HNRNPD regulates the biogenesis of circRNAs and the ratio of mRNAs to circRNAs for a set of genes.

Exonic circular RNAs (ecircRNAs) in animal cells are generated by backsplicing, and the biogenesis of ecircRNAs is regulated by an array of RNA binding proteins (RBPs). HNRNPD is a heterogeneous nuclear ribonucleoprotein family member with both cytoplasmic and nuclear roles, and whether HNRNPD regulates the biogenesis of circRNAs remains unknown. In this study, we examine the role of HNRNPD in the biogenesis of ecircRNAs. The levels of ecircRNAs are primarily increased upon depletion of HNRNPD. HNRNPD preferentially binds to motifs enriched with A and U nucleotides, and the flanking introns of ecircRNAs tend to have more numbers and higher intensity of HNRNPD binding sites. The levels of mRNAs are generally not significantly altered in HNRNPD knockout cells. For a small set of genes, the circRNA:mRNA ratio is substantially affected, and the mRNA levels of some of these genes demonstrate a significant decrease in HNRNPD knockout cells. CDK1 is identified as a key gene modulated by HNRNPD in the context of circRNA biogenesis. HNRNPD suppresses the biogenesis of circCDK1 and favours the generation of CDK1 mRNA, and the CDK1 protein is a critical regulator of the cell cycle and apoptosis. HNRNPD can participate in cellular physiology, including the cell cycle and apoptosis, and plays roles in clear cell renal cell carcinoma (ccRCC) by modulating circRNA biogenesis and the mRNA levels of key genes, such as CDK1.

The amyotrophic lateral sclerosis-linked protein TDP-43 regulates interleukin-6 cytokine production by human brain pericytes.

Amyotrophic lateral sclerosis (ALS) is a fatal movement disorder involving degeneration of motor neurons through dysfunction of the RNA-binding protein TDP-43. Pericytes, the perivascular cells of the blood-brain, blood-spinal cord, and blood-CSF barriers also degenerate in ALS. Indeed, pericytes are among the earliest cell types to show gene expression changes in pre-symptomatic animal models of ALS. This suggests that pericyte degeneration precedes neurodegeneration and may involve pericyte cell-autonomous TDP-43 dysfunction. Here we determined the effect of TDP-43 dysfunction in human brain pericytes on interleukin 6 (IL-6), a critical secreted inflammatory mediator reported to be regulated by TDP 43. Primary human brain pericytes were cultured from biopsy tissue from epilepsy surgeries and TDP-43 was silenced using siRNA. TDP-43 silencing of pericytes stimulated with pro-inflammatory cytokines, interleukin-1ß or tumour necrosis factor alpha, robustly suppressed the induction of IL-6 transcript and protein. IL-6 regulation by TDP-43 did not involve the assembly of TDP-43 nuclear splicing bodies, and did not occur via altered splicing of IL6. Instead, transcriptome-wide analysis by RNA-Sequencing identified a poison exon in the IL6 destabilising factor HNRNPD (AUF1) as a splicing target of TDP-43. Our data support a model whereby TDP-43 silencing favours destabilisation of IL6 mRNA, via enhanced AU-rich element-mediated decay by HNRNP/AUF1. This suggests that cell-autonomous deficits in TDP-43 function in human brain pericytes would suppress their production of IL-6. Given the importance of the blood-brain and blood-spinal cord barriers in maintaining motor neuron health, TDP-43 in human brain pericytes may represent a cellular target for ALS therapeutics.

Further defining the critical genes for the 4q21 microdeletion disorder.

4q21 microdeletion syndrome (MIM: 613509) is a new genomic disorder characterized by intellectual disability, absent or severely delayed speech, growth retardation, hypotonia, variable brain malformation, and facial dysmorphism. The critical genes had been proposed based on an overlapping 1.37 Mb genomic region. No further refinement has been done since year 2010. Here, we present three cases with 4q21 deletion identified by clinical chromosomal microarray analysis. One of the cases have a de novo 761 kb deletion which is the smallest deletion ever reported at this locus. It provides an opportunity to further define the critical regions/genes associated with specific features of the 4q21 microdeletion syndrome. The evidence support the notion that PRKG2 and RASGEF1B are critical genes for intellectual disability and speech defect, and the heterogeneous nuclear ribonucleoprotein HNRNPD and HNRNPDL (previously known as HNRPDL) genes are associated with growth retardation and hypotonia. © 2016 Wiley Periodicals, Inc.

A de novo deletion in a boy with cerebral palsy suggests a refined critical region for the 4q21.22 microdeletion syndrome.

We present an 18-year-old boy with cerebral palsy, intellectual disability, speech delay, and seizures. He carries a likely pathogenic 1.3 Mb de novo heterozygous deletion in the 4q21.22 microdeletion syndrome region. He also carries a 436 kb maternally-inherited duplication impacting the first three exons of CHRNA7. The majority of previously published cases with 4q21.22 syndrome shared common features including growth restriction, muscular hypotonia, and absent or severely delayed speech. Using copy number variation (CNV) data available for other subjects, we defined a minimal critical region of 170.8 kb within the syndromic region, encompassing HNRNPD. We also identified a larger 2 Mb critical region encompassing ten protein-coding genes, of which six (PRKG2, RASGEF1B, HNRNPDL, HNRNPD, LIN54, COPS4) have a significantly low number of truncating loss-of-function mutations. Long-range chromatin interaction data suggest that this deletion may alter chromatin interactions at the 4q21.22 microdeletion region. We suggest that the deletion or misregulation of these genes is likely to contribute to the neurodevelopmental and neuromuscular abnormalities in 4q21.22 syndrome.

A Mammalian Conserved Circular RNA CircLARP1B Regulates Hepatocellular Carcinoma Metastasis and Lipid Metabolism.

Circular RNAs (circRNAs) have emerged as crucial regulators in physiology and human diseases. However, evolutionarily conserved circRNAs with potent functions in cancers are rarely reported. In this study, a mammalian conserved circRNA circLARP1B is identified to play critical roles in hepatocellular carcinoma (HCC). Patients with high circLARP1B levels have advanced prognostic stage and poor overall survival. CircLARP1B facilitates cellular metastatic properties and lipid accumulation through promoting fatty acid synthesis in HCC. CircLARP1B deficient mice exhibit reduced metastasis and less lipid accumulation in an induced HCC model. Multiple lines of evidence demonstrate that circLARP1B binds to heterogeneous nuclear ribonucleoprotein D (HNRNPD) in the cytoplasm, and thus affects the binding of HNRNPD to sensitive transcripts including liver kinase B1 (LKB1) mRNA. This regulation causes decreased LKB1 mRNA stability and lower LKB1 protein levels. Antisense oligodeoxynucleotide complementary to theHNRNPD binding sites in circLARP1B increases the HNRNPD binding to LKB1 mRNA. Through the HNRNPD-LKB1-AMPK pathway, circLARP1B promotes HCC metastasis and lipid accumulation. Results from AAV8-mediated hepatocyte-directed knockdown of circLARP1B or Lkb1 in mouse models also demonstrate critical roles of hepatocytic circLARP1B regulatory pathway in HCC metastasis and lipid accumulation, and indicate that circLARP1B may be potential target of HCC treatment.

Copy number amplification-induced overexpression of lncRNA LOC101927668 facilitates colorectal cancer progression by recruiting hnRNPD to disrupt RBM47/p53/p21 signaling.

BACKGROUND: Somatic copy number alterations (SCNAs) are pivotal in cancer progression and patient prognosis. Dysregulated long non-coding RNAs (lncRNAs), modulated by SCNAs, significantly impact tumorigenesis, including colorectal cancer (CRC). Nonetheless, the functional significance of lncRNAs induced by SCNAs in CRC remains largely unexplored. METHODS: The dysregulated lncRNA LOC101927668, induced by copy number amplification, was identified through comprehensive bioinformatic analyses utilizing multidimensional data. Subsequent in situ hybridization was employed to ascertain the subcellular localization of LOC101927668, and gain- and loss-of-function experiments were conducted to elucidate its role in CRC progression. The downstream targets and signaling pathway influenced by LOC101927668 were identified and validated through a comprehensive approach, encompassing RNA sequencing, RT-qPCR, Western blot analysis, dual-luciferase reporter assay, evaluation of mRNA and protein degradation, and rescue experiments. Analysis of AU-rich elements (AREs) within the mRNA 3' untranslated region (UTR) of the downstream target, along with exploration of putative ARE-binding proteins, was conducted. RNA pull-down, mass spectrometry, RNA immunoprecipitation, and dual-luciferase reporter assays were employed to elucidate potential interacting proteins of LOC101927668 and further delineate the regulatory mechanism between LOC101927668 and its downstream target. Moreover, subcutaneous xenograft and orthotopic liver xenograft tumor models were utilized to evaluate the in vivo impact of LOC101927668 on CRC cells and investigate its correlation with downstream targets. RESULTS: Significantly overexpressed LOC101927668, driven by chr7p22.3-p14.3 amplification, was markedly correlated with unfavorable clinical outcomes in our CRC patient cohort, as well as in TCGA and GEO datasets. Moreover, we demonstrated that enforced expression of LOC101927668 significantly enhanced cell proliferation, migration, and invasion, while its depletion impeded these processes in a p53-dependent manner. Mechanistically, nucleus-localized LOC101927668 recruited hnRNPD and translocated to the cytoplasm, accelerating the destabilization of RBM47 mRNA, a transcription factor of p53. As a nucleocytoplasmic shuttling protein, hnRNPD mediated RBM47 destabilization by binding to the ARE motif within RBM47 3'UTR, thereby suppressing the p53 signaling pathway and facilitating CRC progression. CONCLUSIONS: The overexpression of LOC101927668, driven by SCNAs, facilitates CRC proliferation and metastasis by recruiting hnRNPD, thus perturbing the RBM47/p53/p21 signaling pathway. These findings underscore the pivotal roles of LOC101927668 and highlight its therapeutic potential in anti-CRC interventions.

HNRNPD/MAD2L2 axis facilitates lung adenocarcinoma progression and is a potential prognostic biomarker.

BACKGROUND: Although progress has been made in the treatment of LAUD, the survival rate for patients remains poor. An in-depth grasp of the molecular pathways implicated in LUAD progression is vital for improving diagnosis and treatment strategies. This study aims to explore novel molecular mechanisms driving LUAD progression and identify new potential prognostic biomarkers for LAUD patients. METHODS: Based on mass spectrometry analysis of human LUAD tissues, HNRNPD and MAD2L2 were identified as potential key proteins involved in LUAD progression. Subsequently, the interplay between HNRNPD and MAD2L2 was examined through dual-luciferase reporter assays, RNA-seq analysis, and various molecular biology techniques. Ultimately, the role of the HNRNPD/MAD2L2 axis in LUAD advancement and its potential as a prognostic indicator were investigated utilizing LUAD specimens, cell lines, and xenograft mouse models. RESULTS: In human LAUD tissues and cell lines, elevated levels of HNRNPD and MAD2L2 proteins were discovered. It was determined that HNRNPD binds to the MAD2L2 promoter, forming a regulatory axis at the transcriptional level. Subsequently, both in vitro and in vivo data demonstrated that the downregulation of the HNRNPD/MAD2L2 axis inhibited LUAD progression, while this effect could be rescued by MAD2L2 upregulation. Conversely, the upregulation of the HNRNPD/MAD2L2 axis facilitated LUAD progression, and this outcome could be reversed by MAD2L2 knockdown. Mechanistically, the downregulation of HNRNPD suppressed the promoter activity and transcription of MAD2L2, thus inhibiting the PI3K/HIF1α/ANGPTL4 pathway and tumor angiogenesis. Finally, it was confirmed that LUAD patients with high levels of both HNRNPD and MAD2L2 exhibited the poorest prognosis. Therefore, the HNRNPD/MAD2L2 axis has been identified as a potential predictive indicator for LUAD patients. CONCLUSIONS: The HNRNPD/MAD2L2 axis facilitates LUAD progression and serves as a potential prognostic biomarker.

FAT1 inhibits the proliferation of DLBCL cells via increasing the m6A modification of YAP1 mRNA.

Emerging evidence shows that FAT atypical cadherin 1 (FAT1) mutations occur in lymphoma and are associated with poorer overall survival. Considering that diffuse large B cell lymphoma (DLBCL) is the category of lymphoma with the highest incidence rate, this study aims to explore the role of FAT1 in DLBCL. The findings demonstrate that FAT1 inhibits the proliferation of DLBCL cell lines by downregulating the expression of YAP1 rather than by altering its cellular localization. Mechanistic analysis via meRIP-qPCR/luciferase reporter assays showed that FAT1 increases the m6A modification of YAP1 mRNA 3'UTR and the subsequent binding of heterogeneous nuclear ribonucleoprotein D (HNRNPD) to the m6A modified YAP1 mRNA, thus decreasing the stability of YAP1 mRNA. Furthermore, FAT1 increases YAP1 mRNA 3'UTR m6A modification by decreasing the activity of the TGFß-Smad2/3 pathway and the subsequent expression of ALKBH5, which is regulated at the transcriptional level by Smad2/3. Collectively, these results reveal that FAT1 inhibits the proliferation of DLBCL cells by increasing the m6A modification of the YAP1 mRNA 3'UTR via the TGFß-Smad2/3-ALKBH5 pathway. The findings of this study therefore indicate that FAT1 exerts anti-tumor effects in DLBCL and may represent a novel target in the treatment of this form of lymphoma.

AUF1-RGG peptides up-regulate the VEGF antagonist, soluble VEGF receptor-1 (sFlt-1).

The macrophage is essential to the innate immune response, but also contributes to human disease by aggravating inflammation. Under severe inflammation, macrophages and other immune cells over-produce immune mediators, including vascular endothelial growth factor (VEGF). The VEGF protein stimulates macrophage activation and induces macrophage migration. A natural inhibitor of VEGF, the soluble VEGF receptor (sFlt-1) is also produced by macrophages and sFlt-1 has been used clinically to block VEGF. In macrophages, we have shown that the mRNA regulatory protein AUF1/hnRNP D represses VEGF gene expression by inhibiting translation of AURE-regulated VEGF mRNA. Peptides (AUF1-RGG peptides) that are modeled on the arginine-glycine-glycine (RGG) motif in AUF1 also block VEGF expression. This report shows that the AUF1-RGG peptides reduce two other AURE-regulated genes, TNF and GLUT1. Three alternative splice variants of sFlt-1 contain AURE in their 3'UTR, and in an apparent paradox, AUF1-RGG peptides stimulate expression of these three sFlt-1 Variants. The AUF1-RGG peptides likely act by distinct mechanisms with complimentary effects to repress VEGF gene expression and over-express the endogenous VEGF blocking agent, sFlt-1. The AUF1-RGG peptides are novel reagents that reduce VEGF and other inflammatory mediators, and may be useful tools to suppress severe inflammation.

PFDN2 promotes cell cycle progression via the hnRNPD-MYBL2 axis in gastric cancer.

Gastric cancer (GC) is a major health burden worldwide, but our understanding of GC is limited, and the prognosis is poor. Novel therapeutic strategies and biomarkers are urgently needed to improve GC patient outcomes. Previously, we identified PFDN2 as a novel key gene in gastric cancer based on its differential expression between cancer and normal tissues. However, the role and underlying mechanisms of PFDN2 in GC remain elusive. In this article, we demonstrated that PFDN2 is highly expressed in GC and that upregulation of PFDN2 is associated with the progression of GC. We further found that PFDN2 could promote cell cycle progression by promoting MYBL2 expression. Mechanistically, we demonstrated that PFDN2 could upregulate MYBL2 expression by facilitating the nuclear translocation of hnRNPD, and thus promoting MYBL2 transcriptional program. In conclusion, we found that PFDN2 promotes cell cycle progression via the hnRNPD-MYBL2 axis and may serve as a potential biomarker and therapeutic target for GC.

HNRNPD is a prognostic biomarker in non-small cell lung cancer and affects tumor growth and metastasis via the PI3K-AKT pathway.

Heterogeneous nuclear ribonucleoprotein D (HNRNPD) can regulate expression of key proteins in various cancers. However, the prognostic predictive value and biology function of HNRNPD in non-small cell lung cancer (NSCLC) is unknown. First, we used the TCGA and GEO datasets to determine that HNRNPD predicts the prognosis of NSCLC patients. Following that, we knocked down HNRNPD in NSCLC cell lines in vitro and validated its biological function using CCK-8, transwell assays, wound healing tests, and Western blotting. Finally, we constructed tissue microarrays (TMAs) from 174 NSCLC patients and verified our findings using immunohistochemistry staining for HNRNPD from public databases. In both the public datasets, NSCLC tissues with elevated HNRNPD expression had shorter overall survival (OS). In addition, HNRNPD knockdown NSCLC cell lines showed significantly reduced proliferation, invasion, and metastatic capacity via the PI3K-AKT pathway. Finally, elevated HNRNPD expression in NSCLC TMAs was linked to a poorer prognosis and decreased PD-L1 expression levels. HNRNPD is associated with a poorer prognosis in NSCLC and affects tumor growth and metastasis via the PI3K-AKT pathway.

RNA binding protein AUF1/HNRNPD regulates nuclear export, stability and translation of SNCA transcripts.

Alpha-synuclein (SNCA) accumulation plays a central role in the pathogenesis of Parkinson's disease. Determining and interfering with the mechanisms that control SNCA expression is one approach to limiting disease progression. Currently, most of our understanding of SNCA regulation is protein-based. Post-transcriptional mechanisms directly regulating SNCA mRNA expression via its 3' untranslated region (3'UTR) were investigated here. Mass spectrometry of proteins pulled down from murine brain lysates using a biotinylated SNCA 3'UTR revealed multiple RNA-binding proteins, of which HNRNPD/AUF1 was chosen for further analysis. AUF1 bound both proximal and distal regions of the SNCA 3'UTR, but not the 5'UTR or CDS. In the nucleus, AUF1 attenuated SNCA pre-mRNA maturation and was indispensable for the export of SNCA transcripts. AUF1 destabilized SNCA transcripts in the cytosol, primarily those with shorter 3'UTRs, independently of microRNAs by recruiting the CNOT1-CNOT7 deadenylase complex to trim the polyA tail. Furthermore, AUF1 inhibited SNCA mRNA binding to ribosomes. These data identify AUF1 as a multi-tasking protein regulating maturation, nucleocytoplasmic shuttling, stability and translation of SNCA transcripts.

Evaluation of Heterogeneous Nuclear Ribonucleoprotein D Expression as a Diagnostic Marker for Oral Squamous Cell Carcinoma.

The heterogeneous nuclear ribonucleoprotein D (hnRNPD) serves as a prognostic marker for oral squamous cell carcinoma (OSCC). We evaluated the diagnostic potential of hnRNPD to differentiate between OSCC and normal mucosa. Immunohistochemistry for hnRNPD and a routinely used diagnostic marker deltaNp63 (p40) was performed in 32 normal mucosae and 46 OSCC specimens. Subsequently, receiver-operating characteristic analysis was performed to evaluate the diagnostic potential of hnRNPD in comparison to that of p40. Immunostaining for p40 and hnRNPD was observed in 39 (84.78%) and 38 (82.60%) cases, respectively, in OSCC specimens. The poorly differentiated squamous cell carcinoma displayed 100% (eight cases) immunoreactivity for hnRNPD as compared to 87.5% (seven cases) for p40. Nuclear staining of p40 and hnRNPD was observed in all OSCC specimens. p40 staining was restricted to basal cells, whereas both basal and para-basal cells displayed hnRNPD staining in OSCC specimens. Areas under the curve for p40 and hnRNPD were 0.86 and 0.87, respectively. p40 and hnRNPD showed equal sensitivities (80.95%). However, hnRNPD displayed marginally higher (88.23%) specificity for tumor cells as compared to that of p40 (85.29%). Conclusion: In addition to being a well-established prognostic marker, hnRNPD can serve as a diagnostic marker for OSCC.

Changes to the identity of EndoC-ßH1 beta cells may be mediated by stress-induced depletion of HNRNPD.

BACKGROUND: Beta cell identity changes occur in the islets of donors with diabetes, but the molecular basis of this remains unclear. Protecting residual functional beta cells from cell identity changes may be beneficial for patients with diabetes. RESULTS: A somatostatin-positive cell population was induced in stressed clonal human EndoC-ßH1 beta cells and was isolated using FACS. A transcriptomic characterisation of somatostatin-positive cells was then carried out. Gain of somatostatin-positivity was associated with marked dysregulation of the non-coding genome. Very few coding genes were differentially expressed. Potential candidate effector genes were assessed by targeted gene knockdown. Targeted knockdown of the HNRNPD gene induced the emergence of a somatostatin-positive cell population in clonal EndoC-ßH1 beta cells comparable with that we have previously reported in stressed cells. CONCLUSIONS: We report here a role for the HNRNPD gene in determination of beta cell identity in response to cellular stress. These findings widen our understanding of the role of RNA binding proteins and RNA biology in determining cell identity and may be important for protecting remaining beta cell reserve in diabetes.

Downregulation of miRNA-146a-5p promotes malignant transformation of mesenchymal stromal/stem cells by glioma stem-like cells.

Mesenchymal stromal/stem cells (MSCs) are promising carriers in cell-based therapies against central nervous system diseases, and have been evaluated in various clinical trials in recent years. However, bone marrow-derived MSCs (BMSCs) are reportedly involved in tumorigenesis initiated by glioma stem-like cells (GSCs). We therefore established three different orthotopic models of GSC-MSC interactions in vivo using dual-color fluorescence tracing. Cell sorting and micropipetting techniques were used to obtain highly proliferative MSC monoclones from each model, and these cells were identified as transformed MSC lines 1, 2 and 3. Nineteen miRNAs were upregulated and 24 miRNAs were downregulated in all three transformed MSC lines compared to normal BMSCs. Reduced miR-146a-5p expression in the transformed MSCs was associated with their proliferation, malignant transformation and overexpression of heterogeneous nuclear ribonucleoprotein D. These findings suggest that downregulation of miR-146a-5p leads to overexpression of its target gene, heterogeneous nuclear ribonucleoprotein D, thereby promoting malignant transformation of MSCs during interactions with GSCs. Given the risk that MSCs will undergo malignant transformation in the glioma microenvironment, targeted glioma therapies employing MSCs as therapeutic carriers should be considered cautiously.

METTL3 and ALKBH5 oppositely regulate m6A modification of TFEB mRNA, which dictates the fate of hypoxia/reoxygenation-treated cardiomyocytes.

N6-methyladenosine (m6A) mRNA modifications play critical roles in various biological processes. However, no study addresses the role of m6A in macroautophagy/autophagy. Here, we show that m6A modifications are increased in H/R-treated cardiomyocytes and ischemia/reperfusion (I/R)-treated mice heart. We found that METTL3 (methyltransferase like 3) is the primary factor involved in aberrant m6A modification. Silencing METTL3 enhances autophagic flux and inhibits apoptosis in H/R-treated cardiomyocytes. However, overexpression of METTL3 or inhibition of the RNA demethylase ALKBH5 has an opposite effect, suggesting that METTL3 is a negative regulator of autophagy. Mechanistically, METTL3 methylates TFEB, a master regulator of lysosomal biogenesis and autophagy genes, at two m6A residues in the 3'-UTR, which promotes the association of the RNA-binding protein HNRNPD with TFEB pre-mRNA and subsequently decreases the expression levels of TFEB. Further experiments show that autophagic flux enhanced by METTL3 deficiency is TFEB dependent. In turn, TFEB regulates the expression levels of METTL3 and ALKBH5 in opposite directions: it induces ALKBH5 and inhibits METTL3. TFEB binds to the ALKBH5 promoter and activates its transcription. In contrast, inhibition of METTL3 by TFEB does not involve transcriptional repression but rather downregulation of mRNA stability, thereby establishing a negative feedback loop. Together, our work uncovers a critical link between METTL3-ALKBH5 and autophagy, providing insight into the functional importance of the reversible mRNA m6A methylation and its modulators in ischemic heart disease. Abbreviations: ACTB, actin beta; ALKBH5, alkB homolog 5, RNA demethylase; ANXA5, annexin A5; ATG, autophagy-related; BafA, bafilomycin A1; CASP3, caspase 3; ELAVL1, ELAV like RNA binding protein 1; FTO, FTO, alpha-ketoglutarate dependent dioxygenase; GFP, green fluorescent protein; GST, glutathione S-transferase; HNRNPD, heterogeneous nuclear ribonucleoprotein D; H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; LAD, left anterior descending; m6A, N6-methyladenosine; MEFs, mouse embryo fibroblasts; Mer, mutated estrogen receptor domains; METTL3, methyltransferase like 3; METTL14, methyltransferase like 14; mRFP, monomeric red fluorescent protein; MTORC1, mechanistic target of rapamycin kinase complex 1; NMVCs, neonatal mouse ventricular cardiomyocytes; PCNA, proliferating cell nuclear antigen; PE, phosphatidylethanolamine; PI, propidium iodide; PTMs, post-translational modifications; PVDF, polyvinylidenedifluoride; RIP, RNA-immunoprecipitation; siRNA, small interfering RNA; SQSTM1, sequestosome 1; TFEB, transcription factor EB; TUBA: tublin alpha; WTAP, WT1 associated protein; YTHDF, YTH N6-methyladenosine RNA binding protein.

LINC01354 interacting with hnRNP-D contributes to the proliferation and metastasis in colorectal cancer through activating Wnt/ß-catenin signaling pathway.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been identified to play an important role in the development and progression of various tumors, including colorectal cancer (CRC). However, the regulatory molecular mechanism by lncRNA in CRC initiation and progression has not been fully clarified. METHODS: TCGA database was used to identify the involvement of LINC01354 in CRC. qRT-PCR and western blot were used to determine RNA and protein expression. The gain- and loss-of-function assays were conducted to explore the function of LINC01354 in the progression of CRC. In order to investigate the LINC01354-mediated mRNA in CRC tumorigenesis, we applied the profiling analysis as well as GO and KEGG analysis. Pulldown and RIP assays were applied to detect the interaction of hnRNP-D with LINC01354 and ß-catenin. RESULTS: The upregulation of LINC01354 in CRC and its prognostic significance were identified by TCGA database and confirmed in CRC tissues. Functionally, forced expression of LINC01354 promoted, while knockdown of LINC01354 inhibited cell proliferation, migration and EMT phenotype formation of CRC cells. A significant enrichment of the Wnt/ß-catenin signaling pathway genes under LINC01354 overexpression. In addition, LINC01354 modulated the mRNA stability of ß-catenin through interacting with hnRNP-D, thereby activating Wnt/ß-catenin signaling pathway. CONCLUSIONS: Our investigations proposed novel regulatory axis of LINC01354/hnRNP-D/Wnt/ß-catenin, which might be in favor of exploring novel therapeutic regimens for the clinical treatment of CRC.

Complex interaction between HNRNPD mutations and risk polymorphisms is associated with discordant Crohn's disease in monozygotic twins.

Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) affecting the lining of digestive tracts of the colon and ileum. To investigate the reasons behind the presence of CD phenotype in one of the monozygotic (MZ) twins, we utilized the whole exome sequence (WES) datasets of CD tissue biopsy and CD blood of affected twin and the exome dataset of blood from healthy twin. We report the presence of discordant and rare damaging mutation in HNRNPD and other risk polymorphisms such as, rs12103, rs2241880, rs3810936, rs7076156, rs1042058 and rs1292053. HNRNPD was found carrying two novel heterozygous mutations - a stop gain mutation that truncated the protein at 249th and 268th amino acid position and a single base missense mutation replacing Aspartate with Valine at 300th amino acid. The identified risk polymorphisms were found conferring susceptibility to CD and IBD. Discordant deleterious and damaging mutation was detected in HNRNPD that have been implicated in inflammatory pathways. Integrating these variants led to the elucidation of pathophysiology of CD in the affected twin involving the causal processes of macrophage activation, tissue death, autophagy, immune response, cell-migration and T-cell activation.

Peptides modeled on the RGG domain of AUF1/hnRNP-D regulate 3' UTR-dependent gene expression.

Messenger RNA binding proteins control post-transcriptional gene expression of targeted mRNAs. The RGG (arginine-glycine-glycine) domain of the AUF1/hnRNP-D mRNA binding protein is a regulatory region that is essential for protein function. The AUF1-RGG peptide, modeled on the RGG domain of AUF1, represses expression of the macrophage cytokine, VEGF. This report expands studies on the AUF1-RGG peptide and evaluates the role of post-translational modifications of the AUF1 protein. Results show that a minimal 31-amino acid AUF1-RGG peptide that lacks poly-glutamine and nuclear localization motifs retains suppressive activity on a VEGF-3'UTR reporter. Arginine residues in RGG motifs may be methylated with resulting changes in protein function. Mass spectroscopy analysis was performed on AUF1 expressed in RAW-264.7 cells. In resting cells, arginines in the first and second RGG motifs are monomethylated. Following activation with lipopolysaccharide, the arginines are dimethylated. To evaluate if the arginine residues are essential for AUF1-RGG activity, the methylatable arginines in the AUF1-3RGG peptide were mutated to lysine or alanine. The R→K and R→A mutants lack activity. We also demonstrate that PI3K/AKT inhibitors reduce VEGF gene expression. Although immunoscreening of AUF1 suggests that LPS and PI3K inhibitors alter the phosphorylation status of AUF1-p37, mass spectroscopy results show that the p37 AUF1 isoform is not phosphorylated with or without lipopolysaccharide stimulation. In summary, arginines in the RGG domain of AUF1 are methylated, and AUF1-RGG peptides may be novel reagents that reduce macrophage activation in inflammation.