Tumor Interferon Signaling Is Regulated by a lncRNA INCR1 Transcribed from the PD-L1 Locus.

Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.

hnRNPH1 establishes Sertoli-germ cell crosstalk through cooperation with PTBP1 and AR, and is essential for male fertility in mice.

Spermatogenesis depends on the crosstalk of Sertoli cells (SCs) and germ cells. However, the gene regulatory network establishing the communications between SCs and germ cells remains unclear. Here, we report that heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) in SCs is essential for the establishment of crosstalk between SCs and germ cells. Conditional knockout of hnRNPH1 in mouse SCs leads to compromised blood-testis barrier function, delayed meiotic progression, increased germ cell apoptosis, sloughing of germ cells and, eventually, infertility of mice. Mechanistically, we discovered that hnRNPH1 could interact with the splicing regulator PTBP1 in SCs to regulate the pre-mRNA alternative splicing of the target genes functionally related to cell adhesion. Interestingly, we also found hnRNPH1 could cooperate with the androgen receptor, one of the SC-specific transcription factors, to modulate the transcription level of a group of genes associated with the cell-cell junction and EGFR pathway by directly binding to the gene promoters. Collectively, our findings reveal a crucial role for hnRNPH1 in SCs during spermatogenesis and uncover a potential molecular regulatory network involving hnRNPH1 in establishing Sertoli-germ cell crosstalk.

MACC1 enhances an oncogenic RNA splicing of IRAK1 through interacting with HNRNPH1 in lung adenocarcinoma.

Dysregulation of alternative pre-mRNA splicing plays a critical role in the progression of cancers, yet the underlying molecular mechanisms remain largely unknown. It is reported that metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic and predictive marker in many types of cancers, including lung adenocarcinoma. Here, we reveal that the oncogene MACC1 specifically drives the progression of lung adenocarcinoma through its control over cancer-related splicing events. MACC1 depletion inhibits lung adenocarcinoma progression through triggering IRAK1 from its long isoform, IRAK1-L, to the shorter isoform, IRAK1-S. Mechanistically, MACC1 interacts with splicing factor HNRNPH1 to prevent the production of the short isoform of IRAK1 mRNA. Specifically, the interaction between MACC1 and HNRNPH1 relies on the involvement of MACC1's SH3 domain and HNRNPH1's GYR domain. Further, HNRNPH1 can interact with the pre-mRNA segment (comprising exon 11) of IRAK1, thereby bridging MACC1's regulation of IRAK1 splicing. Our research not only sheds light on the abnormal splicing regulation in cancer but also uncovers a hitherto unknown function of MACC1 in tumor progression, thereby presenting a novel potential therapeutic target for clinical treatment.

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.

A novel HNRNPH1::ERG rearrangement in aggressive acute myeloid leukemia.

FUS::ERG rearrangement is a recurrent abnormality seen in a subgroup of acute myeloid leukemia (AML) with a poor prognosis. We described here a novel HNRNPH1::ERG rearrangement in a de novo AML. The patient was unresponsive to routine chemotherapy and succumbed to the disease just 3 months after diagnosis. Two additional cases of AML with HNRNPH1::ERG rearrangement were discovered by searching a publicly available sequencing database. The three patients share several clinical phenotypes with the FUS::ERG rearranged AML, including high blast count at diagnosis, pediatric or young adult-onset, and poor overall survival. In addition, hnRNPH1 and FUS are both hnRNP family members, a group of RNA-binding proteins functioning in RNA metabolism and transport. Therefore, we suggest that patients with HNRNPH1::ERG or FUS::ERG rearrangement belong to the same distinct clinicopathologic subtype of AML, that is, AML with ERG rearrangement. Based on a previous study showing that FUS::ERG binds to the retinoic acid-responsive elements and that all-trans retinoic acid-induced cell differentiation of AML cells, we support the clinical evaluation of an APL-like therapeutic regimen for AML with ERG rearrangement.

Variants in HNRNPH1 are associated with high myopia in humans and ocular coloboma in zebrafish.

High myopia is one of the most common causes for blindness due to its associated complications. Genetic factor has been considered as the major cause for early-onset high myopia (eoHM), but exact genetic defects for most eoHM are yet to be identified. Through multi-step bioinformatics analysis of our in-house whole exome sequencing dataset from 6397 individuals, variants from 928 probands with eoHM were further compared with those from in-house controls as well as gnomAD database. The results showed that loss-of-function (LoF) variants in a novel gene HNRNPH1 were identified in two of 928 probands with eoHM but in none of 5469 probands with other eye conditions (p = 0.02). LoF variants in HNRNPH1 were extremely rare and intolerant, while two LoF variants in 928 eoHM were statistically higher than their frequency in gnomAD (p = 5.98 × 10-4 ). These two LoF variants, c.2dup/p.(M1?) and c.121dup/p.(Q41Pfs*20), were absent from existing database. Variants in HNRNPH1 have not been associated with any inherited eye disease before. Expression of HNRNPH1 was enriched in ganglion cell layer and inner nuclear layer in humans. Knockdown of hnrnph1 in zebrafish resulted in ocular coloboma. All these suggest that HNRNPH1 is potential contribution to eoHM when mutated.

SRSF3 and HNRNPH1 Regulate Radiation-Induced Alternative Splicing of Protein Arginine Methyltransferase 5 in Hepatocellular Carcinoma.

Protein arginine methyltransferase 5 (PRMT5) is an epigenetic regulator which has been proven to be a potential target for cancer therapy. We observed that PRMT5 underwent alternative splicing (AS) and generated a spliced isoform PRMT5-ISO5 in hepatocellular carcinoma (HCC) patients after radiotherapy. However, the regulatory mechanism and the clinical implications of IR-induced PRMT5 AS are unclear. This work revealed that serine and arginine rich splicing factor 3 (SRSF3) silencing increased PRMT5-ISO5 level, whereas heterogeneous nuclear ribonucleoprotein H 1 (HNRNPH1) silencing reduced it. Then, we found that SRSF3 and HNRNPH1 competitively combined with PRMT5 pre-mRNA located at the region around the 3'- splicing site on intron 2 and the alternative 3'- splicing site on exon 4. IR-induced SRSF3 downregulation led to an elevated level of PRMT5-ISO5, and exogenous expression of PRMT5-ISO5 enhanced cell radiosensitivity. Finally, we confirmed in vivo that IR induced the increased level of PRMT5-ISO5 which in turn enhanced tumor killing and regression, and liver-specific Prmt5 depletion reduced hepatic steatosis and delayed tumor progression of spontaneous HCC. In conclusion, our data uncover the competitive antagonistic interaction of SRSF3 and HNRNPH1 in regulating PRMT5 splicing induced by IR, providing potentially effective radiotherapy by modulating PRMT5 splicing against HCC.

A Mutation in Hnrnph1 That Decreases Methamphetamine-Induced Reinforcement, Reward, and Dopamine Release and Increases Synaptosomal hnRNP H and Mitochondrial Proteins.

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.

HNRNPH1-dependent splicing of a fusion oncogene reveals a targetable RNA G-quadruplex interaction.

The primary oncogenic event in ∼85% of Ewing sarcomas is a chromosomal translocation that generates a fusion oncogene encoding an aberrant transcription factor. The exact genomic breakpoints within the translocated genes, EWSR1 and FLI1, vary; however, in EWSR1, breakpoints typically occur within introns 7 or 8. We previously found that in Ewing sarcoma cells harboring EWSR1 intron 8 breakpoints, the RNA-binding protein HNRNPH1 facilitates a splicing event that excludes EWSR1 exon 8 from the EWS-FLI1 pre-mRNA to generate an in-frame mRNA. Here, we show that the processing of distinct EWS-FLI1 pre-mRNAs by HNRNPH1, but not other homologous family members, resembles alternative splicing of transcript variants of EWSR1 We demonstrate that HNRNPH1 recruitment is driven by guanine-rich sequences within EWSR1 exon 8 that have the potential to fold into RNA G-quadruplex structures. Critically, we demonstrate that an RNA mimetic of one of these G-quadruplexes modulates HNRNPH1 binding and induces a decrease in the growth of an EWSR1 exon 8 fusion-positive Ewing sarcoma cell line. Finally, we show that EWSR1 exon 8 fusion-positive cell lines are more sensitive to treatment with the pan-quadruplex binding molecule, pyridostatin (PDS), than EWSR1 exon 8 fusion-negative lines. Also, the treatment of EWSR1 exon 8 fusion-positive cells with PDS decreases EWS-FLI1 transcriptional activity, reversing the transcriptional deregulation driven by EWS-FLI1. Our findings illustrate that modulation of the alternative splicing of EWS-FLI1 pre-mRNA is a novel strategy for future therapeutics against the EWSR1 exon 8 containing fusion oncogenes present in a third of Ewing sarcoma.

hnRNPH1-MTR4 complex-mediated regulation of NEAT1v2 stability is critical for IL8 expression.

Many long noncoding RNAs (lncRNAs) are localized in the nucleus and play important roles in various biological processes, including cell proliferation, differentiation and antiviral response. Yet, it remains unclear how some nuclear lncRNAs are turned over. Here we show that the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) controls expression levels of NEAT1v2, a lncRNA involved in the formation of nuclear paraspeckles. hnRNPH1 associates, in an RNA-independent manner, with the RNA helicase MTR4/MTREX, an essential co-factor of the nuclear ribonucleolytic RNA exosome. hnRNPH1 localizes in nuclear speckles and depletion of hnRNPH1 enhances NEAT1v2-mediated expression of the IL8 mRNA, encoding a cytokine involved in the innate immune response. Taken together, our results indicate that the hnRNPH1-MTR4 linkage regulates IL8 expression through the degradation of NEAT1v2 RNA.

HNRNPH1-related syndromic intellectual disability: Seven additional cases suggestive of a distinct syndromic neurodevelopmental syndrome.

Pathogenic variants in HNRNPH1 were first reported in 2018. The reported individual, a 13 year old boy with a c.616C>T (p.R206W) variant in the HNRNPH1 gene, was noted to have overlapping symptoms with those observed in HNRNPH2-related X-linked intellectual disability, Bain type (MRXSB), specifically intellectual disability and dysmorphic features. While HNRNPH1 variants were initially proposed to represent an autosomal cause of MRXSB, we report an additional seven cases which identify phenotypic differences from MRXSB. Patients with HNRNPH1 pathogenic variants diagnosed via WES were identified using clinical networks and GeneMatcher. Features unique to individuals with HNRNPH1 variants include distinctive dysmorphic facial features; an increased incidence of congenital anomalies including cranial and brain abnormalities, genitourinary malformations, and palate abnormalities; increased incidence of ophthalmologic abnormalities; and a decreased incidence of epilepsy and cardiac defects compared to those with MRXSB. This suggests that pathogenic variants in HNRNPH1 result in a related, but distinct syndromic cause of intellectual disability from MRXSB, which we refer to as HNRNPH1-related syndromic intellectual disability.

Missense variants in the Arg206 residue of HNRNPH2: Further evidence of causality and expansion of the phenotype.

Missense variants in HNRNPH2 cause Bain type syndromic X-linked intellectual disability (XLID). To date, only six affected females and three affected males have been reported in the literature, and the phenotype has yet to be delineated in detail. Here, we report on a 35-year-old female with a novel de novo variant in HNRNPH2, providing further evidence that missense changes in the nuclear localization sequence cause Bain type XLID and that aminoacid 206 likely represents a mutational hotspot. We expand the phenotype of Bain type XLID to include breathing, sleep and movement disorders, cerebellar vermis hypoplasia, stereotypies, and hypersensitivity to noise. Our data indicate that the phenotype may be broader and more variable than initially reported, and suggest Rett syndrome as a possible differential diagnosis.

Burkitt lymphoma-related TCF3 mutations alter TCF3 alternative splicing by disrupting hnRNPH1 binding.

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.

Evidence for HNRNPH1 being another gene for Bain type syndromic mental retardation.

The HNRNPH2-associated disease (mental retardation, X-linked, syndromic, Bain type [MRXSB, MIM #300986]) is caused by de novo mutations in the X-linked HNRNPH2 gene. MRXSB has been described in six female patients with dysmorphy, developmental delay, intellectual disability, autism, hypotonia and seizures. The reported HNRNPH2 mutations were clustered in the small domain encoding nuclear localization signal; in particular, the p.Arg206Trp was found in four independent de novo events. HNRNPH1 is a conserved autosomal paralogue of HNRNPH2 with a similar function in regulation of pre-mRNAs splicing but so far it has not been associated with human disease. We describe a boy with a disease similar to MRXSB in whom a novel de novo mutation c.616C>T (p.Arg206Trp) in HNRNPH1 was found (ie, the exact paralogue of the recurrent HNRNPH2 mutation). We propose that defective function of HNRNPH2 and HNRNPH1 nuclear localization signal has similar clinical consequences. An important difference between the two diseases is that the HNRNPH1-associated syndrome may occur in boys (as in the case of our proband) which is well explained by the autosomal (chr5q35.3) rather than X-linked localization of the HNRNPH2 gene.

Predicting lymphoma prognosis using machine learning-based genes associated with lactylation.

BACKGROUND: Lactylation, a newly discovered PTM involving lactic acid, is linked to solid tumor proliferation and metastasis. Lymphoma patients exhibit high lactic acid levels, yet lactylation's role in lymphoma is underexplored. This study aimed to identify lactylation-related genes in lymphoma using tumor databases and assess their predictive value in patient prognosis through cell experiments and clinical specimens. METHODS: Using TCGA and GEO datasets, we analyzed the expression levels of lactylation-related genes in diffuse large B-cell lymphoma patients. We also evaluated the prognostic significance of lactylation gene risk scores, exploring their impact on drug sensitivity and tumor immune function. Key lactylation-affecting genes were identified and functionally validated through cell experiments and mouse in vivo experiments. Additionally, the relationship between lactylation and lymphoma prognosis was examined in clinical specimens. RESULTS: We identified 70 genes linked to diffuse large B-cell lymphoma prognosis from the lactylation-related gene set. Using clinical data and a COX regression algorithm, we developed an optimized lactylation Riskscore model. This model significantly correlated with prognosis and showed differences in immune cell infiltration, particularly macrophages. High-risk patients showed resistance to chemotherapy drugs but responded well to immunotherapy. HNRNPH1, a lactylation-related gene, influenced patient prognosis, apoptosis, cell cycle distribution in lymphoma cells, and tumor volume in mice. In lymphoma specimens, lactylation levels correlated with Bcl-2, C-myc, and P53 levels. CONCLUSIONS: Lactylation impacts diffuse large B-cell lymphoma prognosis, tumor immune function, and drug resistance. Our lactylation-based Riskscore model aids in patient stratification and treatment selection. HNRNPH1 regulates lactylation, thereby affecting patient prognosis.

Viral hijacking of hnRNPH1 unveils a G-quadruplex-driven mechanism of stress control.

Viral genomes are enriched with G-quadruplexes (G4s), non-canonical structures formed in DNA or RNA upon assembly of four guanine stretches into stacked quartets. Because of their critical roles, G4s are potential antiviral targets, yet their function remains largely unknown. Here, we characterize the formation and functions of a conserved G4 within the polymerase coding region of orthoflaviviruses of the Flaviviridae family. Using yellow fever virus, we determine that this G4 promotes viral replication and suppresses host stress responses via interactions with hnRNPH1, a host nuclear protein involved in RNA processing. G4 binding to hnRNPH1 causes its cytoplasmic retention with subsequent impacts on G4-containing tRNA fragments (tiRNAs) involved in stress-mediated reductions in translation. As a result, these host stress responses and associated antiviral effects are impaired. These data reveal that the interplay between hnRNPH1 and both host and viral G4 targets controls the integrated stress response and viral replication.

The E3 Ligase TRIM25 Impairs Apoptotic Cell Death in Colon Carcinoma Cells via Destabilization of Caspase-7 mRNA: A Possible Role of hnRNPH1.

Therapy resistance is still a major reason for treatment failure in colorectal cancer (CRC). Previously, we identified the E3 ubiquitin ligase TRIM25 as a novel suppressor of caspase-2 translation which contributes to the apoptosis resistance of CRC cells towards chemotherapeutic drugs. Here, we report the executioner caspase-7 as being a further target of TRIM25. The results from the gain- and loss-of-function approaches and the actinomycin D experiments indicate that TRIM25 attenuates caspase-7 expression mainly through a decrease in mRNA stability. The data from the RNA pulldown assays with immunoprecipitated TRIM25 truncations indicate a direct TRIM25 binding to caspase-7 mRNA, which is mediated by the PRY/SPRY domain, which is also known to be highly relevant for protein-protein interactions. By employing TRIM25 immunoprecipitation, we identified the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) as a novel TRIM25 binding protein with a functional impact on caspase-7 mRNA stability. Notably, the interaction of both proteins was highly sensitive to RNase A treatment and again depended on the PRY/SPRY domain, thus indicating an indirect interaction of both proteins which is achieved through a common RNA binding. Ubiquitin affinity chromatography showed that both proteins are targets of ubiquitin modification. Functionally, the ectopic expression of caspase-7 in CRC cells caused an increase in poly ADP-ribose polymerase (PARP) cleavage concomitant with a significant increase in apoptosis. Collectively, the negative regulation of caspase-7 by TRIM25, which is possibly executed by hnRNPH1, implies a novel survival mechanism underlying the chemotherapeutic drug resistance of CRC cells. The targeting of TRIM25 could therefore offer a promising strategy for the reduction in therapy resistance in CRC patients.

Acute Undifferentiated Leukemia With a Balanced t(5;10)(q35;p12) Resulting in Fusion of HNRNPH1 With MLLT10.

BACKGROUND/AIM: Acute undifferentiated leukemia (AUL) is leukemia which does not express lineage-specific antigens. Such cases are rare, accounting for 2.7% of all acute leukemia. The reported genetic information of AULs is limited to less than 100 cases with abnormal karyotypes and a few cases carrying chimeric genes or point mutation of a gene. We herein present the genetic findings and clinical features of a case of AUL. CASE REPORT: Bone marrow cells obtained at diagnosis from a 31-year-old patient with AUL were genetically investigated. G-Banding karyotyping revealed an abnormal karyotype: 45,X,-Y,t(5;10)(q35;p12),del(12)(p13)[12]/46,XY[5]. Array comparative genomic hybridization examination confirmed the del(12)(p13) seen by G-banding but also detected additional losses from 1q, 17q, Xp, and Xq corresponding to the deletion of approximately 150 genes from these five chromosome arms. RNA sequencing detected six HNRNPH1::MLLT10 and four MLLT10::HNRNPH1 chimeric transcripts, later confirmed by reverse-transcription polymerase chain reaction together with Sanger sequencing. Fluorescence in situ hybridization analysis showed the presence of HNRNPH1::MLLT10 and MLLT10::HNRNPH1 chimeric genes. CONCLUSION: To the best of our knowledge, this is the first AUL in which a balanced t(5;10)(q35;p12) leading to fusion of HNRNPH1 with MLLT10 has been detected. The relative leukemogenic importance of the chimeras and gene losses cannot be reliably assessed, but both mechanisms were probably important in the development of AUL.

A new Karyopherin-ß2 binding PY-NLS epitope of HNRNPH2 linked to neurodevelopmental disorders.

The HNRNPH2 proline-tyrosine nuclear localization signal (PY-NLS) is mutated in HNRNPH2-related X-linked neurodevelopmental disorder, causing the normally nuclear HNRNPH2 to accumulate in the cytoplasm. We solved the cryoelectron microscopy (cryo-EM) structure of Karyopherin-ß2/Transportin-1 bound to the HNRNPH2 PY-NLS to understand importin-NLS recognition and disruption in disease. HNRNPH2 206RPGPY210 is a typical R-X2-4-P-Y motif comprising PY-NLS epitopes 2 and 3, followed by an additional Karyopherin-ß2-binding epitope, we term epitope 4, at residues 211DRP213; no density is present for PY-NLS epitope 1. Disease variant mutations at epitopes 2-4 impair Karyopherin-ß2 binding and cause aberrant cytoplasmic accumulation in cells, emphasizing the role of nuclear import defect in disease. Sequence/structure analysis suggests that strong PY-NLS epitopes 4 are rare and thus far limited to close paralogs of HNRNPH2, HNRNPH1, and HNRNPF. Epitope 4-binidng hotspot Karyopherin-ß2 W373 corresponds to close paralog Karyopherin-ß2b/Transportin-2 W370, a pathological variant site in neurodevelopmental abnormalities, suggesting that Karyopherin-ß2b/Transportin-2-HNRNPH2/H1/F interactions may be compromised in the abnormalities.

The HNRNPF/H RNA binding proteins and disease.

The members of the HNRNPF/H family of heterogeneous nuclear RNA proteins-HNRNPF, HNRNPH1, HNRNPH2, HNRNPH3, and GRSF1, are critical regulators of RNA maturation. Documented functions of these proteins include regulating splicing, particularly alternative splicing, 5' capping and 3' polyadenylation of RNAs, and RNA export. The assignment of these proteins to the HNRNPF/H protein family members relates to differences in the amino acid composition of their RNA recognition motifs, which differ from those of other RNA binding proteins (RBPs). HNRNPF/H proteins typically bind RNA sequences enriched with guanine (G) residues, including sequences that, in the presence of a cation, have the potential to form higher-order G-quadruplex structures. The need to further investigate members of the HNRNPF/H family of RBPs has intensified with the recent descriptions of their involvement in several disease states, including the pediatric tumor Ewing sarcoma and the hematological malignancy mantle cell lymphoma; newly described groups of developmental syndromes; and neuronal-related disorders, including addictive behavior. Here, to foster the study of the HNRNPF/H family of RBPs, we discuss features of the genes encoding these proteins, their structures and functions, and emerging contributions to disease. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.