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PubMed2001

Identification and characterization of RRM-containing coactivator activator (CoAA) as TRBP-interacting protein, and its splice variant as a coactivator modulator (CoAM).

We previously cloned and characterized thyroid hormone receptor-binding protein (TRBP) as an LXXLL-containing general coactivator that associates with coactivator complexes through its C terminus. To identify protein cofactors for TRBP action, a Sos-Ras yeast two-hybrid cDNA library was screened using TRBP C terminus as bait. A novel coactivator was isolated, coactivator activator (CoAA), that specifically associates with TRBP. Human CoAA is composed of 669 amino acids with a TRBP-interacting domain and two highly conserved RNA recognition motifs (RRM) commonly found in ribonucleoproteins. A splice variant lacking the entire TRBP-interacting domain was also isolated as a coactivator modulator (CoAM), a 156-amino acid protein containing only the RRM region. Human CoAA and CoAM mRNAs are encoded by a single gene located on chromosome 11q13; alternative splicing in exon 2 of CoAA yields CoAM. CoAA interacts with both TRBP and p300 in vitro. In addition, CoAA potently coactivates transcription mediated by multiple hormone-response elements and acts synergistically with TRBP and CREB-binding protein (CBP). Furthermore, CoAA is associated with the DNA-dependent protein kinase-poly(ADP-ribose) polymerase complex. Strikingly, CoAM, which lacks a TRBP-interacting domain, strongly represses both TRBP and CBP action suggesting that CoAM may modulate endogenous CoAA function. These data suggest that CoAA may serve as a mediator of coactivators such as TRBP in gene activation.

Alternative Splicing↗
PubMed2001

ZNF265--a novel spliceosomal protein able to induce alternative splicing.

The formation of the active spliceosome, its recruitment to active areas of transcription, and its role in pre-mRNA splicing depends on the association of a number of multifunctional serine/arginine-rich (SR) proteins. ZNF265 is an arginine/serine-rich (RS) domain containing zinc finger protein with conserved pre-mRNA splicing protein motifs. Here we show that ZNF265 immunoprecipitates from splicing extracts in association with mRNA, and that it is able to alter splicing patterns of Tra2-beta1 transcripts in a dose-dependent manner in HEK 293 cells. Yeast two-hybrid analysis and immunoprecipitation indicated interaction of ZNF265 with the essential splicing factor proteins U1-70K and U2AF(35). Confocal microscopy demonstrated colocalization of ZNF265 with the motor neuron gene product SMN, the snRNP protein U1-70K, the SR protein SC35, and with the transcriptosomal components p300 and YY1. Transfection of HT-1080 cells with ZNF265-EGFP fusion constructs showed that nuclear localization of ZNF265 required the RS domain. Alignment with other RS domain-containing proteins revealed a high degree of SR dipeptide conservation. These data show that ZNF265 functions as a novel component of the mRNA processing machinery.

Active Transport, Cell Nucleus↗
PubMed2001

Evidence for the decreased expression of the latent TGF-beta binding protein and its splice form in human liver tumours.

BACKGROUND: Recently, a splice form of the latent TGF-beta binding protein (LTBP-1) was identified in the liver lacking potential important sequences for matrix association and proteinase cleavage (LTBP-1D, -1delta53). For a better understanding of the unknown (patho)physiological role, the expression levels of LTBP-1D and LTBP-1 (full length) were investigated in normal and malignant human liver on the mRNA and protein level. METHODS: Normal liver (5 specimens), hepatocellular carcinoma (4 specimens) and fibrolamellar carcinoma (2 specimens) were examined by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry, for which specific antibodies were generated. RESULTS: The mRNA levels of LTBP-1/-1D in malignant liver tissues are decreased in comparison to normal liver--more so in HCC than in FLC. This finding was confirmed by a strong decrease of immunostaining of LTBP-1/-1D in neoplastic parenchymal cells of HCC and FLC. However, the intensity of LTBP-1 (full length) protein staining was increased in the extracellular matrix of the carcinomas, while LTBP-1D was not detectable in the matrix. CONCLUSION: Since TGF-beta is known to be over-expressed in liver tumours, the results suggest its enhanced synthesis without binding to LTBP-1. This probably influences the availability of bioactive TGF-beta in the tumour tissue. The missing matrix localization of LTBP-1D indicates that the hinge region containing a heparin-binding site is essential for the binding of LTBP-1 in the extracellular matrix. LTBP-1D may fulfil specific functions for the latency of matrix-unbound TGF-beta.

Carcinoma, Hepatocellular↗
PubMed1983

Infection of mouse teratocarcinoma stem cells with influenza virus: normal expression of proteins derived from spliced messenger RNAs.

Mouse teratocarcinoma stem cells before and after induction of differentiation have been infected with influenza virus. This virus is able to infect the cells irrespective of the state of differentiation. In addition, viral-encoded proteins that are known to be translated from spliced messenger RNAs are produced in normal amounts in the infected cells, and this expression is also independent of differentiation. These results indicate that both the stem cells and their differentiated progeny can efficiently splice the precursor RNAs for these influenza virus messengers.

Animals↗
PubMed2005

Chironomus tentans-repressor splicing factor represses SR protein function locally on pre-mRNA exons and is displaced at correct splice sites.

Chironomus tentans-repressor splicing factor (Ct-RSF) represses the activation of splicing by SR proteins in vitro. Ct-RSF colocalizes with the Ser-Arg-rich (SR) protein hrp45 in interchromatin granule clusters and coimmunoprecipitates with hrp45 in nuclear extracts. Ct-RSF and hrp45 can also interact directly in vitro. Ct-RSF and hrp45 are recruited together to transcribing genes and associate with growing pre-mRNAs. Ct-RSF and hrp45 colocalize at a large number of gene loci. Injection of anti-Ct-RSF antibodies into nuclei of living cells blocks association of both Ct-RSF and hrp45 with the growing pre-mRNA, whereas binding of U2 small nuclear ribonucleoprotein particle (snRNP) to the pre-mRNA is unaffected. On the intron-rich Balbiani ring (BR) 3 pre-mRNA, hrp45 as well as U1 and U2 snRNPs bind extensively, whereas relatively little Ct-RSF is present. In contrast, the BR1 and BR2 pre-mRNAs, dominated by exon sequences, bind relatively much Ct-RSF compared with hrp45 and snRNPs. Our data suggest that Ct-RSF represses SR protein function at exons and that the assembly of spliceosomes at authentic splice sites displaces Ct-RSF locally.

Animals↗
PubMed1991

Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators.

SF2 is a protein factor essential for constitutive pre-mRNA splicing in HeLa cell extracts and also activates proximal alternative 5' splice sites in a concentration-dependent manner. This latter property suggests a role for SF2 in preventing exon skipping, ensuring the accuracy of splicing, and regulating alternative splicing. Human SF2 cDNAs have been isolated and overexpressed in bacteria. Recombinant SF2 is active in splicing and stimulates proximal 5' splice sites. SF2 has a C-terminal region rich in arginine-serine dipeptides, similar to the RS domains of the U1 snRNP 70K polypeptide and the Drosophila alternative splicing regulators transformer, transformer-2, and suppressor-of-white-apricot. Like transformer-2 and 70K, SF2 contains an RNP-type RNA recognition motif.

Amino Acid Sequence↗
PubMed2000

ARP3beta, the gene encoding a new human actin-related protein, is alternatively spliced and predominantly expressed in brain neuronal cells.

A cDNA encoding a new human actin-related protein (ARP) was cloned. The corresponding protein is highly conserved with the previously described ARP3 protein, suggesting that it represents a second isoform of the human ARP3 subfamily. This new actin-related protein was subsequently named ARP3beta and represents the second example of multiple isoforms of an actin-related protein in a single organism. The ARP3beta gene was mapped to chromosome band 7q34, centromeric to Sonic Hedgehog. Gene structure analysis revealed that at least part of the observed ARP3beta mRNA heterogeneity is caused by alternative splicing resulting in exon skipping. Transcripts produced after exon 2 skipping are predicted to encode truncated products, whose functionality is still unclear. An ARP3beta pseudogene was detected on chromosome 2p11 by database searching. Several ARP3beta mRNA species were detected by Northern blotting and their abundance varied importantly among tissues: the highest expression levels were detected in fetal and adult brain, whereas lower levels were observed in liver, muscle and pancreas. In contrast, ARP3 mRNAs were detected in all tissues tested. Using in situ hybridization, the expression of ARP3beta in brain was shown to be restricted to neurons and epithelial cells from choroid plexus. This suggests a specific function for ARP3beta in the physiology of the development and/or maintenance of distinct subsets of nerve cells.

Actin-Related Protein 3↗
PubMed2002

Repression of transcription and interference with DNA binding of TATA-binding protein by C-terminal alternatively spliced p53.

The protein encoded by C-terminal alternatively spliced p53 mRNA (p53as) has been shown previously to occur naturally in mouse cells and to bind sequence-specifically to DNA more efficiently than p53 (p53r, regular form). In the current study, p53as and p53r proteins ectopically expressed in p53-deficient cells each transactivated reporter plasmids containing p53 binding sites. However, p53as consistently was more efficient in transcriptional repression of promoters lacking p53 binding sites and in concentration-dependent repression of the p21(WAF1/Cip-l/Sdi) promoter sequence. The p53as protein, like p53r, associated with TATA-binding protein (TBP), indicating that this interaction does not require the last 26 amino acids of p53. Consistent with its stronger repression effects, p53as interfered with TBP binding to a TATA-containing DNA sequence more efficiently than p53r protein. Taken together, these in vitro and in vivo results demonstrate a novel role in transcriptional repression for a naturally occurring C-terminal variant form of mouse p53 protein associated with differences in DNA binding properties and interference with transcription factor binding.

Alternative Splicing↗
PubMed1994

The lamin B receptor-associated protein p34 shares sequence homology and antigenic determinants with the splicing factor 2-associated protein p32.

The lamin B receptor (p58) is an inner nuclear membrane protein that forms an in vivo complex with the nuclear lamins, a nuclear envelope kinase, and two other nuclear proteins with apparent M(r) of 18,000 (p18) and 34,000 (p34). We now report the isolation of p34 by partial dissociation of the immunoaffinity-purified p58 protein complex. Determination of the N-terminal amino acid sequence of purified p34 shows that this polypeptide is homologous to p32, a splicing factor 2 (SF2)-associated protein. The relatedness between p34 and p32 can be further established by showing that antibodies raised against N- and C-terminal peptides of p32 cross-react with purified p34. As the amino acid sequence of p58 contains an arginine/serine (RS)-rich region similar to the RS-rich region found in SF 2, we speculate that these domains provide binding sites for p34 and that this protein may be a linker between the nuclear membrane and intranuclear spliceosomal substructures.

Amino Acid Sequence↗
PubMed2005

Protein modularity of alternatively spliced exons is associated with tissue-specific regulation of alternative splicing.

Recent comparative genomic analysis of alternative splicing has shown that protein modularity is an important criterion for functional alternative splicing events. Exons that are alternatively spliced in multiple organisms are much more likely to be an exact multiple of 3 nt in length, representing a class of "modular" exons that can be inserted or removed from the transcripts without affecting the rest of the protein. To understand the precise roles of these modular exons, in this paper we have analyzed microarray data for 3,126 alternatively spliced exons across ten mouse tissues generated by Pan and coworkers. We show that modular exons are strongly associated with tissue-specific regulation of alternative splicing. Exons that are alternatively spliced at uniformly high transcript inclusion levels or uniformly low levels show no preference for protein modularity. In contrast, alternatively spliced exons with dramatic changes of inclusion levels across mouse tissues (referred to as "tissue-switched" exons) are both strikingly biased to be modular and are strongly conserved between human and mouse. The analysis of different subsets of tissue-switched exons shows that the increased protein modularity cannot be explained by the overall exon inclusion level, but is specifically associated with tissue-switched alternative splicing.

Alternative Splicing↗
PubMed1996

In vitro mutagenesis of the mitochondrial leucyl tRNA synthetase of Saccharomyces cerevisiae shows that the suppressor activity of the mutant proteins is related to the splicing function of the wild-type protein.

The NAM2 gene of Saccharomyces cerevisiae encodes the mitochondrial leucyl tRNA synthetase (mLRS), which is necessary for the excision of the fourth intron of the mitochondrial cytb gene (bI4) and the fourth intron of the mitochondrial coxI gene (aI4), as well as for mitochondrial protein synthesis. Some dominant mutant alleles of the gene are able to suppress mutations that inactivate the bI4 maturase, which is essential for the excision of the introns aI4 and bI4. Here we report mutagenesis studies which focus on the splicing and suppressor functions of the protein. Small deletions in the C-terminal region of the protein preferentially reduce the splicing, but not the synthetase activity; and all the C-terminal deletions tested abolish the suppressor activity. Mutations which increase the volume of the residue at position 240 in the wild-type mLRS without introducing a charge, lead to a suppressor activity. The mutant 238C, which is located in the suppressor region, has a reduced synthetase activity and no detectable splicing activity. These data show that the splicing and suppressor functions are linked and that the suppressor activity of the mutant alleles results from a modification of the wild-type splicing activity.

Endoribonucleases↗
PubMed1996

Asynchronous regulation of splicing events within protein 4.1 pre-mRNA during erythroid differentiation.

Protein 4.1 is an 80-kD structural component of the red blood cell (RBC) cytoskeleton. It is critical for the formation of the spectrin/actin/protein 4.1 junctional complex, the integrity of which is important for the horizontal strength and elasticity of RBCs. We and others have previously shown that multiple protein 4.1 mRNA isoforms are generated from a single genomic locus by several alternative mRNA splicing events, leading to the insertion or skipping of discrete internal sequence motifs. The physiologic significance of these motifs: (1) an upstream 17-nucleotide sequence located at the 5' end of exon 2 that contains an in-frame ATG initiation codon, the inclusion of which by use of an alternative splice acceptor site in exon 2 allows the production of a 135-kD high-molecular-weight isoform present in nonerythroid cells; (2) exon 16, which encodes a 21-amino acid (21aa) segment located in the 10-kD "spectrin/actin binding domain" (SAB), the presence of which is required for junctional complex stability in RBCs. Previous studies by our group and others suggested that, among blood cells, this exon was retained only in mature mRNA in the erythroid lineage. Exon 16 is one of a series of three closely linked alternatively spliced exons, generating eight possible mRNA products with unique configurations of the SAB. In this communication, we report studies of the expression of both the translation initiation region and the SAB region during induced erythroid maturation in mouse erythroleukemia (MEL) cells. We have found that only two of eight possible combinatorial patterns of exon splicing at the SAB region are encountered: the isoform lacking all three exons, present in predifferentiated cells, and the isoform containing only exon 16, which increases in amount during erythroid differentiation. The protein isoform containing the 21aa segment encoded by exon 16 efficiently and exclusively incorporates into the membrane, whereas the isoform lacking this 21aa segment remains in the cytoplasm, as well as the membrane. In contrast with exon 16, the erythroid pattern of exon 2 splicing, i.e., skipping of the 17-base sequence at the 5' end, was found to be already established in the uninduced MEL cells, suggesting strongly that this regulated splicing event occurs at an earlier stage of differentiation. Our results demonstrate asynchronous regulation of two key mRNA splicing events during erythroid cell maturation. These findings also show that the splicing of exon 16 alters the intracellular localization of protein 4.1 in MEL cells, and appears to be essential for its targeting to the plasmalemma.

Alternative Splicing↗
PubMed2004

Alternative splice variants encoding unstable protein domains exist in the human brain.

Alternative splicing has been recognized as a major mechanism by which protein diversity is increased without significantly increasing genome size in animals and has crucial medical implications, as many alternative splice variants are known to cause diseases. Despite the importance of knowing what structural changes alternative splicing introduces to the encoded proteins for the consideration of its significance, the problem has not been adequately explored. Therefore, we systematically examined the structures of the proteins encoded by the alternative splice variants in the HUGE protein database derived from long (>4 kb) human brain cDNAs. Limiting our analyses to reliable alternative splice junctions, we found alternative splice junctions to have a slight tendency to avoid the interior of SCOP domains and a strong statistically significant tendency to coincide with SCOP domain boundaries. These findings reflect the occurrence of some alternative splicing events that utilize protein structural units as a cassette. However, 50 cases were identified in which SCOP domains are disrupted in the middle by alternative splicing. In six of the cases, insertions are introduced at the molecular surface, presumably affecting protein functions, while in 11 of the cases alternatively spliced variants were found to encode pairs of stable and unstable proteins. The mRNAs encoding such unstable proteins are much less abundant than those encoding stable proteins and tend not to have corresponding mRNAs in non-primate species. We propose that most unstable proteins encoded by alternative splice variants lack normal functions and are an evolutionary dead-end.

Alternative Splicing↗
PubMed1998

Parathyroid hormone-related protein (PTHrP) mRNA splicing and parathyroid hormone/PTHrP receptor mRNA expression in human placenta and fetal membranes.

During human pregnancy, parathyroid hormone-related protein (PTHrP) and parathyroid hormone (PTH)/PTHrP receptor are produced by the uterus, placenta, fetal membranes (amnion and chorion) and developing fetus. PTHrP alternative 3' mRNA splicing results in transcripts which encode three PTHrP isoforms and have been identified in amnion. Uteroplacental PTHrP expression is greatest in amnion and increases dramatically during late pregnancy. The aims of this study were to determine PTH/PTHrP receptor mRNA expression at preterm and term gestations and to determine 3' alternative splicing patterns in placenta, amnion and choriodecidua at preterm and term gestations. Using semiquantitative reverse transcription-polymerase chain reaction, PTHrP and PTH/PTHrP receptor transcripts were identified in preterm (n=5) and term (n=7) gestational tissues. PTH/PTHrP receptor mRNA expression did not differ between tissue types or change with advancing gestation. In contrast, PTHrP expression in the same tissues increased with advancing gestation and was significantly greater in amnion than in placenta and choriodecidua. Thus PTHrP, although produced predominantly in amnion, may act in amnion and other tissues including placenta, choriodecidua and myometrium. In amnion over placenta, transcripts encoding PTHrP 1-139 and 1-173 were detected in some preterm and all term samples and those encoding PTHrP 1-141 were detected in all samples. Similar results were obtained for reflected amnion. In placenta and choriodecidua, PTHrP 1-139 and 1-173 transcripts were undetectable or of low abundance. PTHrP 1-141 transcripts were detected in some placenta and choriodecidua samples. In summary, transcripts encoding PTHrP 1-141 appeared to be more abundantly expressed than those encoding PTHrP 1-139 or 1-173. However, the up-regulation of PTHrP expression in amnion at term may involve each of the alternative 3' mRNA splicing pathways since transcripts for each isoform appeared to be more consistently expressed at term.

Alternative Splicing↗
PubMed2002

AMOP, a protein module alternatively spliced in cancer cells.

This article describes a new extracellular domain--AMOP, for adhesion-associated domain in MUC4 and other proteins. This domain occurs in putative cell adhesion molecules and in some splice variants of MUC4. MUC4 splice variants are overexpressed in several tumours; in particular, they are highly expressed in pancreatic carcinomas but not in normal pancreas. The presence of AMOP in cell adhesion molecules could be indicative of a role for this domain in adhesion.

Alternative Splicing↗
PubMed2004

A phosphorylated, carboxy-terminal fragment of beta-amyloid precursor protein localizes to the splicing factor compartment.

Beta-amyloid precursor protein (APP) is implicated in the pathobiology of Alzheimer's disease (AD). To gain insight into its function, we have investigated the proteolytic processing and post-translational modification of APP in relation to its intracellular traffic and localization. The proteolytic processing that generates the amyloid beta-peptide (Abeta) also releases into the cytoplasm the carboxy-terminal fragment of APP, Cgamma. Using the catecholaminergic cell line, CAD, and an antibody to a form of APP that is phosphorylated at Thr668 (pAPP; numbering for APP695), we show that a phosphorylated, carboxy-terminal fragment of APP, probably Cgamma, is present in the nucleus, where it localizes to subnuclear particles. The labeling with anti-pAPP antibody co-localizes with proteins that define the splicing factor compartment (SFC) [e.g. the small nuclear ribonucleoprotein (snRNP), U2B, and serine/arginine-rich (SR) proteins], but is excluded from the coiled bodies and the gems. This distribution of pAPP epitopes was found in CAD cells independent of their state of differentiation, as well as in primary cortical neurons, epithelial cells and fibroblasts. We further show that exogenously expressed Cgamma becomes phosphorylated, and distributes throughout the cell. A fraction of this Cgamma is translocated into the nucleus, where it co-localizes with endogenous pAPP epitopes. Finally, we show that the APP binding, scaffolding protein, Fe65 co-localizes with pAPP epitopes and with expressed Cgamma at intranuclear speckles. These results suggest that phosphorylated Cgamma accumulates at the SFC. Thus, APP may play a role in pre-mRNA splicing, and Fe65 and APP phosphorylation may regulate this function.

Alzheimer Disease↗
PubMed2003

Widespread production of novel soluble protein isoforms by alternative splicing removal of transmembrane anchoring domains.

We have investigated the effects of alternative splicing on transcripts encoding membrane proteins in 1001 human genes. Out of a total of 464 alternatively spliced genes encoding single-pass transmembrane (TM) proteins, in 188 we observed a splice form that specifically removed the TM domain, producing a soluble protein isoform. For example, in syndecan-4, the new alternative splice form closely parallels the proteolytic ectodomain shedding previously shown in this protein, and recognized as an important regulatory mechanism of receptor function. While many of the soluble isoforms produced by alternative splicing have already been validated, most are novel, and in 57 genes showed a statistically significant association (P-value<0.01) with a specific tissue.

Alternative Splicing↗
PubMed1999

Antagonism between RSF1 and SR proteins for both splice-site recognition in vitro and Drosophila development.

Specific recognition of splice sites within metazoan mRNA precursors (pre-mRNAs) is a potential stage for gene regulation by alternative splicing. Splicing factors of the SR protein family play a major role in this regulation, as they are required for early recognition of splice sites during spliceosome assembly. Here, we describe the characterization of RSF1, a splicing repressor isolated from Drosophila, that functionally antagonizes SR proteins. Like the latter, RSF1 comprises an amino-terminal RRM-type RNA-binding domain, whereas its carboxy-terminal part is enriched in glycine (G), arginine (R), and serine (S) residues (GRS domain). RSF1 induces a dose-sensitive inhibition of splicing for several reporter pre-mRNAs, an inhibition that occurs at the level of early splicing complexes formation. RSF1 interacts, through its GRS domain, with the RS domain of the SR protein SF2/ASF and prevents the latter from cooperating with the U1 small nuclear ribonucleoprotein particle (U1 snRNP) in binding pre-mRNA. Furthermore, overproduction of RSF 1 in the fly rescues several developmental defects caused by overexpression of the splicing activator SR protein B52/ SRp55. Therefore, RSF1 may correspond to the prototypical member of a novel family of general splicing repressors that selectively antagonize the effect of SR proteins on 5' splice-site recognition.

Animals↗