SUMOylation modification of HNRNPK at the K422 site promotes invasion in glioblastoma.

Glioblastoma multiforme (GBM) is a highly heterogeneous brain tumor with limited treatment options. Recent studies revealed cellular heterogeneity and the potential for interconversion between distinct cell types on the basis of RNA sequencing and single-cell analyses. The ability of different cell types to adapt to their surrounding environment and undergo transformation significantly complicates the study and treatment of GBM. In this study, we reveal that HNRNPK-SUMO1 expression is predominantly found in the GBM infiltration area. SUMOylation of the K422 residue of HNRNPK interferes with its DNA binding ability, thereby disrupting downstream transcription, and ultimately leading to transitions between different states of glioblastoma stem cells. Although the proneural subtype is considered to have a better prognosis, transitioning towards this state promotes tumor invasion. These findings serve as a reminder to exercise caution when considering treatments targeting specific cellular subtypes.

The methyltransferase SETD3 regulates mRNA alternative splicing through interacting with hnRNPK.

The methyltransferase SETD3 is an enzyme essential for catalyzing histidine-73 methylation on ß-Actin, thereby promoting its polymerization and regulating muscle contraction. Although increasing evidence suggests that SETD3 is involved in multiple physiological or pathological events, its biological functions remain incompletely understood. In this study, we utilize in situ proximity labeling combined with mass spectrometry analysis to detect potential interacting partners of SETD3. Unexpectedly, we find that many splicing factors are associated with SETD3. Genome-wide RNA sequencing reveals that SETD3 regulates pre-mRNA splicing events, predominantly influencing exon skipping. Biochemical and bioinformatic analyses suggest that SETD3 interacts with hnRNPK, and they collaboratively regulate exon skipping in a common subset of genes. Functionally, we demonstrate that SETD3 and hnRNPK are required for retention of exon 7 skipping in the FNIP1 gene. This promotes FNIP1-mediated nuclear translocation of the transcription factor TFEB and the subsequent induction of lysosomal and mitochondrial biogenesis. Overall, this study uncovers a novel function of SETD3 in modulating mRNA exon splicing.

Tumor-suppressive miR-4732-3p is sorted into fucosylated exosome by hnRNPK to avoid the inhibition of lung cancer progression.

BACKGROUND: Aberrant fucosylation observed in cancer cells contributes to an augmented release of fucosylated exosomes into the bloodstream, where miRNAs including miR-4732-3p hold promise as potential tumor biomarkers in our pilot study. However, the mechanisms underlying the sorting of miR-4732-3p into fucosylated exosomes during lung cancer progression remain poorly understood. METHODS: A fucose-captured strategy based on lentil lectin-magnetic beads was utilized to isolate fucosylated exosomes and evaluate the efficiency for capturing tumor-derived exosomes using nanoparticle tracking analysis (NTA). Fluorescence in situ hybridization (FISH) and qRT-PCR were performed to determine the levels of miR-4732-3p in non-small cell lung cancer (NSCLC) tissue samples. A co-culture system was established to assess the release of miRNA via exosomes from NSCLC cells. RNA immunoprecipitation (RIP) and miRNA pull-down were applied to validate the interaction between miR-4732-3p and heterogeneous nuclear ribonucleoprotein K (hnRNPK) protein. Cell functional assays, cell derived xenograft, dual-luciferase reporter experiments, and western blot were applied to examine the effects of miR-4732-3p on MFSD12 and its downstream signaling pathways, and the impact of hnRNPK in NSCLC. RESULTS: We enriched exosomes derived from NSCLC cells using the fucose-captured strategy and detected a significant upregulation of miR-4732-3p in fucosylated exosomes present in the serum, while its expression declined in NSCLC tissues. miR-4732-3p functioned as a tumor suppressor in NSCLC by targeting 3'UTR of MFSD12, thereby inhibiting AKT/p21 signaling pathway to induce cell cycle arrest in G2/M phase. NSCLC cells preferentially released miR-4732-3p via exosomes instead of retaining them intracellularly, which was facilitated by the interaction of miR-4732-3p with hnRNPK protein for selective sorting into fucosylated exosomes. Moreover, knockdown of hnRNPK suppressed NSCLC cell proliferation, with the elevated levels of miR-4732-3p in NSCLC tissues but the decreased expression in serum fucosylated exosomes. CONCLUSIONS: NSCLC cells escape suppressive effects of miR-4732-3p through hnRNPK-mediated sorting of them into fucosylated exosomes, thus supporting cell malignant properties and promoting NSCLC progression. Our study provides a promising biomarker for NSCLC and opens a novel avenue for NSCLC therapy by targeting hnRNPK to prevent the "exosome escape" of tumor-suppressive miR-4732-3p from NSCLC cells.

The Mytilus chilensis Steamer-like Element-1 Retrotransposon Antisense mRNA Harbors an Internal Ribosome Entry Site That Is Modulated by hnRNPK.

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.

Hnrnpk protects against osteoarthritis through targeting WWC1 mRNA and inhibiting Hippo signaling pathway.

Osteoarthritis (OA) is an age-related or post-traumatic degenerative whole joint disease characterized by the rupture of articular cartilage homeostasis, the regulatory mechanisms of which remain elusive. This study identifies the essential role of heterogeneous nuclear ribonucleoprotein K (hnRNPK) in maintaining articular cartilage homeostasis. Hnrnpk expression is markedly downregulated in human and mice OA cartilage. The deletion of Hnrnpk effectively accelerates the development of post-traumatic and age-dependent OA in mice. Mechanistically, the KH1 and KH2 domain of Hnrnpk bind and degrade the mRNA of WWC1. Hnrnpk deletion increases WWC1 expression, which in turn leads to the activation of Hippo signaling and ultimately aggravates OA. In particular, intra-articular injection of LPA and adeno-associated virus serotype 5 expressing WWC1 RNA interference ameliorates cartilage degeneration induced by Hnrnpk deletion, and intra-articular injection of adeno-associated virus serotype 5 expressing Hnrnpk protects against OA. Collectively, this study reveals the critical roles of Hnrnpk in inhibiting OA development through WWC1-dependent downregulation of Hippo signaling in chondrocytes and defines a potential target for the prevention and treatment of OA.

Reciprocal regulation of lncRNA MEF and c-Myc drives colorectal cancer tumorigenesis.

More than half of all cancers demonstrate aberrant c-Myc expression, making this arguably the most important human oncogene. Deregulated long non-coding RNAs (lncRNAs) are also commonly implicated in tumorigenesis, and some limited examples have been established where lncRNAs act as biological tuners of c-Myc expression and activity. Here, we demonstrate that the lncRNA denoted c-Myc Enhancing Factor (MEF) enjoys a cooperative relationship with c-Myc, both as a transcriptional target and driver of c-Myc expression. Mechanistically, MEF functions by binding to and stabilizing the expression of hnRNPK in colorectal cancer cells. The MEF-hnRNPK interaction serves to disrupt binding between hnRNPK and the E3 ubiquitin ligase TRIM25, which attenuates TRIM25-dependent hnRNPK ubiquitination and proteasomal destruction. In turn, the stabilization of hnRNPK through MEF enhances c-Myc expression by augmenting the translation c-Myc. Moreover, modulating the expression of MEF in shRNA-mediated knockdown and overexpression studies revealed that MEF expression is essential for colorectal cancer cell proliferation and survival, both in vitro and in vivo. From the clinical perspective, we show that MEF expression is differentially increased in colorectal cancer tissues compared to normal adjacent tissues. Further, correlations exist between MEF, c-Myc, and hnRNPK suggesting the MEF-c-Myc positive feedback loop is active in patients. Together these data demonstrate that MEF is a pivotal partner of the c-Myc network and propose MEF as a valuable therapeutic target for colorectal cancer.

LINC00571 drives tricarboxylic acid cycle metabolism in triple-negative breast cancer through HNRNPK/ILF2/IDH2 axis.

BACKGROUND: Triple-negative breast cancer is a complex breast malignancy subtype characterized by poor prognosis. The pursuit of effective therapeutic approaches for this subtype is considerably challenging. Notably, recent research has illuminated the key role of the tricarboxylic acid cycle in cancer metabolism and the complex landscape of tumor development. Concurrently, an emerging body of evidence underscores the noteworthy role that long non-coding RNAs play in the trajectory of breast cancer development. Despite this growing recognition, the exploration of whether long non-coding RNAs can influence breast cancer progression by modulating the tricarboxylic acid cycle has been limited. Moreover, the underlying mechanisms orchestrating these interactions have not been identified. METHODS: The expression levels of LINC00571 and IDH2 were determined through the analysis of the public TCGA dataset, transcriptome sequencing, qRT‒PCR, and Western blotting. The distribution of LINC00571 was assessed using RNA fluorescence in situ hybridization. Alterations in biological effects were evaluated using CCK-8, colony formation, EdU, cell cycle, and apoptosis assays and a tumor xenograft model. To elucidate the interaction between LINC00571, HNRNPK, and ILF2, RNA pull-down, mass spectrometry, coimmunoprecipitation, and RNA immunoprecipitation assays were performed. The impacts of LINC00571 and IDH2 on tricarboxylic acid cycle metabolites were investigated through measurements of the oxygen consumption rate and metabolite levels. RESULTS: This study revealed the complex interactions between a novel long non-coding RNA (LINC00571) and tricarboxylic acid cycle metabolism. We validated the tumor-promoting role of LINC00571. Mechanistically, LINC00571 facilitated the interaction between HNRNPK and ILF2, leading to reduced ubiquitination and degradation of ILF2, thereby stabilizing its expression. Furthermore, ILF2 acted as a transcription factor to enhance the expression of its downstream target gene IDH2. CONCLUSIONS: Our study revealed that the LINC00571/HNRNPK/ILF2/IDH2 axis promoted the progression of triple-negative breast cancer by regulating tricarboxylic acid cycle metabolites. This discovery provides a novel theoretical foundation and new potential targets for the clinical treatment of triple-negative breast cancer.

A novel link between circPDE3B and ferroptosis in esophageal squamous cell carcinoma progression.

AIM: To unravel whether ferroptosis involves with the actions by circPDE3B-mediated facilitation of esophageal squamous cell carcinoma (ESCC) progression. METHODS: Human ESCC tissues and cell lines were prepared for the evaluation of ferroptosis. Cellular iron, ROS, GSH, and MDA levels were measured to assess ferroptosis. Flow cytometry was employed to analyze apoptosis and cell cycle. Subcellular fractionation and fluorescence in situ hybridization (FISH) were conducted to validate the localization of circPDE3B. RNA pull-down, RNA immunoprecipitation (RIP), and luciferase assay were subjected to identify the molecular mechanisms. Nude mouse xenograft model was carried out to evaluate the function of circPDE3B/SLC7A11/CBS in vivo. RESULTS: Increased circPDE3B in human ESCC specimens was positively correlated with ferroptosis-related molecules, SLC7A11 and CBS. Functionally, circPDE3B knockdown triggered ferroptosis, apoptosis, and cell cycle arrest in ESCC cells. Whereas, these effects were obviously blocked by miR-516b-5p inhibitor. Mechanistically, not only circPDE3B sponged miR-516b-5p to upregulate CBS, but also directly bound with HNRNPK to stabilize SLC7A11. In mice, depletion of circPDE3B restrained ESCC growth, while this was abolished by overexpression of CBS or SLC7A11. CONCLUSION: In summary, circPDE3B promotes ESCC progression by suppressing ferroptosis through recruiting HNRNPK/SLC7A11 and miR-516b-5p/CBS axes.

Insight into the mechanism of AML del(9q) progression: hnRNP K targets the myeloid master regulators CEBPA (C/EBPα) and SPI1 (PU.1).

Deletions on the long arm of chromosome 9 (del(9q)) are recurrent abnormalities in about 2 % of acute myeloid leukemia cases, which usually involve HNRNPK and are frequently associated with other known aberrations. Based on an Hnrnpk haploinsufficient mouse model, a recent study demonstrated a function of hnRNP K in pathogenesis of myeloid malignancies via the regulation of cellular proliferation and myeloid differentiation programs. Here, we provide evidence that reduced hnRNP K expression results in the dysregulated expression of C/EBPα and additional transcription factors. CyTOF analysis revealed monocytic skewing with increased levels of mature myeloid cells. To explore the role of hnRNP K during normal and pathological myeloid differentiation in humans, we characterized hnRNP K-interacting RNAs in human AML cell lines. Notably, RNA-sequencing revealed several mRNAs encoding key transcription factors involved in the regulation of myeloid differentiation as targets of hnRNP K. We showed that specific sequence motifs confer the interaction of SPI1 and CEBPA 5' and 3'UTRs with hnRNP K. The siRNA mediated reduction of hnRNP K in human AML cells resulted in an increase of PU.1 and C/EBPα that is most pronounced for the p30 isoform. The combinatorial treatment with the inducer of myeloid differentiation valproic acid resulted in increased C/EBPα expression and myeloid differentiation. Together, our results indicate that hnRNP K post-transcriptionally regulates the expression of myeloid master transcription factors. These novel findings can inaugurate novel options for targeted treatment of AML del(9q) by modulation of hnRNP K function.

RNA isoform expression landscape of the human dorsal root ganglion (DRG) generated from long read sequencing.

Splicing is a post-transcriptional RNA processing mechanism that enhances genomic complexity by creating multiple isoforms from the same gene. Diversity in splicing in the mammalian nervous system is associated with neuronal development, synaptic function and plasticity, and is also associated with diseases of the nervous system ranging from neurodegeneration to chronic pain. We aimed to characterize the isoforms expressed in the human peripheral nervous system, with the goal of creating a resource to identify novel isoforms of functionally relevant genes associated with somatosensation and nociception. We used long read sequencing (LRS) to document isoform expression in the human dorsal root ganglia (hDRG) from 3 organ donors. Isoforms were validated in silico by confirming expression in hDRG short read sequencing (SRS) data from 3 independent organ donors. 19,547 isoforms of protein-coding genes were detected using LRS and validated with SRS and strict expression cutoffs. We identified 763 isoforms with at least one previously undescribed splice-junction. Previously unannotated isoforms of multiple pain-associated genes, including ASIC3, MRGPRX1 and HNRNPK were identified. In the novel isoforms of ASIC3, a region comprising ~35% of the 5'UTR was excised. In contrast, a novel splice-junction was utilized in isoforms of MRGPRX1 to include an additional exon upstream of the start-codon, consequently adding a region to the 5'UTR. Novel isoforms of HNRNPK were identified which utilized previously unannotated splice-sites to both excise exon 14 and include a sequence in the 5' end of exon 13. The insertion and deletion in the coding region was predicted to excise a serine-phosphorylation site favored by cdc2, and replace it with a tyrosine-phosphorylation site potentially phosphorylated by SRC. We also independently confirm a recently reported DRG-specific splicing event in WNK1 that gives insight into how painless peripheral neuropathy occurs when this gene is mutated. Our findings give a clear overview of mRNA isoform diversity in the hDRG obtained using LRS. Using this work as a foundation, an important next step will be to use LRS on hDRG tissues recovered from people with a history of chronic pain. This should enable identification of new drug targets and a better understanding of chronic pain that may involve aberrant splicing events.

HnRNPK is involved in stress-induced depression-like behavior via ERK-BDNF pathway in mice.

As a ubiquitous RNA-binding protein, heterogeneous nuclear ribonucleoprotein K (hnRNPK) interacts with numerous nucleic acids and proteins and is involved in various cellular functions. Available literature indicates that it can regulate dendritic spine density through the extracellular signal-regulating kinase (ERK) - brain-derived neurotrophic factor (BDNF) pathway, which is crucial to retain the synaptic plasticity in patients with major depressive disorder (MDD) and mouse depression models. However, ERK upstream regulatory kinase has not been fully elucidated. Furthermore, it remains unexplored whether hnRNPK may impact the depressive condition via the ERK pathway. The present study addressed this issue by integrating approaches of genetics, molecular biology, behavioral testing. We found that hnRNPK in the brain was mainly distributed in the hippocampal neurons; that it was significantly downregulated in mice that displayed stress-induced depression-like behaviors; and that the level of hnRNPK markedly decreased in MDD patients from the GEO database. Further in vivo and in vitro analyses revealed that the changes in the expressions of BDNF and PSD95 and in the phosphorylation of ERK (Thr202/Tyr204) paralleled the variation of hnRNPK levels in the ventral hippocampal neurons in mice with depression-like behaviors. Finally, esketamine treatment significantly increased the level of hnRNPK in mice. These findings evidence that hnRNPK involved in the pathogenesis of depression via the ERK-BDNF pathway, pinpointing hnRNPK as a potential therapeutic target in treating MDD patients.

Keratin 19 binds and regulates cytoplasmic HNRNPK mRNA targets in triple-negative breast cancer.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein K (HNRNPK) regulates pre-mRNA processing and long non-coding RNA localization in the nucleus. It was previously shown that shuttling of HNRNPK to the cytoplasm promotes cell proliferation and cancer metastasis. However, the mechanism of HNRNPK cytoplasmic localization, its cytoplasmic RNA ligands, and impact on post-transcriptional gene regulation remain uncharacterized. RESULTS: Here we show that the intermediate filament protein Keratin 19 (K19) directly interacts with HNRNPK and sequesters it in the cytoplasm. Correspondingly, in K19 knockout breast cancer cells, HNRNPK does not localize in the cytoplasm, resulting in reduced cell proliferation. We comprehensively mapped HNRNPK binding sites on mRNAs and showed that, in the cytoplasm, K19-mediated HNRNPK-retention increases the abundance of target mRNAs bound to the 3' untranslated region (3'UTR) at the expected cytidine-rich (C-rich) sequence elements. Furthermore, these mRNAs protected by HNRNPK in the cytoplasm are typically involved in cancer progression and include the p53 signaling pathway that is dysregulated upon HNRNPK knockdown (HNRNPK KD) or K19 knockout (KRT19 KO). CONCLUSIONS: This study identifies how a cytoskeletal protein can directly regulate gene expression by controlling the subcellular localization of RNA-binding proteins to support pathways involved in cancer progression.

LncRNA GAS5-hnRNPK axis inhibited ovarian cancer progression via inhibition of AKT signaling in ovarian cancer cells.

BACKGROUND: The incidence of ovarian cancer ranks third among gynecologic malignancies, but the mortality rate ranks first. METHODS: The expression of GAS5 is low in ovarian cancer and is associated with the low survival of ovarian cancer patients according to public ovarian cancer databases. GAS5 overexpression inhibited ovarian malignancy by affecting the proliferation and migratory abilities in OVCAR3 and A2780 cells. GAS5 overexpression increased the rate of cell apoptosis, and the cells were blocked in the G1 phase as assessed by flow cytometry. RESULTS: We found that hnRNPK was a potential target gene, which was regulated negatively by GAS5 based on RNA-pulldown and mass spectrometry analysis. Mechanistically, GAS5 affected the inhibition of the PI3K/AKT/mTOR pathways and bound the protein of hnRNPK, which influenced hnRNPK stability. Furthermore, rescue assays demonstrated hnRNPK was significantly involved in the progression of ovarian cancer. CONCLUSIONS: Our study showed one of the mechanisms that GAS5 inhibited ovarian cancer metastasis by down-regulating hnRNPK expression, and GAS5 can be used to predict the prognosis of ovarian cancer patients.

Activation of NLRP2 in Triple-Negative Breast Cancer sensitizes chemotherapeutic therapy through facilitating hnRNPK function.

Nucleotide-binding oligomerization domain (NOD)-like receptor type 2 protein (NLRP2) was reported to inhibit NF-κB in response to inflammatory stimuli, but its role in tumors remains elusive. We screened out NLRP2 from mouse models of breast cancer metastasis. Bioinformatics analysis showed NLRP2 expression was positively correlated with survival rate and negatively correlated with the potential of cancer metastasis. Its significance in Triple-Negative Breast Cancer (TNBC) was investigated by gain- and loss-of-function studies in vivo and vitro. Re-expression of NLRP2 dramatically inhibited the growth and metastasis of the xenograft model of MDA-MB-231 cells. Mechanically, NLRP2 confined hnRNPK within cytoplasm, which in turn blocked vimentin mRNA production. Not only that, NLRP2 further enhanced the H2O2-induced high level of p53&Bax and hence dramatically increased the apoptosis rate (fivefold). Likewise, carboplatin-treated cells showed decreased cell viability, suggesting that patients of TNBC with high level of NLRP2 respond well to chemotherapeutics. Under the stimulus of H2O2, NLRP2-hnRNPK no longer stayed in the cytoplasm, but entered the nucleus to increase the expression of p53 and hence enhanced corresponding apoptosis effect, increasing Bax expression. It suggested that NLRP2 helps p53 enter the nucleus to induce apoptosis. This study revealed a novel function of NLRP2 that modulated oncogenic and anti-oncogenic characteristics of hnRNPK, and provided a new biomarker for TNBC chemotherapy.

Study of FOXO1-interacting proteins using TurboID-based proximity labeling technology.

BACKGROUND: Protein‒protein interactions (PPIs) are the foundation of the life activities of cells. TurboID is a biotin ligase with higher catalytic efficiency than BioID or APEX that reduces the required labeling time from 18 h to 10 min. Since many proteins participate in binding and catalytic events that are very short-lived, it is theoretically possible to find relatively novel binding proteins using the TurboID technique. Cell proliferation, apoptosis, autophagy, oxidative stress and metabolic disorders underlie many diseases, and forkhead box transcription factor 1 (FOXO1) plays a key role in these physiological and pathological processes. RESULTS: The FOXO1-TurboID fusion gene was transfected into U251 astrocytes, and a cell line stably expressing FOXO1 was constructed. While constructing the FOXO1 overexpression plasmid, we also added the gene sequence of TurboID to perform biotin labeling experiments in the successfully fabricated cell line to look for FOXO1 reciprocal proteins. Label-free mass spectrometry analysis was performed, and 325 interacting proteins were found. A total of 176 proteins were identified in the FOXO1 overexpression group, and 227 proteins were identified in the Lipopolysaccharide -treated group (Lipopolysaccharide, LPS). Wild-type U251 cells were used to exclude interference from nonspecific binding. The FOXO1-interacting proteins hnRNPK and RBM14 were selected for immunoprecipitation and immunofluorescence verification. CONCLUSION: The TurboID technique was used to select the FOXO1-interacting proteins, and after removing the proteins identified in the blank group, a large number of interacting proteins were found in both positive groups. This study lays a foundation for further study of the function of FOXO1 and the regulatory network in which it is involved.

A cytosine-patch sequence motif identified in the conserved region of lincRNA-p21 interacts with the KH3 domain of hnRNPK.

Long Intergenic Non-coding RNAs (lincRNAs) are the largest class of long non-coding RNAs in eukaryotes, originating from the genome's intergenic regions. A ∼4 â€‹kb long lincRNA-p21 is derived from a transcription unit next to the p21/Cdkn1a gene locus. LincRNA-p21 plays regulatory roles in p53-dependent transcriptional and translational repression through its physical association with proteins such as hnRNPK and HuR. It is also involved in the aberrant gene expression in different cancers. In this study, we have carried out a bioinformatics-based gene analysis and annotation of lincRNA-p21 to show that it is highly conserved in primates and identified two conserved domains in its sequence at the 5' and 3' terminal regions. hnRNPK has previously been shown to interact specifically with the 5' conserved region of lincRNA-p21. hnRNPK is known to bind preferentially to the pyrimidine-rich (poly C) nucleotide sequences in RNAs. Interestingly, we observed a single occurrence of a cytosine-rich patch (C-patch) consisting of a CUCCCGC sequence in the 5' conserved region of human lincRNA-p21, making it a putative hnRNPK binding motif. Using NMR and ITC experiments, we showed that the single-stranded C-patch containing RNA sequence motif interacts specifically with the KH3 domain of hnRNPK.

Epigenetic Control of Cancer Cell Proliferation and Cell Cycle Progression by HNRNPK via Promoting Exon 4 Inclusion of Histone Code Reader SPIN1.

Heterogeneous nuclear ribonucleoprotein K (HNRNPK, hnRNP K), a multifunctional RNA/DNA binding protein, mainly regulates transcription, translation and RNA splicing, and then plays oncogenic roles in many cancers. However, the related mechanisms remain largely unknown. Here, we found that HNRNPK can partially epigenetically regulate cancer cell proliferation via increasing transcription and exon 4-inclusion of SPIN1, an important oncogenic histone code reader. This exon 4 skipping event of SPIN1 generates a long non-coding RNA, followed by the downregulation of SPIN1 protein. SPIN1 is one of the most significantly co-expressed genes of HNRNPK in thirteen TCGA cancers. Our further studies revealed HNRNPK knockdown significantly inhibited cell growth and cell cycle progression in oral squamous cell carcinoma (OSCC) cells and promoted cell apoptosis. Overexpression of SPIN1 was able to partially rescue the growth inhibition triggered by HNRNPK knockdown. Moreover, CCND1 (Cyclin D1), a key cell cycle regulator and oncogene, epigenetically up-regulated by SPIN1, was also positively regulated by HNRNPK. In addition, we discovered that HNRNPK promoted SPIN1 exon 4 inclusion by interacting with an intronic splicing enhancer in intron 4. Collectively, our study suggests a novel epigenetic regulatory pathway of HNRNPK in OSCC, mediated by controlling the transcription activity and alternative splicing of SPIN1 gene.

BBOX1-AS1 mediates trophoblast cells dysfunction via regulating hnRNPK/GADD45A axis†.

Recurrent pregnancy loss (RPL) is a common pathological problem during pregnancy, and its clinical etiology is complex and unclear. Dysfunction of trophoblasts may cause a series of pregnancy complications, including preeclampsia, fetal growth restriction, and RPL. Recently, lncRNAs have been found to be closely related to the occurrence and regulation of pregnancy-related diseases, but few studies have focused on their role in RPL. In this study, we identified a novel lncRNA BBOX1-AS1 that was significantly upregulated in villous tissues and serum of RPL patients. Functionally, BBOX1-AS1 inhibited proliferation, migration, invasion, tube formation and promoted apoptosis of trophoblast cells. Mechanistically, overexpression of BBOX1-AS1 activated the p38 and JNK MAPK signaling pathways by upregulating GADD45A expression. Further studies indicated that BBOX1-AS1 could increase the stability of GADD45A mRNA by binding hnRNPK and ultimately cause abnormal trophoblast function. Collectively, our study highlights that the BBOX1-AS1/hnRNPK/GADD45A axis plays an important role in trophoblast-induced RPL and that BBOX1-AS1 may serve as a potential target for the diagnosis of RPL.

HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction.

Background: Chloride channel 3 (CLCN3) is regulated by transcription-coactivator, however, it is unclear which core transcription factor regulates CLCN3. The role of CLCN3 in lung adenocarcinoma (LUAD) is unexplored and the relationship between CLCN3 and tumor microenvironment is unknown. Methods: A 5'-biotin-labeled promoter probe of CLCN3 was used to pull down the promoter-binding transcription factor. Further study was investigated using LUAD samples, cell lines, and xenograft mice models, and the mechanism was explored. Results: CLCN3 was upregulated in human LUAD, and CLCN3 knockdown inhibited tumor proliferation and migration in vitro. Next, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was first validated as a CLCN3 promoter-binding transcription factor. Mechanistically, HNRNPK knockdown suppressed the promoter activity of CLCN3, thus regulating CLCN3 expression at the transcriptional level, and the binding motif 'GCGAGG' and binding site '-538/-248 bp' were identified. Subsequently, the RNA-seq data illustrated that the primary functions of HNRNPK were similar to those of CLCN3. The results from in vitro and in vivo trials indicated that the expression and function of CLCN3 were regulated by HNRNPK. By isolating primary cancer-associated fibroblasts (CAFs) from human LUAD, we confirmed that decreased extracellular CLCN3 secretion induced by HNRNPK knockdown inhibited CAFs activation and TGF-ß1 production, thus suppressing nuclear HNRNPK expression and LUAD progression in a feedback way. Furthermore, this phenomenon was rescued after the addition of TGF-ß1, revealing that the HNRNPK/CLCN3 axis facilitated LUAD progression through intercellular interactions. Finally, we identified that CLCN3 and HNRNPK were upregulated and correlated with poor prognosis in LUAD patients. Conclusions: HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction.

An arrayed genome-wide perturbation screen identifies the ribonucleoprotein Hnrnpk as rate-limiting for prion propagation.

A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting cellular host-factors that can modify prion propagation. We exposed prion-infected cells in high-density microplates to 35,364 ternary pools of 52,746 siRNAs targeting 17,582 genes representing the majority of the mouse protein-coding transcriptome. We identified 1,191 modulators of prion propagation. While 1,151 modified the expression of both the pathological prion protein, PrPSc , and its cellular counterpart, PrPC , 40 genes selectively affected PrPSc . Of the latter 40 genes, 20 augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrPC . Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Further, the unexpected identification of a prion-controlling ribonucleoprotein suggests a role for RNA in the generation of infectious prions.