Heterogeneous nuclear ribonucleoprotein F deficiency in mouse podocyte promotes podocytopathy mediated by methyltransferase-like 14 nuclear translocation resulting in Sirtuin 1 gene inhibition.

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 FPOD KO) mice and control (HnRNP Ffl/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 FPOD KO mice were fertile and appeared grossly normal. However, male 20-week-old HnRNP FPOD KO than HnRNP Ffl/fl mice had increased urinary albumin/creatinine ratio, and lower expression of podocyte markers. ADR- or diabetic- HnRNP FPOD KO (vs. HnRNP Ffl/fl) mice had more severe podocytopathy. Moreover, methyltransferase-like 14 (Mettl14) gene expression was increased in podocytes from HnRNP FPOD KO mice, further enhanced in ADR- or diabetic-treated HnRNP FPOD 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.

Drosha-dependent microRNAs modulate FUS-mediated neurodegeneration in vivo.

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 RNA-binding proteins hnRNP H and F regulate splicing of a MYC-dependent HRAS exon in prostate cancer cells.

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.

Rett-like Phenotypes in HNRNPH2-Related Neurodevelopmental Disorder.

Rett Syndrome (RTT) is a neurodevelopmental disorder with a prevalence of 1:10,000 to 15,000 females worldwide. Classic Rett Syndrome presents in early childhood with a period of developmental regression, loss of purposeful hand skills along with hand stereotypies, gait abnormalities, and loss of acquired speech. Atypical RTT is diagnosed when a child shows some but not all the phenotypes of classic RTT, along with additional supporting criteria. Over 95% of classic RTT cases are attributed to pathogenic variants in Methyl-CpG Binding Protein 2 (MECP2), though additional genes have been implicated in other RTT cases, particularly those with the atypical RTT clinical picture. Other genetic etiologies have emerged with similar clinical characteristics to RTT Syndrome. Our team has characterized HNRNPH2-related neurodevelopmental disorder (HNRNPH2-RNDD) in 33 individuals associated with de novo pathogenic missense variants in the X-linked HNRNPH2 gene, characterized by developmental delay, intellectual disability, seizures, autistic-like features, and motor abnormalities. We sought to further characterize RTT clinical features in this group of individuals by using caregiver report. Twenty-six caregivers completed electronic surveys, with only 3 individuals having previously received an atypical RTT diagnosis, and no individuals with a typical RTT diagnosis. Caregivers reported a high number of behaviors and/or phenotypes consistent with RTT, including the major criteria of the syndrome, such as regression of developmental skills and abnormal gait. Based on the survey results, 12 individuals could meet the diagnostic clinical criteria for atypical RTT Syndrome. In summary, individuals with HNRNPH2-RNDD exhibit clinical characteristics that overlap with those of RTT, and therefore, HNRNPH2-RNDD, should be considered on the differential diagnosis list with this clinical picture.

The RNA-binding protein hnRNP F is required for the germinal center B cell response.

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.

N-nitrosamines-mediated downregulation of LncRNA-UCA1 induces carcinogenesis of esophageal squamous by regulating the alternative splicing of FGFR2.

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.

Encoded Conformational Dynamics of the HIV Splice Site A3 Regulatory Locus: Implications for Differential Binding of hnRNP Splicing Auxiliary Factors.

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.

The Drosophila hnRNP F/H homolog Glorund recruits dFMRP to inhibit nanos translation elongation.

Translational control of maternal mRNAs generates spatial and temporal patterns of protein expression necessary to begin animal development. Translational repression of unlocalized nanos (nos) mRNA in late-stage Drosophila oocytes by the hnRNP F/H homolog, Glorund (Glo), is important for embryonic body patterning. While previous work has suggested that repression occurs at both the translation initiation and elongation phases, the molecular mechanism by which Glo regulates nos translation remains elusive. Here, we have identified the Drosophila fragile X mental retardation protein, dFMRP, as a Glo interaction partner with links to the translational machinery. Using an oocyte-based in vitro translation system, we confirmed that Glo regulates both initiation and elongation of a nos translational reporter and showed that dFMRP specifically represses translation elongation and promotes ribosome stalling. Furthermore, we combined mutational analysis and in vivo and in vitro binding assays to show that Glo's qRRM2 domain specifically and directly interacts with dFMRP. Our findings suggest that Glo regulates nos translation elongation by recruiting dFMRP and that Glo's RNA-binding domains can also function as protein-protein interaction interfaces critical for its regulatory functions. Additionally, they reveal a mechanism for targeting dFMRP to specific transcripts.

Interaction of HnRNP F with the guanine-rich segments in viral antigenomic RNA enhances porcine reproductive and respiratory syndrome virus-2 replication.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein (HnRNP) F is a member of HnRNP family proteins that participate in splicing of cellular newly synthesized mRNAs by specifically recognizing tandem guanine-tracts (G-tracts) RNA sequences. Whether HnRNP F could recognize viral-derived tandem G-tracts and affect virus replication remain poorly defined. METHODS: The effect of HnRNP F on porcine reproductive and respiratory syndrome virus (PRRSV) propagation was evaluated by real-time PCR, western blotting, and plaque-forming unit assay. The association between HnRNP F and PRRSV guanine-rich segments (GRS) were analyzed by RNA pulldown and RNA immunoprecipitation. The expression pattern of HnRNP F was investigated by western blotting and nuclear and cytoplasmic fractionation. RESULTS: Knockdown of endogenous HnRNP F effectively blocks the synthesis of viral RNA and nucleocapsid (N) protein. Conversely, overexpression of porcine HnRNP F has the opposite effect. Moreover, RNA pulldown and RNA immunoprecipitation assays reveal that the qRMM1 and qRRM2 domains of HnRNP F recognize the GRS in PRRSV antigenomic RNA. Finally, HnRNP F is redistributed into the cytoplasm and forms a complex with guanine-quadruplex (G4) helicase DHX36 during PRRSV infection. CONCLUSIONS: These findings elucidate the potential functions of HnRNP F in regulating the proliferation of PRRSV and contribute to a better molecular understanding of host-PRRSV interactions.

Variant-specific effects define the phenotypic spectrum of HNRNPH2-associated neurodevelopmental disorders in males.

Bain type of X-linked syndromic intellectual developmental disorder, caused by pathogenic missense variants in HRNRPH2, was initially described in six female individuals affected by moderate-to-severe neurodevelopmental delay. Although it was initially postulated that the condition would not be compatible with life in males, several affected male individuals harboring pathogenic variants in HNRNPH2 have since been documented. However, functional in-vitro analyses of identified variants have not been performed and, therefore, possible genotype-phenotype correlations remain elusive. Here, we present eight male individuals, including a pair of monozygotic twins, harboring pathogenic or likely pathogenic HNRNPH2 variants. Notably, we present the first individuals harboring nonsense or frameshift variants who, similarly to an individual harboring a de novo p.(Arg29Cys) variant within the first quasi-RNA-recognition motif (qRRM), displayed mild developmental delay, and developed mostly autistic features and/or psychiatric co-morbidities. Additionally, we present two individuals harboring a recurrent de novo p.(Arg114Trp), within the second qRRM, who had a severe neurodevelopmental delay with seizures. Functional characterization of the three most common HNRNPH2 missense variants revealed dysfunctional nucleocytoplasmic shuttling of proteins harboring the p.(Arg206Gln) and p.(Pro209Leu) variants, located within the nuclear localization signal, whereas proteins with p.(Arg114Trp) showed reduced interaction with members of the large assembly of splicing regulators (LASR). Moreover, RNA-sequencing of primary fibroblasts of the individual harboring the p.(Arg114Trp) revealed substantial alterations in the regulation of alternative splicing along with global transcriptome changes. Thus, we further expand the clinical and variant spectrum in HNRNPH2-associated disease in males and provide novel molecular insights suggesting the disorder to be a spliceopathy on the molecular level.

Distinct roles of hnRNPH1 low-complexity domains in splicing and transcription.

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.

Cross-sectional, quantitative analysis of motor function in females with HNRNPH2-related disorder.

AIMS: To describe the gross motor function of individuals with HNRNPH2-related disorder (OMIM 300986, Mental Retardation, X-linked, Syndrome, Bain Type; MRXSB) and determine the associations between clinician-measured motor function and caregiver-reported mobility scores. METHODS: Developmental histories of 17 female participants with HNRNPH2-related disorder (mean age 11.2 years, range 2.7-37.1 years) with various genotypes within and adjacent to the nuclear localization sequence (NLS) were analyzed. Participants performed the Gross Motor Function Measure-88 (GMFM-88) and caregivers completed developmental histories and the Pediatric Evaluation of Disability Inventory-Computer Adaptive Test (PEDI-CAT). RESULTS: All participants had measurable and quantifiable motor impairments. A strong positive correlation between the clinician-measured GMFM-88 total score and the caregiver-reported PEDI-CAT mobility domain score was established. Motor deficits were noted more often in individuals who were nonverbal. The 2 participants with genotypes adjacent to the NLS appear to have milder motor phenotypes. CONCLUSIONS: The GMFM-88 and PEDI-CAT are useful and feasible measures of mobility in individuals with HNRNPH2-related disorders. Convergent validity was established between the clinician-measured GMFM-88 raw scores and caregiver-reported PEDI-CAT mobility domain scores. Factors including verbal status and genotype may impact motor abilities.

Long Noncoding RNA RP11-115N4.1 Promotes Inflammatory Responses by Interacting With HNRNPH3 and Enhancing the Transcription of HSP70 in Unexplained Recurrent Spontaneous Abortion.

Background: Unexplained recurrent spontaneous abortion (URSA) is a common pregnancy complication and the etiology is unknown. URSA-associated lncRNAs are expected to be potential biomarkers for diagnosis, and might be related to the disease pathogenesis. Objective: To investigate differential lncRNAs in peripheral blood of non-pregnant URSA patients and matched healthy control women and to explore the possible mechanism of differential lncRNAs leading to URSA. Methods: We profiled lncRNAs expression in peripheral blood from 5 non-pregnant URSA patients and 5 matched healthy control women by lncRNA microarray analysis. Functions of URSA-associated lncRNAs were further investigated in vitro. Results: RP11-115N4.1 was identified as the most differentially expressed lncRNA which was highly upregulated in peripheral blood of non-pregnant URSA patients (P = 3.63E-07, Fold change = 2.96), and this dysregulation was further validated in approximately 26.67% additional patients (4/15). RP11-115N4.1 expression was detected in both lymphocytes and monocytes of human peripheral blood, and in vitro overexpression of RP11-115N4.1 decreased cell proliferation in K562 cells significantly. Furthermore, heat-shock HSP70 genes (HSPA1A and HSPA1B) were found to be significantly upregulated upon RP11-115N4.1 overexpression by transcriptome analysis (HSPA1A (P = 4.39E-08, Fold change = 4.17), HSPA1B (P = 2.26E-06, Fold change = 2.99)). RNA pull down and RNA immunoprecipitation assay (RIP) analysis demonstrated that RP11-115N4.1 bound to HNRNPH3 protein directly, which in turn activate heat-shock proteins (HSP70) analyzed by protein-protein interaction and HNRNPH3 knockdown assays. Most importantly, the high expression of HSP70 was also verified in the serum of URSA patients and the supernatant of K562 cells with RP11-115N4.1 activation, and HSP70 in supernatant can exacerbate inflammatory responses in monocytes by inducing IL-6, IL-1ß, and TNF-α and inhibit the migration of trophoblast cells, which might associate with URSA. Conclusion: Our results demonstrated that the activation of RP11-115N4.1 can significantly increase the protein level of HSP70 via binding to HNRNPH3, which may modulate the immune responses and related to URSA. Moreover, RP11-115N4.1 may be a novel etiological biomarker and a new therapeutic target for URSA.

Deletion of heterogeneous nuclear ribonucleoprotein F in renal tubules downregulates SGLT2 expression and attenuates hyperfiltration and kidney injury in a mouse model of diabetes.

AIMS/HYPOTHESIS: We previously reported that renal tubule-specific deletion of heterogeneous nuclear ribonucleoprotein F (Hnrnpf) results in upregulation of renal angiotensinogen (Agt) and downregulation of sodium-glucose co-transporter 2 (Sglt2) in HnrnpfRT knockout (KO) mice. Non-diabetic HnrnpfRT KO mice develop hypertension, renal interstitial fibrosis and glycosuria with no renoprotective effect from downregulated Sglt2 expression. Here, we investigated the effect of renal tubular Hnrnpf deletion on hyperfiltration and kidney injury in Akita mice, a model of type 1 diabetes. METHODS: Akita HnrnpfRT KO mice were generated through crossbreeding tubule-specific (Pax8)-Cre mice with Akita floxed-Hnrnpf mice on a C57BL/6 background. Male non-diabetic control (Ctrl), Akita, and Akita HnrnpfRT KO mice were studied up to the age of 24 weeks (n = 8/group). RESULTS: Akita mice exhibited elevated systolic blood pressure as compared with Ctrl mice, which was significantly higher in Akita HnrnpfRT KO mice than Akita mice. Compared with Akita mice, Akita HnrnpfRT KO mice had lower blood glucose levels with increased urinary glucose excretion. Akita mice developed kidney hypertrophy, glomerular hyperfiltration (increased glomerular filtration rate), glomerulomegaly, mesangial expansion, podocyte foot process effacement, thickened glomerular basement membranes, renal interstitial fibrosis and increased albuminuria. These abnormalities were attenuated in Akita HnrnpfRT KO mice. Treatment of Akita HnrnpfRT KO mice with a selective A1 adenosine receptor inhibitor resulted in an increase in glomerular filtration rate. Renal Agt expression was elevated in Akita mice and further increased in Akita HnrnpfRT KO mice. In contrast, Sglt2 expression was increased in Akita and decreased in Akita HnrnpfRT KO mice. CONCLUSIONS/INTERPRETATION: The renoprotective effect of Sglt2 downregulation overcomes the renal injurious effect of Agt when these opposing factors coexist under diabetic conditions, at least partly via the activation of tubuloglomerular feedback.

Development and Pilot Screen of Novel High Content Assay for Down Regulators of Expression of Heterogenous Nuclear Ribonuclear Protein H2.

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late-stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al., Cell Physiol Biochem 2019;53:656-686). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H2 that can be used for melanoma therapy and research. METHODS: We established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. RESULTS: All plate-based QC parameters were acceptable: %CV = 6.7±0.3, S/B = 21±2.1, Z' = 0.75±0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis. CONCLUSION: We developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNPH1 and 2 in a large compound collection and, therefore, is amenable to a large-scale screening effort.

HnRNP F and hnRNP H1 regulate mRNA stability of amyloid precursor protein.

Amyloid precursor protein (APP) is a transmembrane protein that plays a crucial role in the production of amyloid-ß peptides. Any disruption in APP protein production, its mRNA decay rate or processing may result in abnormal production of amyloid-ß 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.

C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent nuclear accumulation of Staufen in neurons.

RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Here, we report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP Staufen may be a new pathological feature. We found that in Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS-but not with mutated endogenous NLS-potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/-), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/- is protective. stau+/- also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, our data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.

Amyotrophic Lateral Sclerosis Genes in Drosophila melanogaster.

Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset neurodegenerative disease characterized by the progressive degeneration of upper and lower motoneurons. Most ALS cases are sporadic but approximately 10% of ALS cases are due to inherited mutations in identified genes. ALS-causing mutations were identified in over 30 genes with superoxide dismutase-1 (SOD1), chromosome 9 open reading frame 72 (C9orf72), fused in sarcoma (FUS), and TAR DNA-binding protein (TARDBP, encoding TDP-43) being the most frequent. In the last few decades, Drosophila melanogaster emerged as a versatile model for studying neurodegenerative diseases, including ALS. In this review, we describe the different Drosophila ALS models that have been successfully used to decipher the cellular and molecular pathways associated with SOD1, C9orf72, FUS, and TDP-43. The study of the known fruit fly orthologs of these ALS-related genes yielded significant insights into cellular mechanisms and physiological functions. Moreover, genetic screening in tissue-specific gain-of-function mutants that mimic ALS-associated phenotypes identified disease-modifying genes. Here, we propose a comprehensive review on the Drosophila research focused on four ALS-linked genes that has revealed novel pathogenic mechanisms and identified potential therapeutic targets for future therapy.