple functions in the same cells; however, the precise mechanism through which their pleiotropic functions are determined remains unknown. In this study, we revealed the multiple inhibitory functions of heterogeneous nuclear ribonucleoprotein K (hnRNPK) for myogenic differentiation. We first identified hnRNPK as a lncRNA Myoparr binding protein. Gain- and loss-of-function experiments showed that hnRNPK repressed the expression of myogenin at the transcriptional level. The hnRNPK-binding region of Myoparr was required to repress myogenin expression. Moreover, hnRNPK repressed the expression of a set of genes coding for aminoacyl-tRNA synthetases in a Myoparr-independent manner. Mechanistically, hnRNPK regulated the eIF2α/Atf4 pathway, one branch of the intrinsic pathways of the endoplasmic reticulum sensors, in differentiating myoblasts. Thus, our findings demonstrate that hnRNPK plays lncRNA-associated and -independent multiple roles during myogenic differentiation, indicating that the analysis of lncRNA-binding proteins will be useful for elucidating both the physiological functions of lncRNAs and the multiple functions of RBPs. Full article

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive neoplasm derived from plasmacytoid dendritic cells. While advances in understanding the pathophysiology of the disease have been made, integrated systematic analyses of the spectrum of immunophenotypic and molecular alterations in real-world clinical cases remain limited. We performed mutation profiling of 50 BPDCN cases and assessed our findings in the context of disease immunophenotype, cytogenetics, and clinical characteristics. Patients included 42 men and 8 women, with a median age of 68 years (range, 14–84) at diagnosis. Forty-two (84%) patients had at least one mutation, and 23 (46%) patients had ≥3 mutations. The most common mutations involved TET2 and ASXL1, detected in 28 (56%) and 23 (46%) patients, respectively. Co-existing TET2 and ASXL1 mutations were present in 17 (34%) patients. Other recurrent mutations included ZRSR2 (16%), ETV6 (13%), DNMT3A (10%), NRAS (10%), IKZF1 (9%), SRSF2 (9%), IDH2 (8%), JAK2 (6%), KRAS (4%), NOTCH1 (4%), and TP53 (4%). We also identified mutations that have not been reported previously, including ETNK1, HNRNPK, HRAS, KDM6A, RAD21, SF3A1, and SH2B3. All patients received chemotherapy, and 20 patients additionally received stem cell transplantation. With a median follow-up of 10.5 months (range, 1–71), 21 patients achieved complete remission, 4 had persistent disease, and 24 died. Patients younger than 65 years had longer overall survival compared to those who were ≥65 years (p = 0.0022). Patients who had ≥3 mutations or mutations in the DNA methylation pathway genes had shorter overall survival (p = 0.0119 and p = 0.0126, respectively). Stem cell transplantation significantly prolonged overall survival regardless of mutation status. In conclusion, the majority of patients with BPDCN have somatic mutations involving epigenetic regulators and RNA splicing factors, in addition to ETV6 and IKZF1, which are also frequently mutated. Older age, multiple mutations, and mutations in the DNA methylation pathway are poor prognostic factors. Full article

Prolonged use of opioids can cause opioid-induced hyperalgesia (OIH). The impact of alternative splicing on OIH remains partially characterized. A study of the absolute and relative modes of action of alternative splicing further the understanding of the molecular mechanisms underlying OIH. Differential absolute and relative isoform profiles were detected in the trigeminal ganglia and nucleus accumbens of mice presenting OIH behaviors elicited by chronic morphine administration relative to control mice. Genes that participate in glutamatergic synapse (e.g., Grip1, Grin1, Wnk3), myelin protein processes (e.g., Mbp, Mpz), and axon guidance presented absolute and relative splicing associated with OIH. Splicing of genes in the gonadotropin-releasing hormone receptor pathway was detected in the nucleus accumbens while splicing in the vascular endothelial growth factor, endogenous cannabinoid signaling, circadian clock system, and metabotropic glutamate receptor pathways was detected in the trigeminal ganglia. A notable finding was the prevalence of alternatively spliced transcription factors and regulators (e.g., Ciart, Ablim2, Pbx1, Arntl2) in the trigeminal ganglia. Insights into the nociceptive and antinociceptive modulatory action of Hnrnpk were gained. The results from our study highlight the impact of alternative splicing and transcriptional regulators on OIH and expose the need for isoform-level research to advance the understanding of morphine-associated hyperalgesia. Full article

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA/DNA binding protein involved in diverse cell processes; it is also a p53 coregulator that initiates apoptosis under DNA damage conditions. However, the upregulation of hnRNPK is correlated with cancer transformation, progression, and migration, whereas the regulatory role of hnRNPK in cancer malignancy remains unclear. We previously showed that arginine methylation of hnRNPK attenuated the apoptosis of U2OS osteosarcoma cells under DNA damage conditions, whereas the replacement of endogenous hnRNPK with a methylation-defective mutant inversely enhanced apoptosis. The present study further revealed that an RNA helicase, DDX3, whose C-terminus preferentially binds to the unmethylated hnRNPK and could promote such apoptotic enhancement. Moreover, C-terminus-truncated DDX3 induced significantly less apoptosis than full-length DDX3. Notably, we also identified a small molecule that docks at the ATP-binding site of DDX3, promotes the DDX3-hnRNPK interaction, and induces further apoptosis. Overall, we have shown that the arginine methylation of hnRNPK suppresses the apoptosis of U2OS cells via interfering with DDX3–hnRNPK interaction. On the other hand, DDX3–hnRNPK interaction with a proapoptotic role may serve as a target for promoting apoptosis in osteosarcoma cells. Full article

Circular RNAs (circRNAs) are increasingly recognized as having a role in cancer development. Their expression is modified in numerous cancers, including hepatocellular carcinoma (HCC); however, little is known about the mechanisms of their regulation. The aim of this study was to identify regulators of circRNAome expression in HCC. Using publicly available datasets, we identified RNA binding proteins (RBPs) with enriched motifs around the splice sites of differentially expressed circRNAs in HCC. We confirmed the binding of some of the candidate RBPs using ChIP-seq and eCLIP datasets in the ENCODE database. Several of the identified RBPs were found to be differentially expressed in HCC and/or correlated with the overall survival of HCC patients. According to our bioinformatics analyses and published evidence, we propose that NONO, PCPB2, PCPB1, ESRP2, and HNRNPK are candidate regulators of circRNA expression in HCC. We confirmed that the knocking down the epithelial splicing regulatory protein 2 (ESRP2), known to be involved in the maintenance of the adult liver phenotype, significantly changed the expression of candidate circRNAs in a model HCC cell line. By understanding the systemic changes in transcriptome splicing, we can identify new proteins involved in the molecular pathways leading to HCC development and progression. Full article

Alternative splicing (AS) may increase the number of proteoforms produced by a gene. Alzheimer’s disease (AD) is a neurodegenerative disease with well-characterized AS proteoforms. In this study, we used a proteogenomics strategy to build a customized protein sequence database and identify orthologous AS proteoforms between humans and mice on publicly available shotgun proteomics (MS/MS) data of the corpus callosum (CC) and olfactory bulb (OB). Identical proteotypic peptides of six orthologous AS proteoforms were found in both species: PKM1 (gene PKM/Pkm), STXBP1a (gene STXBP1/Stxbp1), Isoform 3 (gene HNRNPK/Hnrnpk), LCRMP-1 (gene CRMP1/Crmp1), SP3 (gene CADM1/Cadm1), and PKCβII (gene PRKCB/Prkcb). These AS variants were also detected at the transcript level by publicly available RNA-Seq data and experimentally validated by RT-qPCR. Additionally, PKM1 and STXBP1a were detected at higher abundances in a publicly available MS/MS dataset of the AD mouse model APP/PS1 than its wild type. These data corroborate other reports, which suggest that PKM1 and STXBP1a AS proteoforms might play a role in amyloid-like aggregate formation. To the best of our knowledge, this report is the first to describe PKM1 and STXBP1a overexpression in the OB of an AD mouse model. We hope that our strategy may be of use in future human neurodegenerative studies using mouse models. Full article

MEX3A belongs to the MEX3 (Muscle EXcess) protein family consisting of four members (MEX3A-D) in humans. Characteristic for MEX3 proteins is their domain structure with 2 HNRNPK homology (KH) domains mediating RNA binding and a C-terminal really interesting new gene (RING) domain that harbors E3 ligase function. In agreement with their domain composition, MEX3 proteins were reported to modulate both RNA fate and protein ubiquitination. MEX3 paralogs exhibit an oncofetal expression pattern, they are severely downregulated postnatally, and re-expression is observed in various malignancies. Enforced expression of MEX3 proteins in various cancers correlates with poor prognosis, emphasizing their oncogenic potential. The latter is supported by MEX3A’s impact on proliferation, self-renewal as well as migration of tumor cells in vitro and tumor growth in xenograft studies. Full article

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) transcripts are abundant in estrogen receptor (ER)- or progesterone receptor (PR)-positive breast cancer. However, the biological functions of hnRNPK in the ER-mediated signaling pathway have remained largely unknown. Therefore, this study analyzes the functions of hnRNPK expression in the ER-mediated signaling pathway in breast cancer. We initially evaluated hnRNPK expression upon treatment with estradiol (E2) and ICI 182,780 in the ERα-positive breast carcinoma cell line MCF-7. The results revealed that E2 increased hnRNPK; however, hnRNPK expression was decreased with ICI 182,780 treatment, indicating estrogen dependency. We further evaluated the effects of hnRNPK knockdown in the ER-mediated signaling pathway in MCF-7 cells using small interfering RNAs. The results revealed that hnRNPK knockdown decreased ERα expression and ERα target gene pS2 by E2 treatment. As hnRNPK interacts with several other proteins, we explored the interaction between hnRNPK and ERα, which was demonstrated using immunoprecipitation and proximity ligation assay. Subsequently, we immunolocalized hnRNPK in patients with breast cancer, which revealed that hnRNPK immunoreactivity was significantly higher in ERα-positive carcinoma cells and significantly lower in Ki67-positive or proliferative carcinoma cells. These results indicated that hnRNPK directly interacted with ERα and was involved in the ER-mediated signaling pathway in breast carcinoma. Furthermore, hnRNPK expression could be an additional target of endocrine therapy in patients with ERα-positive breast cancer. Full article

RNA-binding proteins (RBPs) have been shown to play a key role in the pathogenesis of a variety of neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS) is an exemplar neurodegenerative disease characterised by rapid progression and relatively selective motor neuron loss. Nuclear-to-cytoplasmic mislocalisation and accumulation of RBPs have been identified as a pathological hallmark of the disease, yet the spatiotemporal responses of RBPs to different extrinsic stressors in human neurons remain incompletely understood. Here, we used healthy induced pluripotent stem cell (iPSC)-derived motor neurons to model how different types of cellular stress affect the nucleocytoplasmic localisation of key ALS-linked RBPs. We found that osmotic stress robustly induced nuclear loss of TDP-43, SPFQ, FUS, hnRNPA1 and hnRNPK, with characteristic changes in nucleocytoplasmic localisation in an RBP-dependent manner. Interestingly, we found that RBPs displayed stress-dependent characteristics, with unique responses to both heat and oxidative stress. Alongside nucleocytoplasmic protein distribution changes, we identified the formation of stress- and RBP-specific nuclear and cytoplasmic foci. Furthermore, the kinetics of nuclear relocalisation upon recovery from extrinsic stressors was also found to be both stress- and RBP-specific. Importantly, these experiments specifically highlight TDP-43 and FUS, two of the most recognised RBPs in ALS pathogenesis, as exhibiting delayed nuclear relocalisation following stress in healthy human motor neurons as compared to SFPQ, hnRNPA1 and hnRNPK. Notably, ALS-causing valosin containing protein (VCP) mutations did not disrupt the relocalisation dynamics of TDP-43 or FUS in human motor neurons following stress. An increased duration of TDP-43 and FUS within the cytoplasm after stress may render the environment more aggregation-prone, which may be poorly tolerated in the context of ALS and related neurodegenerative disorders. In summary, our study addresses stress-specific spatiotemporal responses of neurodegeneration-related RBPs in human motor neurons. The insights into the nucleocytoplasmic dynamics of RBPs provided here may be informative for future studies examining both disease mechanisms and therapeutic strategy. Full article

Background: Breast cancer affects over 2 million women yearly. Its early detection allows for successful treatment, which motivates to research factors that enable an accurate diagnosis. miR-125a is one of them, correlating with different types of cancer. For example, the miR-125a level decreases in breast cancer tissues; polymorphisms in the miR-125a encoding gene are related to prostate cancer and the risk of radiotherapy-induced pneumonitis. Methods: In this work, we investigated two variants of rs12976445 polymorphism in the context of breast cancer. We analyzed the data of 175 blood samples from breast cancer patients and compared them with the control data from 129 control samples. Results: We observed the tendency that in breast cancer cases TT genotype appeared slightly more frequent over CC and CT genotypes (statistically nonsignificant). The TT genotype appeared also to be more frequent among human epidermal growth factor receptor 2 (HER2) positive patients, compared to HER2 negative. In silico modelling showed that the presence of uridine (U) diminished the probability of pri-miR-125a binding to NOVA1 and HNRNPK proteins. We demonstrated that U and C -variants could promote different RNA folding patterns and provoke alternative protein binding. Conclusions: U-variant may imply a lower miR-125a expression in breast cancer. Full article