Histone Deacetylase 3 Unconventional Splicing Mediates Endothelial-to-mesenchymal Transition through Transforming Growth Factor β2

Histone deacetylase 3 (HDAC3) plays a critical role in the maintenance of endothelial integrity and other physiological processes. In this study, we demonstrated that HDAC3 undergoes unconventional splicing during stem cell differentiation. Four different splicing variants have been identified, designated as HD3α, -β, -γ, and -δ, respectively. HD3α was confirmed in stem cell differentiation by specific antibody against the sequences from intron 12. Immunofluorescence staining indicated that the HD3α isoform co-localized with CD31-positive or α-smooth muscle actin-positive cells at different developmental stages of mouse embryos. Overexpression of HD3α reprogrammed human aortic endothelial cells into mesenchymal cells featuring an endothelial-to-mesenchymal transition (EndMT) phenotype. HD3α directly interacts with HDAC3 and Akt1 and selectively activates transforming growth factor β2 (TGFβ2) secretion and cleavage. TGFβ2 functioned as an autocrine and/or paracrine EndMT factor. The HD3α-induced EndMT was both PI3K/Akt- and TGFβ2-dependent. This study provides the first evidence of the role of HDAC3 splicing in the maintenance of endothelial integrity.     

Vitamin A Metabolite,All-trans-retinoic Acid,Mediates Alternative Splicing of Protein Kinase C δVIII (PKCδVIII) Isoform via Splicing Factor SC35

Vitamin A metabolite, all-trans-retinoic acid (RA), induces cell growth, differentiation, and apoptosis and has an emerging role in gene regulation and alternative splicing events. Protein kinase Cδ (PKCδ), a serine/threonine kinase, has a role in cell proliferation, differentiation, and apoptosis. We reported an alternatively spliced variant of human PKCδ, PKCδVIII that functions as a pro-survival protein (1). RA regulates the splicing and expression of PKCδVIII via utilization of a downstream 5′ splice site of exon 10 on PKCδ pre-mRNA. Here, we further elucidate the molecular mechanisms involved in RA regulation of alternative splicing of PKCδVIII mRNA. Overexpression and knockdown of the splicing factor SC35 (i.e. SRp30b) indicated that it is involved in PKCδVIII alternative splicing. To identify the cis-elements involved in 5′ splice site selection we cloned a minigene, which included PKCδ exon 10 and its flanking introns in the pSPL3 splicing vector. Alternative 5′ splice site utilization in the minigene was promoted by RA. Further, co-transfection of SC35 with PKCδ minigene promoted selection of 5′ splice site II. Mutation of the SC35 binding site in the PKCδ minigene abolished RA-mediated utilization of 5′ splice splice II. RNA binding assays demonstrated that the enhancer element downstream of PKCδ exon 10 is a SC35 cis-element. We conclude that SC35 is pivotal in RA-mediated PKCδ pre-mRNA alternative splicing. This study demonstrates how a nutrient, vitamin A, via its metabolite RA, regulates alternative splicing and thereby gene expression of the pro-survival protein PKCδVIII.     

Regulation of vif mRNA Splicing by Human Immunodeficiency Virus Type 1 Requires 5′ Splice Site D2 and an Exonic Splicing Enhancer To Counteract Cellular Restriction Factor APOBEC3G

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5′ splice site (5′ss) D1, to the first splice acceptor, 3′ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5′ss D2, which is 50 nucleotides downstream of 3′ss A1; a GGGG silencer motif proximal to 5′ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5′ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3′ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5′ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5′ss D2 and ESEVif are required for production of sufficient Vif to allow efficient HIV-1 replication in cells expressing relatively high levels of A3G.Human immunodeficiency virus type 1 (HIV-1) Vif is a 23-kDa basic protein (4, 9) that is incorporated into virus particles during productive infection (8-10). Replication of HIV-1 in some T-cell lines is dependent on the expression of a functional Vif protein. Replication of Vif-deleted HIV-1 is restricted in these cells, which are termed nonpermissive, because of the presence of several host deaminases, the most important of which for HIV-1 replication is APOBEC3G (A3G) (25, 26). Human A3G is a single-stranded DNA deaminase that inhibits the replication of HIV-1 as well as other types of retroviruses and retrotransposons (5, 12, 17, 25, 32). HIV-1 Vif forms a complex with A3G and other cellular proteins to promote A3G ubiquitination, resulting in proteasomal degradation of A3G (1, 11, 14, 18, 26). Vif-deleted HIV-1 produced in the presence of A3G packages increased levels of A3G compared to those found in the wild type (WT) and has reduced infectivity in nonpermissive T-cell lines. This reduced infectivity in the absence of Vif has been correlated with the dC-to-dU hypermutation of newly synthesized minus-strand viral DNA by A3G (6, 13, 31, 32). However, other studies have shown that A3G is also able to restrict virus replication without hypermutating viral DNA (7, 19).It has previously been shown that the expression of Vif in infected cells is maintained at a relatively low level compared to levels of the other HIV-1 accessory proteins. One mechanism to explain this phenomenon is that Vif is degraded more rapidly than other accessory proteins by the proteasome (3). Another mechanism is that a relatively low level of vif mRNA is produced by alternative splicing (22). Alternative splicing of HIV-1 RNA results in the production of approximately 40 different mRNA species, which include three different mRNA size classes: 1.8-kb, completely spliced RNAs; 4-kb, incompletely spliced RNAs; and 9-kb, unspliced RNAs (Fig. ​(Fig.1A).1A). The 4-kb mRNA class encodes Vif, Vpr, Tat, Vpu, and Env, and the completely spliced, 1.8-kb mRNA class encodes Tat, Rev, and Nef. Unspliced viral RNA is both packaged into virions as genomic RNA and used as mRNA for Gag and Gag-Pol proteins (2, 27). As shown in Fig. ​Fig.1A,1A, four different 5′ splice donor sites (5′ss) and eight different 3′ splice acceptor sites (3′ss), which are highly conserved among group M HIV-1 strains, are used to produce alternatively spliced HIV-1 mRNAs at different levels in infected cells (22). The efficiencies with which these 5′ss and 3′ss are used are dependent on the presence of suboptimal cis splicing elements within the 5′ss and 3′ss themselves and more-distant elements, which include exonic splicing silencers, an intronic splicing silencer, and exonic splicing enhancers (ESE) (2, 15, 27).Open in a separate windowFIG. 1.HIV-1 splicing pattern and elements regulating vif mRNA splicing. (A) The conserved 5′ss (D1 to D4) and 3′ss (A1 to A7) located within the 9-kb HIV-1 genome are shown. Completely and incompletely spliced HIV-1 mRNAs (∼4 kb and ∼1.8 kb) are shown as open boxes. Spliced mRNAs are denoted by the translated open reading frame and by the exon content. The incompletely spliced mRNAs, denoted with an I, are differentiated from completely spliced mRNAs by inclusion of the intron between 5′ss D4 and 3′ss A7. Either one or both of the noncoding exons 2 and 3 (shown as gray-shaded exons) can be differentially included within all 1.8- and 4.0-kb mRNA species, with the exception of vif mRNA (1.2I) and vpr mRNA, which can include only exon 2 (1.[2].3I). LTR, long terminal repeat. (B) Three elements regulating vif mRNA splicing are shown: positively acting enhancer ESEVif, the 5′ss D2 (underlined), and a negatively acting G4 silencer motif. The locations of noncoding exon 2 and the start site for Vif protein synthesis are also shown. (C) HIV-1 5′ss D2-down mutants used in this study are shown. Sequences were aligned and compared with that of the consensus metazoan 5′ss. The sequence of the ESEVif mutant used in this study is also aligned and compared with the WT sequence. nt, nucleotides.HIV-1 Vif is translated from a low-abundance, incompletely spliced mRNA resulting from splicing of HIV-1 RNA between the major splice donor site (5′ss D1) and 3′ss A1. We have demonstrated that vif mRNA splicing is tightly regulated by the presence of multiple regulatory elements (Fig. ​(Fig.1B).1B). These include a highly conserved suboptimal 5′ss (5′ss D2) 50 nucleotides downstream from 3′ss A1, an SRp75-dependent ESE (ESEVif), and a GGGG silencer element proximal to 5′ss D2 (2). Mutations within the relatively weak 5′ss D2 that increase its homology to a consensus 5′ss result in increased inclusion of the noncoding 50-nucleotide exon defined by 3′ss A1 and 5′ss D2 (exon 2), increased single-spliced vif mRNA levels and Vif expression, and an excessive splicing phenotype in which virion production is reduced to 10 to 25% of that of the WT (16). Conversely, mutations that decrease the homology of 5′ss D2 to a consensus 5′ss inhibit splicing at 3′ss A1 and exon 2 inclusion into both incompletely and completely spliced HIV-1 mRNAs as well as decreased levels of vif mRNA. Virus production, however, is not significantly affected. Mutation of ESEVif resulted in a similar phenotype. We have shown previously that increased or decreased exon 2 inclusion into spliced mRNA does not affect the stability or expression of viral mRNAs (15). Based on these results, we hypothesized that the conserved suboptimal 5′ss D2, which together with 3′ss A1 defines exon 2, and ESEVif are necessary to maintain optimal levels of Gag and Gag-Pol required for HIV-1 replication while producing sufficient Vif to overcome the cellular restriction factor A3G (2). To further test this hypothesis, we examined a panel of HIV-1 mutants producing reduced levels of Vif under permissive and nonpermissive conditions. We also investigated the long-term replication capabilities of these mutant viruses in both permissive and nonpermissive A3G-expressing T-cell lines. Mutant viruses demonstrated increasing sensitivity to A3G, which is inversely proportional to their levels of Vif expression. Our results suggest that the reason 5′ss D2 and ESEVif exist in the HIV-1 genome is to regulate the levels of vif mRNA and Vif protein in infected cells.     

PTB-Associated Splicing Factor (PSF) Is a PPARγ-Binding Protein and Growth Regulator of Colon Cancer Cells

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor that plays an essential role in cell proliferation, apoptosis, and inflammation. It is over-expressed in many types of cancer, including colon, stomach, breast, and lung cancer, suggesting that regulation of PPARγ might affect cancer pathogenesis. Here, using a proteomic approach, we identify PTB-associated splicing factor (PSF) as a novel PPARγ-interacting protein and demonstrate that PSF is involved in several important regulatory steps of colon cancer cell proliferation. To investigate the relationship between PSF and PPARγ in colon cancer, we evaluated the effects of PSF expression in DLD-1 and HT-29 colon cancer cell lines, which express low and high levels of PPARγ, respectively PSF affected the ability of PPARγ to bind, and expression of PSF siRNA significantly suppressed the proliferation of colon cancer cells. Furthermore, PSF knockdown induced apoptosis via activation of caspase-3. Interestingly, DLD-1 cells were more susceptible to PSF knockdown-induced cell death than HT-29 cells. Our data suggest that PSF is an important regulator of cell death that plays critical roles in the survival and growth of colon cancer cells. The PSF-PPARγ axis may play a role in the control of colorectal carcinogenesis. Taken together, this study is the first to describe the effects of PSF on cell proliferation, tumor growth, and cell signaling associated with PPARγ.     

mTOR�� Splicing Isoform Promotes Cell Proliferation and Tumorigenesis

The mTOR (mammalian target of rapamycin) promotes growth in response to nutrients and growth factors and is deregulated in numerous pathologies, including cancer. The mechanisms by which mTOR senses and regulates energy metabolism and cell growth are relatively well understood, whereas the molecular events underlining how it mediates survival and proliferation remain to be elucidated. Here, we describe the existence of the mTOR splicing isoform, TORβ, which, in contrast to the full-length protein (mTORα), has the potential to regulate the G₁ phase of the cell cycle and to stimulate cell proliferation. mTORβ is an active protein kinase that mediates downstream signaling through complexing with Rictor and Raptor proteins. Remarkably, overexpression of mTORβ transforms immortal cells and is tumorigenic in nude mice and therefore could be a proto-oncogene.     

Splicing Factor Transformer-2β (Tra2β) Regulates the Expression of Regulator of G Protein Signaling 4 (RGS4) Gene and Is Induced by Morphine

Regulator of G protein signaling 4 (RGS4) is a critical modulator of G protein-coupled receptor (GPCR)-mediated signaling and plays important roles in many neural process and diseases. Particularly, drug-induced alteration in RGS4 protein levels is associated with acute and chronic effects of drugs of abuse. However, the precise mechanism underlying the regulation of RGS4 expression is largely unknown. Here, we demonstrated that the expression of RGS4 gene was subject to regulation by alternative splicing of the exon 6. Transformer-2β (Tra2β), an important splicing factor, bound to RGS4 mRNA and increased the relative level of RGS4-1 mRNA isoform by enhancing the inclusion of exon 6. Meanwhile, Tra2β increased the expression of full-length RGS4 protein. In rat brain, Tra2β was co-localized with RGS4 in multiple opioid action-related brain regions. In addition, the acute and chronic morphine treatment induced alteration in the expression level of Tra2β in rat locus coerulus (LC) in parallel to that of RGS4 proteins. It suggests that induction of this splicing factor may contribute to the change of RGS4 level elicited by morphine. Taken together, the results provide the evidence demonstrating the function of Tra2β as a new mediator in opioid-induced signaling pathway via regulating RGS4 expression.     

Novel Function and Intracellular Localization of Methionine Adenosyltransferase 2β Splicing Variants

Human methionine adenosyltransferase 2β (MAT2β) encodes for two major splicing variants, V1 and V2, which are differentially expressed in normal tissues. Both variants are induced in human liver cancer and positively regulate growth. The aim of this work was to identify interacting proteins of V1 and V2. His-tagged V1 and V2 were overexpressed in Rosetta pLysS cells, purified, and used in a pulldown assay to identify interacting proteins from human colon cancer cell line RKO cell lysates. The eluted lysates were subjected to Western blot and in solution proteomic analyses. HuR, an mRNA-binding protein known to stabilize the mRNA of several cyclins, was identified to interact with V1 and V2. Immunoprecipitation and Western blotting confirmed their interaction in both liver and colon cancer cells. These variant proteins are located in both nucleus and cytoplasm in liver and colon cancer cells and, when overexpressed, increased the cytoplasmic HuR content. This led to increased expression of cyclin D1 and cyclin A, known targets of HuR. When endogenous expression of V1 or V2 is reduced by small interference RNA, cytoplasmic HuR content fell and the expression of these HuR target genes also decreased. Knockdown of cyclin D1 or cyclin A blunted, whereas knockdown of HuR largely prevented, the ability of V1 or V2 overexpression to induce growth. In conclusion, MAT2β variants reside mostly in the nucleus and regulate HuR subcellular content to affect cell proliferation.     

Transforming Growth Factor β Regulates P-Body Formation through Induction of the mRNA Decay Factor Tristetraprolin

Transforming growth factor β (TGF-β) is a potent growth regulator and tumor suppressor in normal intestinal epithelium. Likewise, epithelial cell growth is controlled by rapid decay of growth-related mRNAs mediated through 3′ untranslated region (UTR) AU-rich element (ARE) motifs. We demonstrate that treatment of nontransformed intestinal epithelial cells with TGF-β inhibited ARE-mRNA expression. This effect of TGF-β was promoted through increased assembly of cytoplasmic RNA processing (P) bodies where ARE-mRNA localization was observed. P-body formation was dependent on TGF-β/Smad signaling, as Smad3 deletion abrogated P-body formation. In concert with increased P-body formation, TGF-β induced expression of the ARE-binding protein tristetraprolin (TTP), which colocalized to P bodies. TTP expression was necessary for TGF-β-dependent P-body formation and promoted growth inhibition by TGF-β. The significance of this was observed in vivo, where colonic epithelium deficient in TGF-β/Smad signaling or TTP expression showed attenuated P-body levels. These results provide new insight into TGF-β''s antiproliferative properties and identify TGF-β as a novel mRNA stability regulator in intestinal epithelium through its ability to promote TTP expression and subsequent P-body formation.     

Inhibition of Pre-mRNA Splicing by a Synthetic Blom7α-Interacting Small RNA

Originally the novel protein Blom7α was identified as novel pre-mRNA splicing factor that interacts with SNEV^Prp19/Pso4, an essential protein involved in extension of human endothelial cell life span, DNA damage repair, the ubiquitin-proteasome system, and pre-mRNA splicing. Blom7α belongs to the heteronuclear ribonucleoprotein K homology (KH) protein family, displaying 2 KH domains, a well conserved and widespread RNA-binding motif. In order to identify specific sequence binding motifs, we here used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) with a synthetic RNA library. Besides sequence motifs like (U/A)_1–4 C_2–6 (U/A)_1–5, we identified an AC-rich RNA-aptamer that we termed AK48 (Aptamer KH-binding 48), binding to Blom7α with high affinity. Addition of AK48 to pre-mRNA splicing reactions in vitro inhibited the formation of mature spliced mRNA and led to a slight accumulation of the H complex of the spliceosome. These results suggest that the RNA binding activity of Blom7α might be required for pre-mRNA splicing catalysis. The inhibition of in-vitro splicing by the small RNA AK48 indicates the potential use of small RNA molecules in targeting the spliceosome complex as a novel target for drug development.