Polypyrimidine tract-binding protein represses splicing of a fibroblast growth factor receptor-2 gene alternative exon through exon sequences

The fibroblast growth factor receptor (FGFR)-2 gene contains two mutually exclusive exons, K-SAM and BEK. We made a cell line designed to become drug-resistant on repression of BEK exon splicing. One drug-resistant derivative of this line carried an insertion within the BEK exon of a sequence containing at least two independent splicing silencers. One silencer was a pyrimidine-rich sequence, which markedly increased binding of polypyrimidine tract-binding protein to the BEK exon. The BEK exon binds to polypyrimidine tract-binding protein even in the silencer's absence. Several exonic pyrimidine runs are required for this binding, and they are also required for overexpression of polypyrimidine tract-binding protein to repress BEK exon splicing. These results show that binding of polypyrimidine tract-binding protein to exon sequences can repress splicing. In epithelial cells, the K-SAM exon is spliced in preference to the BEK exon, whose splicing is repressed. Mutation of the BEK exon pyrimidine runs decreases this repression. If this mutation is combined with the deletion of a sequence in the intron upstream from the BEK exon, a complete switch from K-SAM to BEK exon splicing ensues. Binding of polypyrimidine tract binding protein to the BEK exon thus participates in the K-SAM/BEK alternative splicing choice.     

Enhancer elements activate the weak 3' splice site of alpha-tropomyosin exon 2.

We have identified four purine-rich sequences that act as splicing enhancer elements to activate the weak 3' splice site of alpha-tropomyosin exon 2. These elements also activate the splicing of heterologous substrates containing weak 3' splice sites or mutated 5' splice sites. However, they are unique in that they can activate splicing whether they are placed in an upstream or downstream exon, and the two central elements can function regardless of their position relative to one another. The presence of excess RNAs containing these enhancers could effectively inhibit in vitro pre-mRNA splicing reactions in a substrate-dependent manner and, at lower concentrations of competitor RNA, the addition of SR proteins could relieve the inhibition. However, when extracts were depleted by incubation with biotinylated exon 2 RNAs followed by passage over streptavidin agarose, SR proteins were not sufficient to restore splicing. Instead, both SR proteins and fractions containing a 110-kD protein were necessary to rescue splicing. Using gel mobility shift assays, we show that formation of stable enhancer-specific complexes on alpha-tropomyosin exon 2 requires the presence of both SR proteins and the 110-kD protein. By analogy to the doublesex exon enhancer elements in Drosophila, our results suggest that assembly of mammalian exon enhancer complexes requires both SR and non-SR proteins to activate selection of weak splice sites.     

Role of an inhibitory pyrimidine element and polypyrimidine tract binding protein in repression of a regulated alpha-tropomyosin exon.

Splicing of exons 2 and 3 of a-tropomyosin (TM) involves mutually exclusive selection of either exon 3, which occurs in most cells, or of exon 2 in smooth muscle (SM) cells. The SM-specific selection of exon 2 results from the inhibition of exon 3. At least two essential cis-acting elements are required for exon 3 inhibition, the upstream and downstream regulatory elements (URE and DRE). These elements are essential for repression of TM exon 3 in SM cells, and also mediate a low level of repression of exon 3 in an in vitro 5' splice site competition assay in HeLa extracts. Here, we show that the DRE consists of at least two discrete components, a short region containing a number of UGC motifs, and an essential pyrimidine-rich tract (DY). We show that the specific sequence of the DY element is important and that DY is able to bind to factors in HeLa nuclear extracts that mediate a low background level of exon 3 skipping. Deletion of a sequence within DY identified as an optimal binding site for PTB impairs (1) regulation of splicing in vivo, (2) skipping of exon 3 in an in vitro 5' splice site competition, (3) the ability of DY competitors to affect the 5' splice site competition in vitro, and (4) binding of PTB to DY. Addition of recombinant PTB to in vitro splicing reactions is able to partially reverse the effects of the DY competitor RNA. The data are consistent with a model for regulation of TM splicing that involves the participation of both tissue-specific and general inhibitory factors and in which PTB plays a role in repressing both splice sites of exon 3.     

XGef is a CPEB-interacting protein involved in Xenopus oocyte maturation

XGef was isolated in a screen for proteins interacting with CPEB, a regulator of mRNA translation in early Xenopus development. XGef is a Rho-family guanine nucleotide exchange factor and activates Cdc42 in mammalian cells. Endogenous XGef (58 kDa) interacts with recombinant CPEB, and recombinant XGef interacts with endogenous CPEB in Xenopus oocytes. Injection of XGef antibodies into stage VI Xenopus oocytes blocks progesterone-induced oocyte maturation and prevents the polyadenylation and translation of c-mos mRNA; injection of XGef rescues these events. Overexpression of XGef in oocytes accelerates progesterone-induced oocyte maturation and the polyadenylation and translation of c-mos mRNA. Overexpression of a nucleotide exchange deficient version of XGef, which retains the ability to interact with CPEB, no longer accelerates oocyte maturation or Mos synthesis, suggesting that XGef exchange factor activity is required for the influence of overexpressed XGef on oocyte maturation. XGef overexpression continues to accelerate c-mos polyadenylation in the absence of Mos protein, but does not stimulate MAPK phosphorylation, MPF activation, or oocyte maturation, indicating that XGef may function through the Mos pathway to influence oocyte maturation. These results suggest that XGef may be an early acting component of the progesterone-induced oocyte maturation pathway.     

Polypyrimidine tract-binding protein enhances the internal ribosomal entry site-dependent translation of p27Kip1 mRNA and modulates transition from G1 to S phase

The p27(Kip1) protein plays a critical role in the regulation of cell proliferation through the inhibition of cyclin-dependent kinase activity. Translation of p27(Kip1) is directed by an internal ribosomal entry site (IRES) in the 5' nontranslated region of p27(Kip1) mRNA. Here, we report that polypyrimidine tract-binding protein (PTB) specifically enhances the IRES activity of p27(Kip1) mRNA through an interaction with the IRES element. We found that addition of PTB to an in vitro translation system and overexpression of PTB in 293T cells augmented the IRES activity of p27(Kip1) mRNA but that knockdown of PTB by introduction of PTB-specific small interfering RNAs (siRNAs) diminished the IRES activity of p27(Kip1) mRNA. Moreover, the G(1) phase in the cell cycle (which is maintained in part by p27(Kip1)) was shortened in cells depleted of PTB by siRNA knockdown. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced differentiation in HL60 cells was used to examine PTB-induced modulation of p27(Kip1) protein synthesis during differentiation. The IRES activity of p27(Kip1) mRNA in HL60 cells was increased by TPA treatment (with a concomitant increase in PTB protein levels), but the levels of p27(Kip1) mRNA remained unchanged. Together, these data suggest that PTB modulates cell cycle and differentiation, at least in part, by enhancing the IRES activity of p27(Kip1) mRNA.     

Zic3 is involved in the left-right specification of the Xenopus embryo

Establishment of left-right (L-R) asymmetry is fundamental to vertebrate development. Several genes involved in L-R asymmetry have been described. In the Xenopus embryo, Vg1/activin signals are implicated upstream of asymmetric nodal related 1 (Xnr1) and Pitx2 expression in L-R patterning. We report here that Zic3 carries the left-sided signal from the initial activin-like signal to determinative factors such as Pitx2. Overexpression of Zic3 on the right side of the embryo altered the orientation of heart and gut looping, concomitant with disturbed laterality of expression of Xnr1 and Pitx2, both of which are normally expressed in the left lateral plate mesoderm. The results indicate that Zic3 participates in the left-sided signaling upstream of Xnr1 and Pitx2. At early gastrula, Zic3 was expressed not only in presumptive neuroectoderm but also in mesoderm. Correspondingly, overexpression of Zic3 was effective in the L-R specification at the early gastrula stage, as revealed by a hormone-inducible Zic3 construct. The Zic3 expression in the mesoderm is induced by activin (beta) or Vg1, which are also involved in the left-sided signal in L-R specification. These findings suggest that an activin-like signal is a potent upstream activator of Zic3 that establishes the L-R axis. Furthermore, overexpression of the zinc-finger domain of Zic3 on the right side is sufficient to disturb the L-R axis, while overexpression of the N-terminal domain on the left side affects the laterality. These results suggest that Zic3 has at least two functionally important domains that play different roles and provide a molecular basis for human heterotaxy, which is an L-R pattern anomaly caused by a mutation in human ZIC3.     

5' exon requirement for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA and identification of a cryptic 5' splice site in the 3' exon

The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro. The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5' exon for the accuracy and efficiency of splicing. Three plasmids were constructed with nine, four and two nucleotides of the natural 5' exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3' exon. RNA was transcribed from these plasmids in vitro and tested for self-splicing activity. The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5' exon sequence is replaced with plasmid sequences. Accurate splicing persists even when only four nucleotides of the natural 5' exon sequence remain. When only two nucleotides of the natural exon remain, no ligated exons are observed. As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts. The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA. Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3' splice site. The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA. The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5' splice site. This observation led to a model where where the CUCU sequence in the 3' exon acts as a cryptic 5' splice site. The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3' exon. A small circular RNA was isolated and partially sequenced and found to support the model. So, a cryptic 5' splice site can function even if it is located downstream from the 3' splice site. Precursor RNA labeled at its 5' end, presumably by a GTP exchange reaction mediated by the IVS, is also described.     

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.     

RETRACTED: Polypyrimidine tract-binding protein regulates the cell cycle through IRES-dependent translation of CDK11p58 in mouse embryonic stem cells

This article has been retracted     

Polypyrimidine tract‐binding protein is required for the repression of gene expression by all‐trans retinoic acid

All‐trans retinoic acid is a key regulator of early development. High concentrations of retinoic acid interfere with differentiation and migration of neural crest cells. Here we report that a dinucleotide repeat in the cis‐element of Snail2 (previously known as Slug) gene plays a role in repression by all‐trans retinoic acid. We analyzed the cis‐acting regulatory regions of the Xenopus Snail2 gene, whose expression is repressed by all‐trans retinoic acid. The analysis identified a TG/CA repeat as a necessary element for the repression. By performing a yeast one‐hybrid screen, we found that a polypyrimidine tract‐binding protein (PTB), which is known to be a regulator of the alternative splicing of pre‐messenger RNA, binds to the TG/CA repeat. Overexpression and knockdown experiments for PTB in HEK293 cells and Xenopus embryos indicated that PTB is required for repression by retinoic acid. The green fluorescent protein‐PTB fusion protein was localized in the nucleus of 293T cells. In situ hybridization for PTB in Xenopus embryos showed that PTB is expressed at the regions including neural crest at the early stages. Our results indicate that PTB plays a role in the repression of gene expression by retinoic acid through binding to the TG/CA repeats.     

The ORF3 protein of porcine circovirus type 2 is involved in viral pathogenesis in vivo

Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome. We previously showed that a novel identified protein, ORF3, was not essential for PCV2 replication in cultured PK15 cells and played a major role in virus-induced apoptosis. To evaluate the role of the ORF3 protein in viral pathogenesis in vivo, we inoculated 8-week-old BALB/c mice that have been developed for PCV2 replication with ORF3-deficient mutant PCV2 (mPCV2). By 42 days postinoculation, all of the mice inoculated with the mPCV2, as well as with wild-type PCV2 (wPCV2), had seroconverted to PCV2 capsid antibody, and the mutant induced levels of PCV2 antibodies that were higher than those of the wPCV2. The PCV2 genomic copy numbers in serum were significantly higher (P<0.05) in the wPCV2-inoculated mice than in mice inoculated with the mPCV2. Also, the wPCV2 caused microscopic lesions characterized by lymphocyte depletion with histiocytic infiltration of lymphoid organs, but the mutant virus failed to induce any obvious pathological lesions. In situ hybridization and immunohistochemical analyses also showed that larger amounts of viral DNA and antigens were detected in the lymph nodes of the wPCV2-inoculated than mPCV2-inoculated mice. Furthermore, animals of the wPCV2-inoculated group showed significant downshifts of CD8(+) T-cell subsets of peripheral blood lymphocytes compared to the control mice (P<0.05) at various time points postinoculation. Also, the proportions of the CD4(+) and CD4(+) CD8(+) cells were significantly reduced in wPCV2-inoculated mice at some time points postinoculation. In contrast, there are some reductions in the proportions of these subsets in the mutant virus-inoculated mice, but the proportions do not decrease significantly. Taken together, these results demonstrate that the ORF3 protein is also dispensable for viral replication in vivo and that it plays an important role in viral pathogenesis.     

A cyclic adenosine 3',5'-monophosphate-dependent protein kinase C activation is involved in the hyperactivation of boar spermatozoa

An intracellular cAMP-PKA signaling plays a pivotal role in the expression of fertilizing ability in mammalian spermatozoa. The aim of this study is to disclose biological function of serine/threonine protein kinases that are activated by the action of the cAMP-PKA signaling in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog) at 38.5 degrees C up to 180 min, and then they were used for biochemical analyses of PKCs by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The incubation of spermatozoa with cBiMPS gradually activated PKCs in the connecting piece. The activation of sperm PKCs was accompanied with changes of their electrophoretic mobility by the PKA-mediated serine/threonine phosphorylation. In coincidence with the PKC activation, the cBiMPS-incubated spermatozoa were capable of exhibiting hyperactivation of flagellar movement. Moreover, the cBiMPS-induced hyperactivation was dramatically suppressed by the addition of either of specific PKC inhibitors (Ro-32-0432 and bisindolylmaleimide I) to the sperm suspensions. On the other hand, experiments using a calcium-deficient medium showed that the cBiMPS-induced hyperactivation of flagellar movement and activation of PKCs required the extracellular calcium. Based on the obtained data, we have concluded that a cAMP-PKA signaling can induce activation of calcium-sensitive PKCs that is leading to the hyperactivation of flagellar movement in boar spermatozoa. Moreover, the cAMP may have a unique role as the up-regulator of PKCs during the expression of fertilizing ability in boar spermatozoa.