Search

The mRNAs encoding the human papillomavirus type 16 (HPV16) E6 and E7 oncogene mRNAs are subjected to extensive alternative RNA splicing at multiple regulated splice sites. One of the most extensively used 5'-splice sites in the HPV16 genome is named SD880 and is located immediately downstream of the E7 open reading frame. Here, we show that a cluster of three GGG-motifs adjacent to HPV16 SD880 interacts with heterogeneous nuclear ribonucleoprotein (hnRNP) H that cooperates with SD880 to stimulate splicing to the upstream HPV16 3'-splice site SA742. This splice site is located in the E7 coding region and is required for the production of the HPV16 226^742 mRNA that encodes the E6^E7 fusion protein. Enhancement of HPV16 E6^E7 mRNA production by hnRNP H occurred at the expense of the intron-retained E6 mRNAs and the spliced E7 mRNAs, demonstrating that hnRNP H controls the relative levels of E6, E7, and E6^E7 proteins. Unexpectedly, overexpression of hnRNP H also promoted retention of the downstream E1 encoding intron and enhanced E1 protein production. We concluded that hnRNP H plays an important role in the HPV16 gene expression program.IMPORTANCEHere, we show that hnRNP H binds to multiple GGG-motifs downstream of human papillomavirus type 16 (HPV16) splice site SD880 and acts in concert with SD880 to promote expression of the HPV16 E6^E7 mRNA. The E6^E7 protein has been shown previously to stabilize the HPV16 E6 and E7 oncoproteins and may as such contribute to the carcinogenic properties of HPV16. In its capacity of major regulator of HPV16 oncogene expression, hnRNP H may be exploited as a target for antiviral drugs to HPV16., Competing Interests: The authors declare no conflict of interest.

Nuclear localization of the metabolic enzyme PKM2 is widely observed in various cancer types. We identify nuclear PKM2 as a non-canonical RNA-binding protein (RBP) that specifically interacts with folded RNA G-quadruplex (rG4) structures in precursor mRNAs (pre-mRNAs). PKM2 occupancy at rG4s prevents the binding of repressive RBPs, such as HNRNPF, and promotes the expression of rG4-containing pre-mRNAs (the "rG4ome"). We observe an upregulation of the rG4ome during epithelial-to-mesenchymal transition and a negative correlation of rG4 abundance with patient survival in different cancer types. By preventing the nuclear accumulation of PKM2, we could repress the rG4ome in triple-negative breast cancer cells and reduce migration and invasion of cancer cells in vitro and in xenograft mouse models. Our data suggest that the balance of folded and unfolded rG4s controlled by RBPs impacts gene expression during tumor progression., Competing Interests: Declaration of interests J.R. is on the scientific advisory board and has an equity interest in Pearl Bio. J.R. is a co-founder and has an equity interest in Kapis Biosciences., (Published by Elsevier Inc.)

Heterogeneous nuclear ribonucleoprotein F (HnRNP F) is a key regulator for nucleic acid metabolism; however, whether HnRNP F expression is important in maintaining podocyte integrity is unclear. Nephroseq analysis from a registry of human kidney biopsies was performed. Age- and sex-matched podocyte-specific HnRNP F knockout (HnRNP F POD KO) mice and control (HnRNP F fl/fl ) were studied. Podocytopathy was induced in male mice (more susceptible) either by adriamycin (ADR)- or low-dose streptozotocin treatment for 2 or 8 weeks. The mouse podocyte cell line (mPODs) was used in vitro. Nephroseq data in three human cohorts were varied greatly. Both sexes of HnRNP F POD KO mice were fertile and appeared grossly normal. However, male 20-week-old HnRNP F POD KO than HnRNP F fl/fl mice had increased urinary albumin/creatinine ratio, and lower expression of podocyte markers. ADR- or diabetic- HnRNP F POD KO (vs. HnRNP F fl/fl ) mice had more severe podocytopathy. Moreover, methyltransferase-like 14 (Mettl14) gene expression was increased in podocytes from HnRNP F POD KO mice, further enhanced in ADR- or diabetic-treated HnRNP F POD KO mice. Consequently, this elevated Mettl14 expression led to sirtuin1 (Sirt1) inhibition, associated with podocyte loss. In mPODs, knock-down of HnRNP F promoted Mettl14 nuclear translocation, which was associated with podocyte dysmorphology and Sirt1 inhibition-mediated podocyte loss. This process was more severe in ADR- or high glucose- treated mPODs. Conclusion: HnRNP F deficiency in podocytes promotes podocytopathy through activation of Mettl14 expression and its nuclear translocation to inhibit Sirt1 expression, underscoring the protective role of HnRNP F against podocyte injury., Competing Interests: Declaration of competing interest All authors have nothing to disclose., (Copyright © 2024. Published by Elsevier Inc.)

Mutations in the Fused in Sarcoma (FUS) gene cause the familial and progressive form of amyotrophic lateral sclerosis (ALS). FUS is a nuclear RNA-binding protein involved in RNA processing and the biogenesis of a specific set of microRNAs. Here we report that Drosha and two previously uncharacterized Drosha-dependent miRNAs are strong modulators of FUS expression and prevent the cytoplasmic segregation of insoluble mutant FUS in vivo. We demonstrate that depletion of Drosha mitigates FUS-mediated degeneration, survival and motor defects in Drosophila. Mutant FUS strongly interacts with Drosha and causes its cytoplasmic mis-localization into the insoluble FUS inclusions. Reduction in Drosha levels increases the solubility of mutant FUS. Interestingly, we found two Drosha dependent microRNAs, miR-378i and miR-6832-5p, which differentially regulate the expression, solubility and cytoplasmic aggregation of mutant FUS in iPSC neurons and mammalian cells. More importantly, we report different modes of action of these miRNAs against mutant FUS. Whereas miR-378i may regulate mutant FUS inclusions by preventing G3BP-mediated stress granule formation, miR-6832-5p may affect FUS expression via other proteins or pathways. Overall, our research reveals a possible association between ALS-linked FUS mutations and the Drosha-dependent miRNA regulatory circuit, as well as a useful perspective on potential ALS treatment via microRNAs., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core pre-mRNA splicing machinery and causes misregulation of alternative splicing. However, our understanding of how splicing changes are directed by MYC is limited. We performed a signaling pathway-guided splicing analysis to identify MYC-dependent splicing events. These included an HRAS cassette exon repressed by MYC across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA-binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of HNRNPH gene expression with MYC hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, HNRNPF expression showed a positive correlation with MYC hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal mechanisms for MYC-dependent regulation of splicing and point to possible therapeutic targets in prostate cancers.

The T cell-dependent (TD) antibody response involves the generation of high affinity, immunoglobulin heavy chain class-switched antibodies that are generated through germinal center (GC) response. This process is controlled by coordinated transcriptional and post-transcriptional gene regulatory mechanisms. RNA-binding proteins (RBPs) have emerged as critical players in post-transcriptional gene regulation. Here we demonstrate that B cell-specific deletion of RBP hnRNP F leads to diminished production of class-switched antibodies with high affinities in response to a TD antigen challenge. B cells deficient in hnRNP F are characterized by defective proliferation and c-Myc upregulation upon antigenic stimulation. Mechanistically, hnRNP F directly binds to the G-tracts of Cd40 pre-mRNA to promote the inclusion of Cd40 exon 6 that encodes its transmembrane domain, thus enabling appropriate CD40 cell surface expression. Furthermore, we find that hnRNP A1 and A2B1 can bind to the same region of Cd40 pre-mRNA but suppress exon 6 inclusion, suggesting that these hnRNPs and hnRNP F might antagonize each-other's effects on Cd40 splicing. In summary, our study uncovers an important posttranscriptional mechanism regulating the GC response., (© 2023. The Author(s).)

Concerns are raised over the risk to digestive system's tumors from the N-nitrosamines (NAs) exposure in drinking water. Albeit considerable studies are conducted to explore the underlying mechanism responsible for NAs-induced esophageal squamous cell carcinoma (ESCC), the exact molecular mechanisms remain largely unknown, especially at the epigenetic regulation level. In this study, it is revealed that the urinary concentration of N-Nitrosodiethylamine is higher in high incidence area of ESCC, and the lncRNA-UCA1(UCA1) is significantly decreased in ESCC tissues. In vitro and in vivo experiments further show that UCA1 is involved in the malignant transformation of Het-1A cells and precancerous lesions of the rat esophagus induced by N-nitrosomethylbenzylamine (NMBzA). Functional gain and loss experiments verify UCA1 can affect the proliferation, migration, and invasion of ESCC cells in vitro and in vivo. Mechanically, through binding to heterogeneous nuclear ribonucleoprotein F (hnRNP F) protein, UCA1 regulates alternative splicing of fibroblast growth factor receptor 2 (FGFR2), which promotes the FGFR2IIIb isoform switching to FGFR2 IIIc isoform, and the latter activates epithelial-mesenchymal transition via PI3K-AKT signaling pathways impacting tumorigenesis. Therefore, NAs-mediated downregulation of UCA1 promotes ESCC progression through targeting hnRNP F/FGFR2/PI3k-AKT axis, which provides a new chemical carcinogenic target and establishes a previously unknown mechanism for NAs-induced ESCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Alternative splicing of the HIV transcriptome is controlled through cis regulatory elements functioning as enhancers or silencers depending on their context and the type of host RNA binding proteins they recruit. Splice site acceptor A3 (ssA3) is one of the least used acceptor sites in the HIV transcriptome and its activity determines the levels of tat mRNA. Splice acceptor 3 is regulated by a combination of cis regulatory sequences, auxiliary splicing factors, and presumably RNA structure. The mechanisms by which these multiple regulatory components coordinate to determine the frequency in which ssA3 is utilized is poorly understood. By NMR spectroscopy and phylogenetic analysis, we show that the ssA3 regulatory locus is conformationally heterogeneous and that the sequences that encompass the locus are conserved across most HIV isolates. Despite the conformational heterogeneity, the major stem loop (A3SL1) observed in vitro folds to base pair the Polypyrimdine Tract (PPyT) to the Exon Splicing Silencer 2p (ESS2p) element and to a conserved downstream linker. The 3D structure as determined by NMR spectroscopy further reveals that the A3 consensus cleavage site is embedded within a unique stereochemical environment within the apical loop, where it is surrounded by alternating base-base interactions. Despite being described as a receptor for hnRNP H, the ESS2p element is sequestered by base pairing to the 3' end of the PPyT and within this context it cannot form a stable complex with hnRNP H. By comparison, hnRNP A1 directly binds to the A3 consensus cleavage site located within the apical loop, suggesting that it can directly modulate U2AF assembly. Sequence mutations designed to destabilize the PPyT:ESS2p helix results in an increase usage of ssA3 within HIV-infected cells, consistent with the PPyT becoming more accessible for U2AF recognition. Additional mutations introduced into the downstream ESS2 element synergize with ESS2p to cause further increases in ssA3 usage. When taken together, our work provides a unifying picture by which cis regulatory sequences, splicing auxiliary factors and RNA structure cooperate to provide stringent control over ssA3. We describe this as the pair-and-lock mechanism to restrict access of the PPyT, and posit that it operates to regulate a subset of the heterogenous structures encompassing the ssA3 regulatory locus., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins that control key events in RNA biogenesis under both normal and diseased cellular conditions. The low-complexity (LC) domain of hnRNPs can become liquid-like droplets or reversible amyloid-like polymers by phase separation. Yet, whether phase separation of the LC domains contributes to physiological functions of hnRNPs remains unclear. hnRNPH1 contains two LC domains, LC1 and LC2. Here, we show that reversible phase separation of the LC1 domain is critical for both interaction with different kinds of RNA-binding proteins and control of the alternative-splicing activity of hnRNPH1. Interestingly, although not required for phase separation, the LC2 domain contributes to the robust transcriptional activation of hnRNPH1 when fused to the DNA-binding domain, as found recently in acute lymphoblastic leukemia. Our data suggest that the ability of the LC1 domain to phase-separate into reversible polymers or liquid-like droplets is essential for function of hnRNPH1 as an alternative RNA-splicing regulator, whereas the LC2 domain may contribute to the aberrant transcriptional activity responsible for cancer transformation., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Amyloid precursor protein (APP) is a transmembrane protein that plays a crucial role in the production of amyloid-β peptides. Any disruption in APP protein production, its mRNA decay rate or processing may result in abnormal production of amyloid-β peptides and subsequent development of protein aggregation diseases. Therefore, the equilibrium is crucial for neuronal function. An association study of heterogeneous nuclear ribonucleoprotein (hnRNP)-F and hnRNP H1 with APP was carried out in Neuro-2a (N2a) cells. In the present study, we found that hnRNP F and hnRNP H1 were significantly upregulated in the hippocampus of APP/PS1 mice. The changes in APP expression were positively associated with hnRNP F and hnRNP H1 when hnRNP F and hnRNP H1 were depleted or increased in N2a cells. Importantly, cross-linked RNA immunoprecipitation demonstrated binding affinities of hnRNP F and hnRNP H1 for App mRNA. Mechanistically, mRNA stability assay revealed that overexpression of hnRNP F or hnRNP H1 increases the APP level by stabilizing App mRNA half-life, implying that levels of hnRNP F and hnRNP H1 can change the production of APP. Further understanding of the regulatory mechanism of APP expression in association with hnRNP F and hnRNP H1 would provide insights into the mechanism underlying the maintenance of brain health and cognition. This study provides a theoretical basis for the development of hnRNP-stabilizing compounds to regulate APP., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

mRNA transport in neurons requires formation of transport granules containing many protein components, and subsequent alterations in phosphorylation status can release transcripts for translation. Further, mutations in a structurally disordered domain of the transport granule protein hnRNPA2 increase its aggregation and cause hereditary proteinopathy of neurons, myocytes, and bone. We examine in vitro hnRNPA2 granule component phase separation, partitioning specificity, assembly/disassembly, and the link to neurodegeneration. Transport granule components hnRNPF and ch-TOG interact weakly with hnRNPA2 yet partition specifically into liquid phase droplets with the low complexity domain (LC) of hnRNPA2, but not FUS LC. In vitro hnRNPA2 tyrosine phosphorylation reduces hnRNPA2 phase separation, prevents partitioning of hnRNPF and ch-TOG into hnRNPA2 LC droplets, and decreases aggregation of hnRNPA2 disease variants. The expression of chimeric hnRNPA2 D290V in Caenorhabditis elegans results in stress-induced glutamatergic neurodegeneration; this neurodegeneration is rescued by loss of tdp-1, suggesting gain-of-function toxicity. The expression of Fyn, a tyrosine kinase that phosphorylates hnRNPA2, reduces neurodegeneration associated with chimeric hnRNPA2 D290V. These data suggest a model where phosphorylation alters LC interaction specificity, aggregation, and toxicity., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Viral and cellular RNA-binding proteins regulate numerous key steps in the replication of diverse virus genera. Viruses efficiently co-opt the host cell machinery for purposes such as transcription, splicing and subcellular localization of viral genomes. Though viral RNAs often need to resemble cellular RNAs to effectively utilize the cellular machinery, they still retain unique sequence and structural features for recognition by viral proteins for processes such as RNA polymerization, RNA export and selective packaging into virus particles. While beneficial for virus replication, distinct features of viral nucleic acids can also be recognized as foreign by several host defense proteins. Development of the crosslinking immunoprecipitation coupled with sequencing (CLIP) approach has allowed the study of viral and cellular RNA binding proteins that regulate critical aspects of viral replication in unprecedented detail. By combining immunoprecipitation of covalently crosslinked protein-RNA complexes with high-throughput sequencing, CLIP provides a global account of RNA sequences bound by RNA-binding proteins of interest in physiological settings and at near-nucleotide resolution. Here, we describe the step-by-step application of the CLIP methodology within the context of two cellular splicing regulatory proteins, hnRNP A1 and hnRNP H1 that regulate HIV-1 splicing. In principle, this versatile protocol can be applied to many other viral and cellular RNA-binding proteins., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Burkitt lymphoma (BL) is an aggressive B-cell lymphoma characterized by translocation and deregulation of the proto-oncogene c-MYC. Transcription factor 3 (TCF3) has also been shown to be involved in BL pathogenesis. In BL, TCF3 is constitutively active, and/or expression of its transcriptional targets are altered as a result of BL-associated mutations. Here, we found that BL-related TCF3 mutations affect TCF3 alternative splicing, in part by reducing binding of the splicing regulator hnRNPH1 to exon 18b. This leads to greater exon 18b inclusion, thereby generating more of the mutated E47 isoform of TCF3. Interestingly, upregulation of E47 dysregulates the expression of TCF3 targets PTPN6 , and perhaps CCND3 , which are known to be involved in BL pathogenesis. Our findings thus reveal a mechanism by which TCF3 somatic mutations affect multilayered gene regulation underlying BL pathogenesis.

Heterogeneous nuclear ribonucleoprotein (hnRNP) H is a member of hnRNP H/F protein subfamily of hnRNPs that regulate the maturation and post-transcriptional processing of pre-mRNA. As a component of an mRNA export complex, hnRNP H shuttles mature mRNA from the nucleus to the cytoplasm. Although hnRNP H is primarily a nuclear protein, it can accumulate in the cytoplasm in certain tissues and cell types; however, the physiological relevance of hnRNP H cytoplasmic accumulation is unknown. Here we show that under cellular stress hnRNP H accumulates in the cytoplasm and is required for efficient recovery from cellular stress. Moreover, we find that cytoplasmic hnRNP H localizes to stress granules and that the RRM3 domain of hnRNP H is necessary for this localization. Together, our results demonstrate that hnRNP H accumulates in the cytoplasm under cellular stress and is recruited to stress granules., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The oncogenic properties of heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) have been reported, although the tumor-promoting mechanism remains unclear. We herein report the mechanism underlying colorectal cancer cell progression mediated by hnRNP H1. The growth of colorectal cancer cells was suppressed by hnRNP H1 downregulation. A terminal deoxynucleotidyl transferase dUTP nick-end labeling assay revealed the anti-apoptotic effect of hnRNP H1 in colorectal cancer cells. An RNA immunoprecipitation assay revealed that hnRNP H1 bound to sphingosine-1-phosphate lyase 1 (SGPL1). Reverse transcription-polymerase chain reaction revealed the high expression of hnRNP H1 mRNA in colorectal cancer cells and Spearman's rank correlation coefficient showed a strong positive correlation between hnRNP H1 mRNA and SGPL1 mRNA. An siRNA of hnRNP H1 decreased SGPL1 mRNA expression in colorectal cancer cells, but not in non-tumorous cells. These findings suggested that hnRNP H1 increased SGPL1 mRNA expression specifically in cancer cells through direct binding. Targeted knockdown of hnRNP H1 or SGPL1 with siRNAs upregulated p53 phosphorylation and p53-associated molecules, resulting in cell growth inhibition, while hnRNP H1 upregulated the mRNA of SGPL1 and inhibited p53 activation, thereby promoting tumor cell growth. This is a novel mechanism underlying colorectal cancer cell progression mediated by hnRNP H1-SGPL1 mRNA stabilization.

Human telomerase RNA component hTERC comprises multiple motifs that contribute to hTERC biogenesis, holoenzyme activity, and enzyme recruitment to telomeres. hTERC contains several guanine tracts (G-tracts) at its 5'-end, but its associated proteins and potential roles in telomerase function are still poorly understood. The heterogeneous nuclear ribonucleoproteins F, H1, and H2 (hnRNP F/H) are splicing factors that preferentially bind to poly(G)-rich sequences RNA. Here, we demonstrate that hnRNP F/H associate with both hTERC and telomerase holoenzyme to regulate telomerase activity. We reveal hnRNP F/H bind to the 5'-end region of hTERC in vitro and in vivo, and identify the first three G-tracts of hTERC and qRRM1 domain of hnRNP F/H are required for their interaction. Furthermore, hnRNP F/H also directly interact with telomerase holoenzyme. Functionally, we show that hnRNP F/H plays important roles in modulating telomerase activity and telomere length. Moreover, hnRNP F/H deletion greatly impair cancer and stem cell proliferation, and induce stem cell senescence, while hnRNP F/H overexpression delay stem cell senescence. Collectively, our findings unveil a novel role of hnRNP F/H as the binding partners of hTERC and telomerase holoenzyme to regulate telomerase function.

RNA G-quadruplexes (RG4s) are four-stranded structures known to control mRNA translation of cancer relevant genes. RG4 formation is pervasive in vitro but not in cellulo, indicating the existence of poorly characterized molecular machinery that remodels RG4s and maintains them unfolded. Here, we performed a quantitative proteomic screen to identify cytosolic proteins that interact with a canonical RG4 in its folded and unfolded conformation. Our results identified hnRNP H/F as important components of the cytoplasmic machinery modulating the structural integrity of RG4s, revealed their function in RG4-mediated translation and uncovered the underlying molecular mechanism impacting the cellular stress response linked to the outcome of glioblastoma.

p53 is one of the most important tumor suppressor genes, and the exploration of p53-target genes is important for elucidation of its functional mechanisms. In this study, we identified Armadillo Repeat gene deleted in Velo-Cardio-Facial syndrome (ARVCF) as a direct target of p53 through ChIP-sequencing analysis. Activated p53 protein was found to bind to two distinct sites in the ARVCF gene, resulting in induction of ARVCF expression at both the mRNA and protein levels. We revealed that the knockdown of ARVCF inhibited p53-induced apoptosis. Interestingly, ARVCF interacted with hnRNPH2, which is involved in pre-mRNA splicing, and ARVCF knockdown induced dynamic changes in alternative splicing patterns. These results suggest that p53-induced ARVCF indirectly, but not directly, regulates p53 target selectivity through splicing alterations of specific genes. Thus, we demonstrated that the induction of ARVCF expression contributed to the tumor suppressive function of p53. Recently, it has been reported that many tumors have thousands of alternative splicing events that are not detectable in normal samples. ARVCF may play a role in alternative splicing events in cancer and may provide clues to explore novel approaches for cancer diagnosis and therapy.

It is critical to identify biomarkers and functional networks associated with aggressive thyroid cancer to anticipate disease progression and facilitate personalized patient management. We performed miRNome sequencing of 46 thyroid tumors enriched with advanced disease patients with a median follow-up of 96 months. MiRNome profiles correlated with tumor-specific histopathological and molecular features, such as stromal cell infiltration and tumor driver mutation. Differential expression analysis revealed a consistent hsa-miR-139-5p downexpression in primary carcinomas from patients with recurrent/metastatic disease compared to disease-free patients, sustained in paired local metastases and validated in publicly available thyroid cancer series. Exogenous expression of hsa-miR-139-5p significantly reduced migration and proliferation of anaplastic thyroid cancer cells. Proteomic analysis indicated RICTOR, SMAD2/3 and HNRNPF as putative hsa-miR-139-5p targets in our cell system. Abundance of HNRNPF mRNA, encoding an alternative splicing factor involved in cryptic exon inclusion/exclusion, inversely correlated with hsa-miR-139-5p expression in human tumors. RNA sequencing analysis revealed 174 splicing events differentially regulated upon HNRNPF repression in our cell system, affecting genes involved in RTK/RAS/MAPK and PI3K/AKT/MTOR signaling cascades among others. These results point at the hsa-miR-139-5p/HNRNPF axis as a novel regulatory mechanism associated with the modulation of major thyroid cancer signaling pathways and tumor virulence., (© 2019 UICC.)

Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying decreased methamphetamine-induced locomotor activity in mice. Here, we showed that mice (both females and males) with a heterozygous mutation in the first coding exon of Hnrnph1 (H1 +/- ) showed reduced methamphetamine reinforcement and intake and dose-dependent changes in methamphetamine reward as measured via conditioned place preference. Furthermore, H1 +/- mice showed a robust decrease in methamphetamine-induced dopamine release in the NAc with no change in baseline extracellular dopamine, striatal whole-tissue dopamine, dopamine transporter protein, dopamine uptake, or striatal methamphetamine and amphetamine metabolite levels. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for TH did not reveal any major changes in staining intensity, cell number, or forebrain puncta counts. Surprisingly, there was a twofold increase in hnRNP H protein in the striatal synaptosome of H1 +/- mice with no change in whole-tissue levels. To gain insight into the mechanisms linking increased synaptic hnRNP H with decreased methamphetamine-induced dopamine release and behaviors, synaptosomal proteomic analysis identified an increased baseline abundance of several mitochondrial complex I and V proteins that rapidly decreased at 30 min after methamphetamine administration in H1 +/- mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in WT mice showed a rapid increase. We conclude that H1 +/- decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function. SIGNIFICANCE STATEMENT Methamphetamine dependence is a significant public health concern with no FDA-approved treatment. We discovered a role for the RNA binding protein hnRNP H in methamphetamine reward and reinforcement. Hnrnph1 mutation also blunted methamphetamine-induced dopamine release in the NAc, a key neurochemical event contributing to methamphetamine addiction liability. Finally, Hnrnph1 mutants showed a marked increase in basal level of synaptosomal hnRNP H and mitochondrial proteins that decreased in response to methamphetamine, whereas WT mice showed a methamphetamine-induced increase in synaptosomal mitochondrial proteins. Thus, we identified a potential role for hnRNP H in basal and dynamic mitochondrial function that informs methamphetamine-induced cellular adaptations associated with reduced addiction liability., (Copyright © 2020 the authors.)

Long non-coding RNAs (lncRNAs) have structural and regulatory effects on RNA-binding proteins (RBPs). However, the mechanisms by which lncRNAs regulate the neurodegenerative-causative RBP like FUS protein remain poorly understood. Here, we show that knockdown of the Drosophila lncRNA hsrω causes a shift in the methylation status of human FUS from mono- (MMA) to di-methylated (DMA) arginine via upregulation of the arginine methyltransferase 5 (PRMT5, known as ART5 in flies). We found this novel regulatory role to be critical for FUS toxicity since the PRMT5-dependent dimethylation of FUS is required for its proteasomal degradation and causes a reduction of high levels of FUS. Moreover, we show that an increase of FUS causes a decline of both PRMT1 (known as ART1 in flies) and PRMT5 transcripts, leading to an accumulation of neurotoxic MMA-FUS. Therefore, overexpression of either PRMT1 or PRMT5 is able to rescue the FUS toxicity. These results highlight a novel role of lncRNAs in post-translation modification (PTM) of FUS and suggest a causal relationship between lncRNAs and dysfunctional PRMTs in the pathogenesis of FUSopathies., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Alternative splicing of HIV-1 mRNAs increases viral coding potential and controls the levels and timing of gene expression. HIV-1 splicing is regulated in part by heterogeneous nuclear ribonucleoproteins (hnRNPs) and their viral target sequences, which typically repress splicing when studied outside their native viral context. Here, we determined the location and extent of hnRNP binding to HIV-1 mRNAs and their impact on splicing in a native viral context. Notably, hnRNP A1, hnRNP A2, and hnRNP B1 bound to many dispersed sites across viral mRNAs. Conversely, hnRNP H1 bound to a few discrete purine-rich sequences, a finding that was mirrored in vitro hnRNP H1 depletion and mutation of a prominent viral RNA hnRNP H1 binding site decreased the use of splice acceptor A1, causing a deficit in Vif expression and replicative fitness. This quantitative framework for determining the regulatory inputs governing alternative HIV-1 splicing revealed an unexpected splicing enhancer role for hnRNP H1 through binding to its target element. IMPORTANCE Alternative splicing of HIV-1 mRNAs is an essential yet quite poorly understood step of virus replication that enhances the coding potential of the viral genome and allows the temporal regulation of viral gene expression. Although HIV-1 constitutes an important model system for general studies of the regulation of alternative splicing, the inputs that determine the efficiency with which splice sites are utilized remain poorly defined. Our studies provide an experimental framework to study an essential step of HIV-1 replication more comprehensively and in much greater detail than was previously possible and reveal novel cis -acting elements regulating HIV-1 splicing., (Copyright © 2019 American Society for Microbiology.)

Plant photoreceptors tightly regulate gene expression to control photomorphogenic responses. Although gene expression is modulated by photoreceptors at various levels, the regulatory mechanism at the pre-mRNA splicing step remains unclear. Alternative splicing, a widespread mechanism in eukaryotes that generates two or more mRNAs from the same pre-mRNA, is largely controlled by splicing regulators, which recruit spliceosomal components to initiate pre-mRNA splicing. The red/far-red light photoreceptor phytochrome participates in light-mediated splicing regulation, but the detailed mechanism remains unclear. Here, using protein-protein interaction analysis, we demonstrate that in the moss Physcomitrella patens , phytochrome4 physically interacts with the splicing regulator heterogeneous nuclear ribonucleoprotein H1 (PphnRNP-H1) in the nucleus, a process dependent on red light. We show that PphnRNP-H1 is involved in red light-mediated phototropic responses in P. patens and that it binds with higher affinity to the splicing factor pre-mRNA-processing factor39-1 (PpPRP39-1) in the presence of red light-activated phytochromes. Furthermore, PpPRP39-1 associates with the core component of U1 small nuclear RNP in P. patens Genome-wide analyses demonstrated the involvement of both PphnRNP-H1 and PpPRP39-1 in light-mediated splicing regulation. Our results suggest that phytochromes target the early step of spliceosome assembly via a cascade of protein-protein interactions to control pre-mRNA splicing and photomorphogenic responses., (© 2019 American Society of Plant Biologists. All rights reserved.)

Ultraviolet (UV) irradiation from the sunlight is a major etiologic factor for premature skin aging. Long noncoding RNAs (lncRNAs) are involved in various biological processes, and their roles in UV irradiation-induced skin aging have recently been described. Previously, we found that the lncRNA RP11-670E13.6 was up-regulated and delayed cellular senescence in UVB-irradiated primary human dermal fibroblasts. Here, we performed further investigations of RP11-670E13.6 function. The results showed that this lncRNA directly bound to miR-663a and functioned as a sponge for miR-663a to modulate the derepression of Cdk4 and Cdk6, thereby delaying cellular senescence during UV irradiation-induced skin photoaging. Moreover, we found that RP11-670E13.6 may facilitate DNA damage repair by increasing ATM and γH2A.X levels. In addition, heterogeneous nuclear ribonucleoprotein H physically interacted with RP11-670E13.6 and blocked its expression. Collectively, our results suggested that the RP11-670E13.6/miR-663a / CDK4 and RP11-670E13.6/miR-663a / CDK6 axis, which may function as competitive endogenous RNA networks, played important roles in UVB-induced cellular senescence.

The splicing factor Sam68 is upregulated in many human cancers, including prostate cancer (PCa) where it promotes cell proliferation and survival. Nevertheless, in spite of its frequent upregulation in cancer, the mechanism(s) underlying its expression are largely unknown. Herein, bioinformatics analyses identified the promoter region of the Sam68 gene (KHDRBS1) and the proto-oncogenic transcription factor c-MYC as a key regulator of Sam68 expression. Upregulation of Sam68 and c-MYC correlate in PCa patients. c-MYC directly binds to and activates the Sam68 promoter. Furthermore, c-MYC affects productive splicing of the nascent Sam68 transcript by modulating the transcriptional elongation rate within the gene. Importantly, c-MYC-dependent expression of Sam68 is under the tight control of external cues, such as androgens and/or mitogens. These findings uncover an unexpected coordination of transcription and splicing of Sam68 by c-MYC, which may represent a key step in PCa tumorigenesis., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

MicroRNAs are small endogenous RNAs that pair and bind to sites on mRNAs to direct post-transcriptional repression. However, there is a possibility that microRNAs directly influence protein structure and activity, and this influence can be termed post-translational riboregulation. This conceptual review explores the literature on neurodegenerative disorders. Research on the association between neurodegeneration and RNA-repeat toxicity provides data that support a protein-RNA recognition code. For example, this code explains why hnRNP H and SFPQ proteins, which are involved in amyotrophic lateral sclerosis, are sequestered by the (GGGGCC)n repeat sequence. Similarly, it explains why MNBL proteins and (CTG)n repeats in RNA, which are involved in myotonic dystrophy, are sequestered into RNA foci. Using this code, proteins involved in diseases can be identified. A simple protein BLAST search of the human genome for amino acid repeats that correspond to the nucleotide repeats reveals new proteins among already known proteins that are involved in diseases. For example, the (CAG)n repeat sequence, when transcribed into possible peptide sequences, leads to the identification of PTCD3, Rem2, MESP2, SYPL2, WDR33, COL23A1, and others. After confirming this approach on RNA repeats, in the next step, the code was used in the opposite manner. Proteins that are involved in diseases were compared with microRNAs involved in those diseases. For example, a reasonable correspondence of microRNA 9 and 107 with amyloid-β-peptide (Aβ42) was identified. In the last step, a miRBase search for micro-nucleotides, obtained by transcription of a prion amino acid sequence, revealed new microRNAs and microRNAs that have previously been identified as involved in prion diseases. This concept provides a useful key for designing RNA or peptide probes.

We investigated the molecular mechanism(s) by which insulin prevents Bcl2-modifying factor (Bmf)-induced renal proximal tubular cell (RPTC) apoptosis and loss in diabetic mice. Transgenic mice (Tg) mice specifically overexpressing human BMF in RPTCs and non-Tg littermates were studied at 10 to 20 weeks of age. Non-diabetic littermates, diabetic Akita mice +/- insulin implant, Akita Tg mice specifically overexpressing heterogeneous nuclear ribonucleoprotein F (hnRNP F) in their RPTCs and immortalized rat renal proximal tubular cells (IRPTCs) were also studied. BMF-Tg mice exhibited higher systolic blood pressure, urinary albumin/creatinine ratio, RPTC apoptosis and urinary RPTCs than non-Tg mice. Insulin treatment in Akita mice and Akita mice overexpressing hnRNP F suppressed Bmf expression and RPTC apoptosis. In hyperinsulinemic-euglycemic wild type mice, renal Bmf expression was down-regulated with up-regulation of hnRNP F. In vitro, insulin inhibited high glucose-stimulation of Bmf expression, predominantly via p44/42 mitogen-activated protein kinase (MAPK) signaling. Transfection of p44/42 MAPK or hnRNP F small interfering RNA (siRNA) prevented insulin inhibition of Bmf expression. HnRNP F inhibited Bmf transcription via hnRNP F-responsive element in the Bmf promoter. Our results demonstrate that hnRNP F suppression of Bmf transcription is an important mechanism by which insulin protects RPTCs from apoptosis in diabetes.

Background: The biology function of antisense intronic long noncoding RNA (Ai-lncRNA) is still unknown. Meanwhile, cancer patients with paclitaxel resistance have limited therapeutic options in the clinic. However, the potential involvement of Ai-lncRNA in paclitaxel sensitivity remains unclear in human cancer., Methods: Whole transcriptome sequencing of 33 breast specimens was performed to identify Ai-lncRNA EGOT. Next, the role of EGOT in regulation of paclitaxel sensitivity was investigated. Moreover, the mechanism of EGOT enhancing autophagy sensitizes paclitaxel cytotoxicity via upregulation of ITPR1 expression by RNA-RNA and RNA-protein interactions was investigated in detail. Furthermore, upstream transcriptional regulation of EGOT expression was also investigated by co-immunoprecipitation and chromatin immunoprecipitation. Finally, clinical breast specimens in our cohort, TCGA and ICGC were applied to validate the role of EGOT in enhancing of paclitaxel sensitivity., Results: EGOT enhances autophagosome accumulation via the up-regulation of ITPR1 expression, thereby sensitizing cells to paclitaxel toxicity. Mechanistically, on one hand, EGOT upregulates ITPR1 levels via formation of a pre-ITPR1/EGOT dsRNA that induces pre-ITPR1 accumulation to increase ITPR1 protein expression in cis. On the other hand, EGOT recruits hnRNPH1 to enhance the alternative splicing of pre-ITPR1 in trans via two binding motifs in EGOT segment 2 (324-645 nucleotides) in exon 1. Moreover, EGOT is transcriptionally regulated by stress conditions. Finally, EGOT expression enhances paclitaxel sensitivity via assessment of cancer specimens., Conclusions: These findings broaden comprehensive understanding of the biology function of Ai-lncRNAs. Proper regulation of EGOT may be a novel synergistic strategy for enhancing paclitaxel sensitivity in cancer therapy.

piRNAs, small non-coding RNAs, were considered to be restricted to germline cells. Although they have recently been detected in somatic cells including neurons, it remains unclear how piRNA biogenesis is involved in neuronal diseases. We herein examined the possible roles of Aubergine (Aub), a Piwi-family protein (PIWI) responsible for piRNA biogenesis, in the neuronal disorders, using the Cabeza (Caz) knockdown Drosophila. Caz is a Drosophila homologue of FUS, which is one of the genes causing amyotrophic lateral sclerosis (ALS). Aub overexpression enhanced the mobility defects accompanied by anatomical defects in motoneurons at neuromuscular junctions induced by the neuron-specific knockdown of Caz. In order to elucidate the underlying mechanisms, we examined pre-piRNA and mature-size piRNA levels under these conditions. qRT-PCR and RNA-seq analyses revealed that the Caz knockdown increased pre-piRNA levels, but reduced mature-size piRNA levels in the central nervous system (CNS), suggesting a role in the pre-piRNAs production. Aub overexpression did not increase mature-size piRNA levels. These results suggest that the accumulated pre-piRNAs are abnormal abortive pre-piRNAs that cannot be further processed by slicers, including Aub. We also demonstrated a relationship between Caz and pre-piRNAs in the CNS by RNA immunoprecipitation. Aub overexpression induced the abnormal cytoplasmic localization of Caz. Based on these results, we propose a model in which Caz knockdown-induced abnormal pre-piRNAs associate with Caz, then translocate and accumulate in the cytoplasm, a process that may be mediated by Aub. The novel roles for Caz and Aub demonstrated herein using the Caz-knockdown fly will contribute to a deeper understanding of the pathogenesis of ALS., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Human IL12RB1 is an autosomal gene that is essential for mycobacterial disease resistance and T cell differentiation. Using primary human tissue and PBMCs, we demonstrate that lung and T cell IL12RB1 expression is allele-biased, and the extent to which cells express one IL12RB1 allele is unaffected by activation. Furthermore following its expression the IL12RB1 pre-mRNA is processed into either IL12RB1 Isoform 1 (IL12Rβ1, a positive regulator of IL12 responsiveness) or IL12RB1 Isoform 2 (a protein of heretofore unknown function). T cells choice to process pre-mRNA into Isoform 1 or Isoform 2 is controlled by intragenic competition of IL12RB1 exon 9-10 splicing with IL12RB1 exon 9b splicing, as well as an IL12RB1 exon 9b-associated polyadenylation site. Heterogeneous nuclear ribonucleoprotein H (hnRNP H) binds near the regulated polyadenylation site, but is not required for exon 9b polyadenylation. Finally, microRNA-mediated knockdown experiments demonstrated that IL12RB1 Isoform 2 promotes T cell IL12 responses. Collectively, our data support a model wherein tissue expression of human IL12RB1 is allele-biased and produces an hnRNP H-bound pre-mRNA, the processing of which generates a novel IL12 response regulator.

TAR DNA-binding protein-43 (TDP-43) is a ubiquitously expressed DNA-/RNA-binding protein that has been linked to numerous aspects of the mRNA life cycle. Similar to many RNA-binding proteins, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. Cell function and survival depend on the strict control of TDP-43 protein levels. TDP-43 has been identified as the major constituent of ubiquitin-positive inclusions in patients with Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Several observations argue for a pathogenic role of elevated TDP-43 levels in these disorders. Modulation of the cycle of TDP-43 production might therefore provide a new therapeutic strategy. Using a Drosophila model mimicking key features of the TDP-43 autoregulatory feedback loop, we identified CG42724 as a genetic modulator of TDP-43 production in vivo. We found that CG42724 protein influences qualitatively and quantitatively the TDP-43 mRNA transcript pattern. CG42724 overexpression promotes the production of transcripts that can be efficiently released into the cytoplasm for protein translation. Importantly, we showed that TCERG1, the human homolog of the Drosophila CG42724 protein, also caused an increase of TDP-43 protein steady-state levels in mammalian cells. Therefore, our data suggest the possibility that targeting TCERG1 could be therapeutic in TDP-43 proteinopathies.

The Hippo tumor-suppressor pathway regulates organ growth, cell proliferation, and stem cell biology. Defects in Hippo signaling and hyperactivation of its downstream effectors-Yorkie (Yki) in Drosophila and YAP/TAZ in mammals-result in progenitor cell expansion and overgrowth of multiple organs and contribute to cancer development. Deciphering the mechanisms that regulate the activity of the Hippo pathway is key to understanding its function and for therapeutic targeting. However, although the Hippo kinase cascade and several other upstream inputs have been identified, the mechanisms that regulate Yki/YAP/TAZ activity are still incompletely understood. To identify new regulators of Yki activity, we screened in Drosophila for suppressors of tissue overgrowth and Yki activation caused by overexpression of atypical protein kinase C (aPKC), a member of the apical cell polarity complex. In this screen, we identified mutations in the heterogeneous nuclear ribonucleoprotein Hrb27C that strongly suppressed the tissue defects induced by ectopic expression of aPKC. Hrb27C was required for aPKC-induced tissue growth and Yki target gene expression but did not affect general gene expression. Genetic and biochemical experiments showed that Hrb27C affects Yki phosphorylation. Other RNA-binding proteins known to interact with Hrb27C for mRNA transport in oocytes were also required for normal Yki activity, although they suppressed Yki output. Based on the known functions of Hrb27C, we conclude that Hrb27C-mediated control of mRNA splicing, localization, or translation is essential for coordinated activity of the Hippo pathway., Competing Interests: The authors declare no conflict of interest.

FUS (fused in sarcoma) proteinopathy is a group of neurodegenerative diseases characterized by the formation of inclusion bodies containing the FUS protein, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Previous studies show that mitochondrial damage is an important aspect of FUS proteinopathy. However, the molecular mechanisms by which FUS induces mitochondrial damage remain to be elucidated. Our biochemical and genetic experiments demonstrate that FUS interacts with the catalytic subunit of mitochondrial ATP synthase (ATP5B), disrupts the formation of ATP synthase complexes, and inhibits mitochondrial ATP synthesis. FUS expression activates the mitochondrial unfolded protein response (UPR mt ). Importantly, down-regulating expression of ATP5B or UPR mt genes in FUS transgenic flies ameliorates neurodegenerative phenotypes. Our data show that mitochondrial impairment is a critical early event in FUS proteinopathy, and provide insights into the pathogenic mechanism of FUS-induced neurodegeneration., Competing Interests: The authors declare no conflict of interest.

RNA binding proteins are a diverse class of proteins that regulate all aspects of RNA metabolism. Accumulating studies indicate that heterogeneous nuclear ribonucleoproteins are associated with cellular adaptations in response to drugs of abuse. We recently mapped and validated heterogeneous nuclear ribonucleoprotein H1 (Hnrnph1) as a quantitative trait gene underlying differential behavioral sensitivity to methamphetamine. The molecular mechanisms by which hnRNP H1 alters methamphetamine behaviors are unknown but could involve pre- and/or post-synaptic changes in protein localization and function. Methamphetamine initiates post-synaptic D1 dopamine receptor signaling indirectly by binding to pre-synaptic dopamine transporters and vesicular monoamine transporters of midbrain dopaminergic neurons which triggers reverse transport and accumulation of dopamine at the synapse. Here, we examined changes in neuronal localization of hnRNP H in primary rat cortical neurons that express dopamine receptors that can be modulated by the D1 or D2 dopamine receptor agonists SKF38393 and (-)-Quinpirole HCl, respectively. Basal immunostaining of hnRNP H was localized primarily to the nucleus. D1 dopamine receptor activation induced an increase in hnRNP H nuclear immunostaining as detected by immunocytochemistry with a C-domain directed antibody containing epitope near the glycine-rich domain but not with an N-domain specific antibody. Although there was no change in hnRNP H protein in the nucleus or cytoplasm, there was a decrease in Hnrnph1 transcript following D1 receptor stimulation. Taken together, these results suggest that D1 receptor activation increases availability of the hnRNP H C-terminal epitope, which could potentially reflect changes in protein-protein interactions. Thus, D1 receptor signaling could represent a key molecular post-synaptic event linking Hnrnph1 polymorphisms to drug-induced behavior., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Alternative splicing (AS) plays important roles in embryonic stem cell (ESC) differentiation. In this study, we first identified transcripts that display specific AS patterns in pluripotent human ESCs (hESCs) relative to differentiated cells. One of these encodes T-cell factor 3 (TCF3), a transcription factor that plays important roles in ESC differentiation. AS creates two TCF3 isoforms, E12 and E47, and we identified two related splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNPs) H1 and F (hnRNP H/F), that regulate TCF3 splicing. We found that hnRNP H/F levels are high in hESCs, leading to high E12 expression, but decrease during differentiation, switching splicing to produce elevated E47 levels. Importantly, hnRNP H/F knockdown not only recapitulated the switch in TCF3 AS but also destabilized hESC colonies and induced differentiation. Providing an explanation for this, we show that expression of known TCF3 target E-cadherin, critical for maintaining ESC pluripotency, is repressed by E47 but not by E12., (© 2018 Yamazaki et al.; Published by Cold Spring Harbor Laboratory Press.)

Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Here, we establish a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enables us to identify more than 1000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON∆165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations. Moreover, we highlight heterogeneous nuclear ribonucleoprotein H (HNRNPH) as a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, we pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11. Our results thereby offer insights into splicing regulation and the impact of mutations on alternative splicing in cancer.

Mutations in the nuclear localization signal of the RNA binding protein FUS cause both Frontotemporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS). These mutations result in a loss of FUS from the nucleus and the formation of FUS-containing cytoplasmic aggregates in patients. To better understand the role of cytoplasmic FUS mislocalization in the pathogenesis of ALS, we identified a population of cholinergic neurons in Drosophila that recapitulate these pathologic hallmarks. Expression of mutant FUS or the Drosophila homolog, Cabeza (Caz), in class IV dendritic arborization neurons results in cytoplasmic mislocalization and axonal transport to presynaptic terminals. Interestingly, overexpression of FUS or Caz causes the progressive loss of neuronal projections, reduction of synaptic mitochondria, and the appearance of large calcium transients within the synapse. Additionally, we find that overexpression of mutant but not wild type FUS results in a reduction in presynaptic Synaptotagmin, an integral component of the neurotransmitter release machinery, and mutant Caz specifically disrupts axonal transport and induces hyperexcitability. These results suggest that FUS/Caz overexpression disrupts neuronal function through multiple mechanisms, and that ALS-causing mutations impair the transport of synaptic vesicle proteins and induce hyperexcitability., (Copyright © 2018 Elsevier B.V. All rights reserved.)

FOXP3 promotes the development and function of regulatory T cells mainly through regulating the transcription of target genes. RNA alternative splicing has been implicated in a wide range of physiological and pathophysiological processes. We report here that FOXP3 associates with heterogeneous nuclear ribonucleoprotein (hnRNP) F through the exon 2-encoded region of FOXP3 and the second quasi-RNA recognition motif (qRRM) of hnRNPF. FOXP3 represses the ability of hnRNPF to bind to its target pre-mRNA and thus modulates RNA alternative splicing. Furthermore, overexpression of mouse hnRNPF in in vitro -differentiated regulatory T cells (Tregs) reduced their suppressive function. Thus, our studies identify a novel mechanism by which FOXP3 regulates mRNA alternative splicing to modulate the function of regulatory T cells., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Formation of the androgen receptor splicing variant 7 (AR-V7) is one of the major mechanisms by which resistance of prostate cancer to androgen deprivation therapy occurs. The histone demethylase JMJD1A (Jumonji domain containing 1A) functions as a key coactivator for AR by epigenetic regulation of H3K9 methylation marks. Here, we describe a role for JMJD1A in AR-V7 expression. While JMJD1A knockdown had no effect on full-length AR (AR-FL), it reduced AR-V7 levels in prostate cancer cells. Reexpression of AR-V7 in the JMJD1A-knockdown cells elevated expression of select AR targets and partially rescued prostate cancer cell growth in vitro and in vivo. The AR-V7 protein level correlated positively with JMJD1A in a subset of human prostate cancer specimens. Mechanistically, we found that JMJD1A promoted alternative splicing of AR-V7 through heterogeneous nuclear ribonucleoprotein F (HNRNPF), a splicing factor known to regulate exon inclusion. Knockdown of JMJD1A or HNRNPF inhibited splicing of AR-V7, but not AR-FL, in a minigene reporter assay. JMJD1A was found to interact with and promote the recruitment of HNRNPF to a cryptic exon 3b on AR pre-mRNA for the generation of AR-V7. Taken together, the role of JMJD1A in AR-FL coactivation and AR-V7 alternative splicing highlights JMJD1A as a potentially promising target for prostate cancer therapy., Competing Interests: The authors declare no conflict of interest.

The human ortholog of the Drosophila ecdysoneless gene (ECD) is required for embryonic development and cell-cycle progression; however, its role in cancer progression and metastasis remains unclear. Here, we found that ECD is frequently overexpressed in gastric cancer (GC), especially in metastatic GC, and is correlated with poor clinical outcomes in GC patients. Silencing ECD inhibited GC migration and invasion in vitro and metastasis in vivo, while ECD overexpression promoted GC migration and invasion. ECD promoted GC invasion and metastasis by protecting hnRNP F from ubiquitination and degradation. We identified ZFP91 as the E3 ubiquitin ligase that is responsible for hnRNP F ubiquitination at Lys 185 and proteasomal degradation. ECD competitively bound to hnRNP F via the N-terminal STG1 domain (13-383aa), preventing hnRNP F from interacting with ZFP91, thus preventing ZFP91-mediated hnRNP F ubiquitination and proteasomal degradation. Collectively, our findings indicate that ECD promotes cancer invasion and metastasis by preventing E3 ligase ZFP91-mediated hnRNP F ubiquitination and degradation, suggesting that ECD may be a marker for poor prognosis and a potential therapeutic target for GC patients.

Aggregation of fused in sarcoma (FUS) protein, and mutations in FUS gene, are causative to a range of neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. To gain insights into the molecular mechanism whereby FUS causes neurodegeneration, we generated transgenic Drosophila melanogaster overexpressing human FUS in the photoreceptor neurons, which exhibited mild retinal degeneration. Expression of familial ALS-mutant FUS aggravated the degeneration, which was associated with an increase in cytoplasmic localization of FUS. A carboxy-terminally truncated R495X mutant FUS also was localized in cytoplasm, whereas the degenerative phenotype was diminished. Double expression of R495X and wild-type FUS dramatically exacerbated degeneration, sequestrating wild-type FUS into cytoplasmic aggregates. Notably, replacement of all tyrosine residues within the low-complexity domain, which abolished self-assembly of FUS, completely eliminated the degenerative phenotypes. Taken together, we propose that self-assembly of FUS through its low-complexity domain contributes to FUS-induced neurodegeneration.

Ubiquitin-mediated protein degradation plays essential roles in proteostasis and is involved in the pathogenesis of neurodegenerative diseases in which ubiquitin-positive aberrant proteins accumulate. However, how such aberrant proteins are processed inside cells has not been fully explored. Here, we show that the product of CG5445 , a previously uncharacterized Drosophila gene, prevents the accumulation of aggregate-prone ubiquitinated proteins. We found that ubiquitin conjugates were associated with CG5445, the knockdown of which caused the accumulation of detergent-insoluble ubiquitinated proteins. Furthermore, CG5445 rescued eye degeneration caused by the amyotrophic lateral sclerosis (ALS)-linked mutant TAR DNA-binding protein of 43 kDa (TDP-43), which often forms ubiquitin-positive aggregates in cells through the capacity of CG5445 to bind to ubiquitin chains. Biochemically, CG5445 inhibited the accumulation of insoluble forms and promoted their clearance. Our results demonstrate a new possible mechanism by which cells maintain ubiquitinated aggregation-prone proteins in a soluble form to decrease their cytotoxicity until they are degraded., (Copyright © 2018 American Society for Microbiology.)

Myeloid cell leukemia-1 (Mcl -1) is one of the most frequently amplified genes in cancer, and its overexpression is associated with poor prognosis and drug resistance. As a member of the Bcl-2 family it is involved in the control of the mitochondrial (intrinsic) cell death pathway. Alternative splicing of the (Mcl-1) gene results in the expression of two functionally distinct proteins, the anti-apoptotic Mcl-1 L (exon 2 included) and the pro-apoptotic Mcl-1 S (exon 2 skipped). Our data shows that transfecting siRNAs that target hnRNP K and the hnRNP F/H family result in a switch in splicing towards the pro-apoptotic Mcl-1 S . Specific binding sites for these and other Mcl-1 splicing factors were investigated and identified by RNA immunoprecipitation and through construction of a Mcl-1 minigene construct. Moreover, this study shows up to a 30 fold change in the levels of Mcl-1 S can be achieved through double and triple knockdowns of the most significant RNA binding proteins involved in Mcl-1 splicing, as well as activation of the mitochondrial cell death pathway. Targeting the splicing process of Mcl-1 along with other apoptotic regulators provides an exciting new therapeutic target in cancer cells, and may provide a way to overcome therapy resistance.

Heterogeneous nuclear ribonucleoprotein F (hnRNP F) controls the expression of various genes through regulating the alternative splicing of pre-mRNAs in the nucleus. It uses three quasi-RNA recognition motifs (qRRMs) to recognize G-tract RNA which contains at least three consecutive guanines. The structures containing qRRMs of hnRNP F in complex with G-tract RNA have been determined by nuclear magnetic resonance (NMR) spectroscopy, shedding light on the recognition mechanism of qRRMs with G-tract RNA. However, knowledge of the recognition details is still lacking. To investigate how qRRMs specifically bind with G-tract RNA and how the mutations of any guanine to an adenine in the G-tract affect the binding, molecular dynamics simulations with binding free energy analysis were performed based on the NMR structure of qRRM2 in complex with G-tract RNA. Simulation results demonstrate that qRRM2 binds strongly with G-tract RNA, but any mutation of the G-tract leads to a drastic reduction of the binding free energy. Further comparisons of the energetic components reveal that van der Waals and non-polar interactions play essential roles in the binding between qRRM2 and G-tract RNA, but the interactions are weakened by the effect of RNA mutations. Structural and dynamical analyses indicate that when qRRM2 binds with G-tract RNA, both qRRM2 and G-tract maintain stabilized structures and dynamics; however, the stability is disrupted by the mutations of the G-tract. These results provide novel insights into the recognition mechanism of qRRM2 with G-tract RNA that are not elucidated by the NMR technique., (Copyright © 2017 Elsevier Inc. All rights reserved.)

It is generally thought that splicing factors regulate alternative splicing through binding to RNA consensus sequences. In addition to these linear motifs, RNA secondary structure is emerging as an important layer in splicing regulation. Here we demonstrate that RNA elements with G-quadruplex-forming capacity promote exon inclusion. Destroying G-quadruplex-forming capacity while keeping G tracts intact abrogates exon inclusion. Analysis of RNA-binding protein footprints revealed that G quadruplexes are enriched in heterogeneous nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated alternatively spliced exons in the human transcriptome. Moreover, hnRNPF regulates an epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a G-quadruplex-dependent manner, which results in inhibition of EMT. Mining breast cancer TCGA (The Cancer Genome Atlas) data sets, we demonstrate that hnRNPF negatively correlates with an EMT gene signature and positively correlates with patient survival. These data suggest a critical role for RNA G quadruplexes in regulating alternative splicing. Modulation of G-quadruplex structural integrity may control cellular processes important for tumor progression., (© 2017 Huang et al.; Published by Cold Spring Harbor Laboratory Press.)

Emerging evidence links exosomes to cancer progression by the trafficking of oncogenic factors and neoplastic reprogramming of stem cells. This necessitates identification and integration of functionally validated exosome-targeting therapeutics into current cancer management regimens. We employed quantitative high throughput screen on two libraries to identify exosome-targeting drugs; a commercially available collection of 1280 pharmacologically active compounds and a collection of 3300 clinically approved compounds. Manumycin-A (MA), a natural microbial metabolite, was identified as an inhibitor of exosome biogenesis and secretion by castration-resistant prostate cancer (CRPC) C4-2B, but not the normal RWPE-1, cells. While no effect was observed on cell growth, MA attenuated ESCRT-0 proteins Hrs, ALIX and Rab27a and exosome biogenesis and secretion by CRPC cells. The MA inhibitory effect is primarily mediated via targeted inhibition of the Ras/Raf/ERK1/2 signaling. The Ras-dependent MA suppression of exosome biogenesis and secretion is partly mediated by ERK-dependent inhibition of the oncogenic splicing factor hnRNP H1. Our findings suggest that MA is a potential drug candidate to suppress exosome biogenesis and secretion by CRPC cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We investigated the mechanism of heterogeneous nuclear ribonucleoprotein F (hnRNP F) renoprotective action in a type 2 diabetes (T2D) mouse model ( db / db ). Immortalized rat renal proximal tubular cells (IRPTCs) and kidneys from humans with T2D were also studied. The db / db mice developed hyperglycemia, oxidative stress, and nephropathy at age 20 weeks compared with their db / m littermates. These abnormalities, with the exception of hyperglycemia, were attenuated in db / db hnRNP F -transgenic (Tg) mice specifically overexpressing hnRNP F in their RPTCs. Sirtuin-1, Foxo3α, and catalase expression were significantly decreased in RPTCs from db / db mice and normalized in db / db hnRNP F -Tg mice. In vitro, hnRNP F overexpression stimulated Sirtuin-1 and Foxo3α with downregulation of acetylated p53 expression and prevented downregulation of Sirtuin-1 and Foxo3α expression in IRPTCs by high glucose plus palmitate. Transfection of Sirtuin-1 small interfering RNA prevented hnRNP F stimulation of Foxo3α and downregulation of acetylated p53 expression. hnRNP F stimulated Sirtuin-1 transcription via hnRNP F -responsive element in the Sirtuin-1 promoter. Human T2D kidneys exhibited more RPTC apoptosis and lower expression of hnRNP F, SIRTUIN-1, and FOXO3α than nondiabetic kidneys. Our results demonstrate that hnRNP F protects kidneys against oxidative stress and nephropathy via stimulation of Sirtuin-1 expression and signaling in diabetes., (© 2017 by the American Diabetes Association.)

The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, we used mutational analysis guided by crystal structures of Glo's RNA-binding domains and identified two discrete RNA-binding surfaces that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, we show that a subset of Glo's functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. Our findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Oxidative stress induces endogenous antioxidants via nuclear factor erythroid 2-related factor 2 (Nrf2), potentially preventing tissue injury. We investigated whether insulin affects renal Nrf2 expression in type 1 diabetes (T1D) and studied its underlying mechanism. Insulin normalized hyperglycemia, hypertension, oxidative stress, and renal injury; inhibited renal Nrf2 and angiotensinogen (Agt) gene expression; and upregulated heterogeneous nuclear ribonucleoprotein F and K (hnRNP F and hnRNP K) expression in Akita mice with T1D. In immortalized rat renal proximal tubular cells, insulin suppressed Nrf2 and Agt but stimulated hnRNP F and hnRNP K gene transcription in high glucose via p44/42 mitogen-activated protein kinase signaling. Transfection with small interfering RNAs of p44/42 MAPK, hnRNP F, or hnRNP K blocked insulin inhibition of Nrf2 gene transcription. Insulin curbed Nrf2 promoter activity via a specific DNA-responsive element that binds hnRNP F/K, and hnRNP F/K overexpression curtailed Nrf2 promoter activity. In hyperinsulinemic-euglycemic mice, renal Nrf2 and Agt expression was downregulated, whereas hnRNP F/K expression was upregulated. Thus, the beneficial actions of insulin in diabetic nephropathy appear to be mediated, in part, by suppressing renal Nrf2 and Agt gene transcription and preventing Nrf2 stimulation of Agt expression via hnRNP F/K. These findings identify hnRNP F/K and Nrf2 as potential therapeutic targets in diabetes., (Copyright © 2017 Endocrine Society.)