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.

Heterogeneous ribonucleoprotein F regulates YAP expression via a G-tract in 3'UTR.

The Yes-associated protein (YAP) is a transcription coactivator that plays crucial roles in organ size control and tumorigenesis, and was demonstrated to be inhibited by the Hippo signaling pathway. To date, the molecular mechanisms regulating the expression of YAP in human cells remain unknown. In the present study, we found that hnRNP F and hnRNP U negatively regulate YAP expression. We also showed that downregulation of YAP expression by hnRNP F and hnRNP U was not at the transcriptional level. Knockdown of hnRNP F or hnRNP U increased YAP mRNA stability, suggesting the downregulation of YAP expression was by a post-transcriptional mechanism. A putative hnRNP F binding site was identified in the YAP 3'UTR at 685 to 698, and deletion of this putative hnRNP F element abolished the down-regulation effect of YAP mRNA stability by hnRNP F. Binding of the hnRNP F to the YAP 3'UTR was demonstrated by Cross-linked RNA Immunoprecipitation. mRNA stability is a possible secondary effect of alternative splicing or other nuclear process. Understanding the regulation of YAP expression would provide insights into the mechanisms underlying the maintenance of tissue size homeostasis and tumorigenesis.

FUS causes synaptic hyperexcitability in Drosophila dendritic arborization neurons.

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.

High-throughput analyses of hnRNP H1 dissects its multi-functional aspect.

hnRNPs are polyvalent RNA binding proteins that have been implicated in a range of regulatory roles including splicing, mRNA decay, translation, and miRNA metabolism. A variety of genome wide studies have taken advantage of methods like CLIP and RIP to identify the targets and binding sites of RNA binding proteins. However, due to the complex nature of RNA-binding proteins, these studies are incomplete without assays that characterize the impact of RBP binding on mRNA target expression. Here we used a suite of high-throughput approaches (RIP-Seq, iCLIP, RNA-Seq and shotgun proteomics) to provide a comprehensive view of hnRNP H1s ensemble of targets and its role in splicing, mRNA decay, and translation. The combination of RIP-Seq and iCLIP allowed us to identify a set of 1,086 high confidence target transcripts. Binding site motif analysis of these targets suggests the TGGG tetramer as a prevalent component of hnRNP H1 binding motif, with particular enrichment around intronic hnRNP H1 sites. Our analysis of the target transcripts and binding sites indicates that hnRNP H1s involvement in splicing is 2-fold: it directly affects a substantial number of splicing events, but also regulates the expression of major components of the splicing machinery and other RBPs with known roles in splicing regulation. The identified mRNA targets displayed function enrichment in MAPK signaling and ubiquitin mediated proteolysis, which might be main routes by which hnRNP H1 promotes tumorigenesis.

Post-transcriptional regulation of mu-opioid receptor: role of the RNA-binding proteins heterogeneous nuclear ribonucleoprotein H1 and F.

Classical opioids have been historically used for the treatment of pain and are among the most widely used drugs for both acute severe pain and long-term pain. Morphine and endogenous mu-opioid peptides exert their pharmacological actions mainly through the mu-opioid receptor (MOR). However, the expression of opioid receptor (OR) proteins is controlled by extensive transcriptional and post-transcriptional processing. Previously, the 5'-untranslated region (UTR) of the mouse MOR was found to be important for post-transcriptional regulation of the MOR gene in neuronal cells. To identify proteins binding to the 5'-UTR as potential regulators of the mouse MOR gene, affinity column chromatography using 5'-UTR-specific RNA oligonucleotides was performed using neuroblastoma NS20Y cells. Chromatography was followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified two heterogeneous ribonucleoproteins (hnRNPs) that bound to RNA sequences of interest: hnRNP H1 and hnRNP F. Binding of these proteins to the RNA region was M4-region sequence-specific as confirmed by Western-blot analysis and RNA supershift assay. Furthermore, a cotransfection study showed that the presence of hnRNP H1 and F resulted in repressed expression of the mouse MOR. Our data suggest that hnRNP H1 and F can function as repressors of MOR translation dependent on the M4 (-75 to -71 bp upstream of ATG) sequences. We demonstrate for the first time a role of hnRNPs as post-transcriptional repressors in MOR gene regulation.

Autoregulatory circuit of human rpL3 expression requires hnRNP H1, NPM and KHSRP.

Alternative pre-mRNA splicing (AS) is a major mechanism that allows proteomic variability in eukaryotic cells. However, many AS events result in mRNAs containing a premature termination codon, which are degraded by nonsense-mediated mRNA decay (NMD) pathway. We have previously demonstrated that human rpL3 autoregulates its expression through the association of AS with NMD. In fact, overexpression of rpL3 promotes downregulation of canonical splicing and upregulation of alternative splicing that produces an NMD-targeted mRNA isoform. The result of these events is a decreased production of rpL3. We have also identified heterogeneous nuclear ribonucleoprotein (hnRNP) H1 as a splicing factor involved in the regulation of rpL3 alternative splicing and identified its regulatory cis-elements within intron 3 transcript. Here, we report that NPM and KHSRP are two newly identified proteins involved in the regulation of rpL3 gene expression via AS-NMD. We demonstrate that hnRNP H1, KHSRP and NPM can be found associated, and present also in ribonucleoproteins (RNPs) including rpL3 and intron 3 RNA in vivo, and describe protein-protein and RNA-protein interactions. Moreover, our data provide an insight on the crucial role of hnRNP H1 in the regulation of the alternative splicing of the rpL3 gene.

Heterogeneous nuclear ribonucleoprotein H blocks MST2-mediated apoptosis in cancer cells by regulating A-Raf transcription.

A-Raf belongs to the family of oncogenic Raf kinases that are involved in mitogenic signaling by activating the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway. Low kinase activity of A-Raf toward MEK suggested that A-Raf might have alternative functions. Here, we show that A-Raf prevents cancer cell apoptosis contingent on the expression of the heterogeneous nuclear ribonucleoprotein H (hnRNP H) splice factor, which is required for the correct transcription and expression of a-raf. Apoptosis was prevented by A-Raf through sequestration and inactivation of the proapoptotic MST2 kinase. Small interfering RNA-mediated knockdown of hnRNP H or A-Raf resulted in MST2-dependent apoptosis. In contrast, enforced expression of either hnRNP H or A-Raf partially counteracted apoptosis induced by etoposide. In vivo expression studies of colon specimens corroborated the overexpression of hnRNP H in malignant tissues and its correlation with A-Raf levels. Our findings define a novel mechanism that is usurped in tumor cells to escape naturally imposed apoptotic signals.

hnRNP A1 and hnRNP H can collaborate to modulate 5' splice site selection.

The mammalian proteins hnRNP A1 and hnRNP H control many splicing decisions in viral and cellular primary transcripts. To explain some of these activities, we have proposed that self-interactions between bound proteins create an RNA loop that represses internal splice sites while simultaneously activating the external sites that are brought in closer proximity. Here we show that a variety of hnRNP H binding sites can affect 5' splice site selection. The addition of two sets of hnRNP H sites in a model pre-mRNA modulates 5' splice site selection cooperatively, consistent with the looping model. Notably, binding sites for hnRNP A1 and H on the same pre-mRNA can similarly collaborate to modulate 5' splice site selection. The C-terminal portion of hnRNP H that contains the glycine-rich domains (GRD) is essential for splicing activity, and it can be functionally replaced by the GRD of hnRNP A1. Finally, we used the bioluminescence resonance energy transfer (BRET) technology to document the existence of homotypic and heterotypic interactions between hnRNP H and hnRNP A1 in live cells. Overall, our study suggests that interactions between different hnRNP proteins bound to distinct locations on a pre-mRNA can change its conformation to affect splicing decisions.

Polypyrimidine tract binding protein induces human papillomavirus type 16 late gene expression by interfering with splicing inhibitory elements at the major late 5' splice site, SD3632.

We have initiated a screen for cellular factors that can induce human papillomavirus type 16 (HPV-16) late gene expression in human cancer cells. We report that the overexpression of polypyrimidine tract binding protein (PTB), also known as heterologous nuclear ribonucleoprotein I (hnRNP I), induces HPV-16 late gene expression in cells transfected with subgenomic HPV-16 plasmids or with full-length HPV-16 genomes and in persistently HPV-16-infected cells. In contrast, other hnRNPs such as hnRNP B1/A2, hnRNP F, and hnRNP Q do not induce HPV-16 late gene expression. PTB activates SD3632, the only 5' splice site on the HPV-16 genome that is used exclusively by late mRNAs. PTB interferes with splicing inhibitory sequences located immediately upstream and downstream of SD3632, thereby activating late gene expression. One AU-rich PTB-responsive element was mapped to a 198-nucleotide sequence located downstream of SD3632. The deletion of this element induced HPV-16 late gene expression in the absence of PTB. Our results suggest that the overexpression of PTB interferes with cellular factors that interact with the inhibitory sequences. One may speculate that an increase in PTB levels or a reduction in the concentration of a PTB antagonist is required for the activation of HPV-16 late gene expression during the viral life cycle.

Complex splicing control of the human Thrombopoietin gene by intronic G runs.

The human thrombopoietin (THPO) gene displays a series of alternative splicing events that provide valuable models for studying splicing mechanisms. The THPO region spanning exon 1-4 presents both alternative splicing of exon 2 and partial intron 2 (IVS2) retention following the activation of a cryptic 3' splice site 85 nt upstream of the authentic acceptor site. IVS2 is particularly rich in stretches of 3-5 guanosines (namely, G1-G10) and we have characterized the role of these elements in the processing of this intron. In vivo studies show that runs G7-G10 work in a combinatorial way to control the selection of the proper 3' splice site. In particular, the G7 element behaves as the splicing hub of intron 2 and its interaction with hnRNP H1 is critical for the splicing process. Removal of hnRNP H1 by RNA interference promoted the usage of the cryptic 3' splice site so providing functional evidence that this factor is involved in the selection of the authentic 3' splice site of THPO IVS2.

Activation of alpha-tropomyosin exon 2 is regulated by the SR protein 9G8 and heterogeneous nuclear ribonucleoproteins H and F.

The inclusion of exons 2 and 3 of alpha-tropomyosin is governed through tissue-specific alternative splicing. These exons are mutually exclusive, with exon 2 included in smooth muscle cells and exon 3 included in nearly all other cell types. Several cis-acting sequences contribute to this splicing decision: the branchpoints and pyrimidine tracts upstream of both exons, UGC-repeat elements flanking exon 3, and a series of purine-rich enhancers in exon 2. Previous work showed that proteins rich in serine-arginine (SR) dipeptides act through the exon 2 enhancers, but the specific proteins responsible for such activation remained unknown. Here we show that a 35-kDa member of the SR protein family, 9G8, can activate the splicing of alpha-tropomyosin exon 2. Using RNA affinity chromatography and cross-linking competition assays, we also demonstrate that the heterogeneous nuclear ribonucleoproteins (hnRNPs) H and F bind to and compete for the same elements. Overexpression of hnRNPs H and F blocked 9G8-mediated splicing both in vivo and in vitro, and small interfering RNA-directed depletion of H and F led to an increase in exon 2 splicing. These data suggest that the activation of exon 2 is dependent on the antagonistic activities of 9G8 and hnRNPs H and F.

Heterogeneous nuclear ribonucleoprotein H is required for optimal U11 small nuclear ribonucleoprotein binding to a retroviral RNA-processing control element: implications for U12-dependent RNA splicing.

An RNA-processing element from Rous sarcoma virus, the negative regulator of splicing (NRS), represses splicing to generate unspliced RNA that serves as mRNA and as genomic RNA for progeny virions and also promotes polyadenylation of the unspliced RNA. Integral to NRS function is the binding of U1 small nuclear ribonucleoprotein (snRNP), but its binding is controlled by U11 snRNP that binds to an overlapping site. U11 snRNP, the U1 counterpart for splicing of U12-dependent introns, binds the NRS remarkably well and requires G-rich elements just downstream of the consensus U11 binding site. We present evidence that heterogeneous nuclear ribonucleoprotein (hnRNP) H binds to the NRS G-rich elements and that hnRNP H is required for optimal U11 binding in vitro. It is further shown that hnRNP H (but not hnRNP F) can promote U11 binding and splicing from the NRS in vivo when tethered to the RNA as an MS2 fusion protein. Interestingly, 17% of the naturally occurring U12-dependent introns have at least two potential hnRNP H binding sites positioned similarly to the NRS. For two such introns from the SCN4A and P120 genes, we show that hnRNP H binds to each in a G-tract-dependent manner, that G-tract mutations strongly reduce splicing of minigene RNA, and that tethered hnRNP H restores splicing to mutant RNA. In support of a role for hnRNP H in both splicing pathways, hnRNP H antibodies co-precipitate U1 and U11 small nuclear ribonucleoproteins. These results indicate that hnRNP H is an auxiliary factor for U11 binding to the NRS and that, more generally, hnRNP H is a splicing factor for a subset of U12-dependent introns that harbor G-rich elements.

HnRNP H inhibits nuclear export of mRNA containing expanded CUG repeats and a distal branch point sequence.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disorder associated with a (CUG)n expansion in the 3'-untranslated region of the DMPK (DM1 protein kinase) gene. Mutant DMPK mRNAs containing the trinucleotide expansion are retained in the nucleus of DM1 cells and form discrete foci. The nuclear sequestration of RNA binding proteins and associated factors binding to the CUG expansions is believed to be responsible for several of the splicing defects observed in DM1 patients and could ultimately be linked to DM1 muscular pathogenesis. Several RNA binding proteins capable of co-localizing with the nuclear-retained mutant DMPK mRNAs have already been identified but none can account for the nuclear retention of the mutant transcripts. Here, we have employed a modified UV crosslinking assay to isolate proteins bound to mutant DMPK-derived RNA and have identified hnRNP H as an abundant candidate. The specific binding of hnRNP H requires not only a CUG repeat expansion but also a splicing branch point distal to the repeats. Suppression of hnRNP H expression by RNAi rescued nuclear retention of RNA with CUG repeat expansions. The identification of hnRNP H as a factor capable of binding and possibly modulating nuclear retention of mutant DMPK mRNA may prove to be an important link in our understanding of the molecular mechanisms that lead to DM1 pathogenesis.

A downstream polyadenylation element in human papillomavirus type 16 L2 encodes multiple GGG motifs and interacts with hnRNP H.

Production of human papillomavirus type 16 (HPV-16) virus particles is totally dependent on the differentiation-dependent induction of viral L1 and L2 late gene expression. The early polyadenylation signal in HPV-16 plays a major role in the switch from the early to the late, productive stage of the viral life cycle. Here, we show that the L2 coding region of HPV-16 contains RNA elements that are necessary for polyadenylation at the early polyadenylation signal. Consecutive mutations in six GGG motifs located 174 nucleotides downstream of the polyadenylation signal resulted in a gradual decrease in polyadenylation at the early polyadenylation signal. This caused read-through into the late region, followed by production of the late mRNAs encoding L1 and L2. Binding of hnRNP H to the various triple-G mutants correlated with functional activity of the HPV-16 early polyadenylation signal. In addition, the polyadenylation factor CStF-64 was also found to interact specifically with the region in L2 located 174 nucleotides downstream of the early polyadenylation signal. Staining of cervix epithelium with anti-hnRNP H-specific antiserum revealed high expression levels of hnRNP H in the lower layers of cervical epithelium and a loss of hnRNP H production in the superficial layers, supporting a model in which a differentiation-dependent down regulation of hnRNP H causes a decrease in HPV-16 early polyadenylation and an induction of late gene expression.