Genome-Wide Analysis of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) Binding to HIV-1 RNA Reveals a Key Role for hnRNP H1 in Alternative Viral mRNA Splicing.

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.

Molecular Dissection of FUS Points at Synergistic Effect of Low-Complexity Domains in Toxicity.

RNA-binding protein aggregation is a pathological hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To gain better insight into the molecular interactions underlying this process, we investigated FUS, which is mutated and aggregated in both ALS and FTLD. We generated a Drosophila model of FUS toxicity and identified a previously unrecognized synergistic effect between the N-terminal prion-like domain and the C-terminal arginine-rich domain to mediate toxicity. Although the prion-like domain is generally considered to mediate aggregation of FUS, we find that arginine residues in the C-terminal low-complexity domain are also required for maturation of FUS in cellular stress granules. These data highlight an important role for arginine-rich domains in the pathology of RNA-binding proteins.

Self-assembly of FUS through its low-complexity domain contributes to neurodegeneration.

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.

Molecular insights into the specific recognition between the RNA binding domain qRRM2 of hnRNP F and G-tract RNA: A molecular dynamics study.

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.

The Drosophila hnRNP F/H Homolog Glorund Uses Two Distinct RNA-Binding Modes to Diversify Target Recognition.

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.

Variants in HNRNPH2 on the X Chromosome Are Associated with a Neurodevelopmental Disorder in Females.

Via whole-exome sequencing, we identified six females from independent families with a common neurodevelopmental phenotype including developmental delay, intellectual disability, autism, hypotonia, and seizures, all with de novo predicted deleterious variants in the nuclear localization signal of Heterogeneous Nuclear Ribonucleoprotein H2, encoded by HNRNPH2, a gene located on the X chromosome. Many of the females also have seizures, psychiatric co-morbidities, and orthopedic, gastrointestinal, and growth problems as well as common dysmorphic facial features. HNRNPs are a large group of ubiquitous proteins that associate with pre-mRNAs in eukaryotic cells to produce a multitude of alternatively spliced mRNA products during development and play an important role in controlling gene expression. The failure to identify affected males, the severity of the neurodevelopmental phenotype in females, and the essential role of this gene suggests that male conceptuses with these variants may not be viable.

The hnRNP F/H homologue of Trypanosoma brucei is differentially expressed in the two life cycle stages of the parasite and regulates splicing and mRNA stability.

Trypanosomes are protozoan parasites that cycle between a mammalian host (bloodstream form) and an insect host, the Tsetse fly (procyclic stage). In trypanosomes, all mRNAs are trans-spliced as part of their maturation. Genome-wide analysis of trans-splicing indicates the existence of alternative trans-splicing, but little is known regarding RNA-binding proteins that participate in such regulation. In this study, we performed functional analysis of the Trypanosoma brucei heterogeneous nuclear ribonucleoproteins (hnRNP) F/H homologue, a protein known to regulate alternative splicing in metazoa. The hnRNP F/H is highly expressed in the bloodstream form of the parasite, but is also functional in the procyclic form. Transcriptome analyses of RNAi-silenced cells were used to deduce the RNA motif recognized by this protein. A purine rich motif, AAGAA, was enriched in both the regulatory regions flanking the 3' splice site and poly (A) sites of the regulated genes. The motif was further validated using mini-genes carrying wild-type and mutated sequences in the 3' and 5' UTRs, demonstrating the role of hnRNP F/H in mRNA stability and splicing. Biochemical studies confirmed the binding of the protein to this proposed site. The differential expression of the protein and its inverse effects on mRNA level in the two lifecycle stages demonstrate the role of hnRNP F/H in developmental regulation.

The high kinetic stability of a G-quadruplex limits hnRNP F qRRM3 binding to G-tract RNA.

The RNA binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) F is involved in telomeres maintenance and pre-mRNA processing, such as alternative splicing and polyadenylation. It specifically recognizes RNA containing three consecutive guanines (G-tracts) that have the potential to assemble into G-quadruplexes. We have proposed recently that hnRNP F could regulate alternative splicing by remodeling RNA structures, such as G-quadruplexes. However, the exact mechanism of hnRNP F binding to such RNA sequences remains unknown. Here, we have studied the binding of the third RNA binding domain of hnRNP F [quasi-RNA recognition motif 3 (qRRM3)] to G-tract RNA using isothermal titration calorimetry, circular dichroism and nuclear magnetic resonance spectroscopy. Our results show that qRRM3 binds specifically exclusively to single-stranded G-tracts (ssRNA), in contrast to previous reports stating that the G-quadruplex was recognized as well. Furthermore, we demonstrate that the pre-existent ssRNA/G-quadruplex equilibrium slows down the formation of the protein-ssRNA complex. Based on in vitro transcription assays, we show that the rate of the protein-RNA complex formation is faster than that of the G-quadruplex. We propose a model according to which hnRNP F could bind RNA co-transcriptionally and prevents G-quadruplex formation.

Differential RNA-binding activity of the hnRNP G protein correlated with the sex genotype in the amphibian oocyte.

A proteomic approach has enabled the identification of an orthologue of the splicing factor hnRNP G in the amphibians Xenopus tropicalis, Ambystoma mexicanum, Notophthalmus viridescens and Pleurodeles walt, which shows a specific RNA-binding affinity similar to that of the human hnRN G protein. Three isoforms of this protein with a differential binding affinity for a specific RNA probe were identified in the P. walt oocyte. In situ hybridization to lampbrush chromosomes of P. waltl revealed the presence of a family of hnRNP G genes, which were mapped on the Z and W chromosomes and one autosome. This indicates that the isoforms identified in this study are possibly encoded by a gene family linked to the evolution of sex chromosomes similarly to the hnRNP G/RBMX gene family in mammals.

Involvement of heterogeneous ribonucleoprotein F in the regulation of cell proliferation via the mammalian target of rapamycin/S6 kinase 2 pathway.

The S6 kinases (S6Ks) have been linked to a number of cellular processes, including translation, insulin metabolism, cell survival, and RNA splicing. Signaling via the phosphotidylinositol 3-kinase and mammalian target of rapamycin (mTOR) pathways is critical in regulating the activity and subcellular localization of S6Ks. To date, nuclear functions of both S6K isoforms, S6K1 and S6K2, are not well understood. To better understand S6K nuclear roles, we employed affinity purification of S6Ks from nuclear preparations followed by mass spectrometry analysis for the identification of novel binding partners. In this study, we report that in contrast to S6K1, the S6K2 isoform specifically associates with a number of RNA-binding proteins, including heterogeneous ribonucleoproteins (hnRNPs). We focused on studying the mechanism and physiological relevance of the S6K2 interaction with hnRNP F/H. Interestingly, the S6K2-hnRNP F/H interaction was not affected by mitogenic stimulation, whereas mTOR binding to hnRNP F/H was induced by serum stimulation. In addition, we define a new role of hnRNP F in driving cell proliferation, which could be partially attenuated by rapamycin treatment. S6K2-driven cell proliferation, on the other hand, could be blocked by small interfering RNA-mediated down-regulation of hnRNP F. These results demonstrate that the specific interaction between mTOR and S6K2 with hnRNPs is implicated in the regulation of cell proliferation.

Heterogeneous nuclear ribonucleoproteins H and F regulate the proteolipid protein/DM20 ratio by recruiting U1 small nuclear ribonucleoprotein through a complex array of G runs.

In this study, we sought to investigate the mechanism by which heterogeneous nuclear ribonucleoprotein (hnRNP) H and F regulate proteolipid protein (PLP)/DM20 alternative splicing. G-rich sequences in exon 3B, G1 and M2, are required for hnRNPH- and F-mediated regulation of the PLP/DM20 ratio and, when placed between competing 5' splice sites in an alpha-globin minigene, direct hnRNPH/F-regulated alternative splicing. In contrast, the activity of the intronic splicing enhancer, which is necessary for PLP splicing, is only modestly reduced by removal of hnRNPH/F both in PLP and alpha-globin gene context. In vivo, hnRNPH reversed reduction of DM20 splicing induced by hnRNPH/F removal, whereas hnRNPF had little effect. Tethering of the MS2-hnRNPH fusion protein downstream of the DM20 5' splice site increased DM20 splicing, whereas MS2-hnRNPF did not. Binding of U1 small nuclear ribonucleoprotein (U1snRNP) to DM20 is greatly impaired by mutation of G1 and M2 and depletion of hnRNPH and F. Reconstitution of hnRNPH/F-depleted extracts with either hnRNPH or F restored U1snRNP binding. We conclude that hnRNPH and F regulate DM20 splicing by recruiting U1snRNP and that hnRNPH plays a primary role in DM20 splice site selection in vivo. Decreased expression of hnRNPH/F in differentiated oligodendrocytes may regulate the PLP/DM20 ratio by reducing DM20 5' splice site recognition by U1snRNP.

The secondary structure of the human immunodeficiency virus type 1 transcript modulates viral splicing and infectivity.

Splicing of human immunodeficiency virus type 1 (HIV-1) exon 6D is regulated by the presence of a complex splicing regulatory element (SRE) sequence that interacts with the splicing factors hnRNP H and SC35. In this work, we show that, in the context of the wild-type viral sequence, hnRNP H acts as a repressor of exon 6D inclusion independent of its binding to the SRE. However, hnRNP H binding to the SRE acts as an enhancer of exon 6D inclusion in the presence of a critical T-to-C mutation. These seemingly contrasting functional properties of hnRNP H appear to be caused by a change in the RNA secondary structure induced by the T-to-C mutation that affects the spatial location of bound hnRNP H with respect to the exon 6D splicing determinants. We propose a new regulatory mechanism mediated by RNA folding that may also explain the dual properties of hnRNP H in splicing regulation.

hnRNP H and hnRNP F complex with Fox2 to silence fibroblast growth factor receptor 2 exon IIIc.

The heterogeneous nuclear ribonucleoprotein H (hnRNP) family of proteins has been shown to activate exon inclusion by binding intronic G triplets. Much less is known, however, about how hnRNP H and hnRNP F silence exons. In this study, we identify hnRNP H and hnRNP F proteins as being novel silencers of fibroblast growth factor receptor 2 exon IIIc. In cells that normally include this exon, we show that the overexpression of either hnRNP H1 or hnRNP F resulted in the dramatic silencing of exon IIIc. In cells that normally skip exon IIIc, skipping was disrupted when RNA interference was used to knock down both hnRNP H and hnRNP F. We show that an exonic GGG motif overlapped a critical exonic splicing enhancer, which was predicted to bind the SR protein ASF/SF2. Furthermore, the expression of ASF/SF2 reversed the silencing of exon IIIc caused by the expression of hnRNP H1. We show that hnRNP H and hnRNP F proteins are present in a complex with Fox2 and that the presence of Fox allows hnRNP H1 to better compete with ASF/SF2 for binding to exon IIIc. These results establish hnRNP H and hnRNP F as being repressors of exon inclusion and suggest that Fox proteins enhance their ability to antagonize ASF/SF2.

Members of the heterogeneous nuclear ribonucleoprotein H family activate splicing of an HIV-1 splicing substrate by promoting formation of ATP-dependent spliceosomal complexes.

In this study we analyzed members of the heterogeneous nuclear ribonucleoprotein (hnRNP) H protein family to determine their RNA binding specificities and roles in splicing regulation. Our data indicate that hnRNPs H, H', F, 2H9, and GRSF-1 bind the consensus motif DGGGD (where D is U, G, or A) and aggregate in a multimeric complex. We analyzed the role of these proteins in the splicing of a substrate derived from the HIV-1 tat gene and have shown that hnRNP H family members are required for efficient splicing of this substrate. The hnRNP H protein family members activated splicing of the viral substrate by promoting the formation of ATP-dependent spliceosomal complexes. Mutational analysis of six consensus motifs present within the intron of the substrate indicated that only one of these motifs acts as an intronic splicing enhancer.

(1)H, (13)C and (15)N resonance assignment of the first N-terminal RNA recognition motif (RRM) of the human heterogeneous nuclear ribonucleoprotein H (hnRNP H).

Human heterogeneous nuclear ribonucleoprotein H (hnRNP H) regulates alternative splicing of HIV-1 Tat pre-mRNA. The structure of the first N-terminal domain (residues 1-104) of hnRNP H was solved and its binding to an exonic splicing silencer (pESS2) studied. For this, all backbone and 85% of side-chain resonance frequencies were assigned.

NMR structure of the three quasi RNA recognition motifs (qRRMs) of human hnRNP F and interaction studies with Bcl-x G-tract RNA: a novel mode of RNA recognition.

The heterogeneous nuclear ribonucleoprotein (hnRNP) F belongs to the hnRNP H family involved in the regulation of alternative splicing and polyadenylation and specifically recognizes poly(G) sequences (G-tracts). In particular, hnRNP F binds a G-tract of the Bcl-x RNA and regulates its alternative splicing, leading to two isoforms, Bcl-x(S) and Bcl-x(L), with antagonist functions. In order to gain insight into G-tract recognition by hnRNP H members, we initiated an NMR study of human hnRNP F. We present the solution structure of the three quasi RNA recognition motifs (qRRMs) of hnRNP F and identify the residues that are important for the interaction with the Bcl-x RNA by NMR chemical shift perturbation and mutagenesis experiments. The three qRRMs exhibit the canonical betaalphabetabetaalphabeta RRM fold but additional secondary structure elements are present in the two N-terminal qRRMs of hnRNP F. We show that qRRM1 and qRRM2 but not qRRM3 are responsible for G-tract recognition and that the residues of qRRM1 and qRRM2 involved in G-tract interaction are not on the beta-sheet surface as observed for the classical RRM but are part of a short beta-hairpin and two adjacent loops. These regions define a novel interaction surface for RNA recognition by RRMs.

Heterogeneous nuclear ribonucleoprotein F/H proteins modulate the alternative splicing of the apoptotic mediator Bcl-x.

Bcl-x is a member of the Bcl-2 family of proteins that are key regulators of apoptosis. The Bcl-x pre-mRNA is alternatively spliced to yield Bcl-x(S) and Bcl-x(L), two isoforms that have been associated, respectively, with the promotion and the prevention of apoptosis. We have investigated some of the elements and factors involved in the production of these two splice variants. Deletion mutagenesis using a human Bcl-x minigene identifies two regions in exon 2 that modulate Bcl-x 5'-splice site selection in human HeLa cells. One region (B3) is located upstream of the Bcl-x(L) 5'-splice site and enforces Bcl-x(L) production in cells and splicing extracts. The other region (B2) is located immediately downstream of the 5'-splice site of Bcl-x(S) and favors Bcl-x(S) production in vivo and in vitro. A 30-nucleotide G-rich element (B2G) is responsible for the activity of the B2 element. We show that recombinant heterogeneous nuclear ribonucleoprotein (hnRNP) F and H proteins bind to B2G, and mutating the G stretches abolishes binding. Moreover, the addition of hnRNP F to a HeLa extract improved the production of the Bcl-x(S) variant in a manner that was dependent on the integrity of the G stretches in B2G. Consistent with the in vitro results, small interfering RNA-mediated RNA interference targeting hnRNP F and H decreased the Bcl-x(S)/Bcl-x(L) ratio of plasmid-derived and endogenously produced Bcl-x transcripts. Our results document a positive role for the hnRNP F/H proteins in the production of the proapoptotic regulator Bcl-x(S.).