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At least 217 records · Page 12Linked to original sources
PubMed1999

An alternatively spliced surfactant protein B mRNA in normal human lung: disease implication.

We identified an alternatively-spliced surfactant protein B (SP-B) mRNA from normal human lung with a 12 nt deletion at the beginning of exon 8. This deletion causes a loss of four amino acids in the SP-B precursor protein. Sequence comparison of the 3' splice sites reveals only one difference in the frequency of U/C in the 11 predominantly-pyrimidine nucleotide tract, 73% for the normal and 45% for the alternatively-spliced SP-B mRNA (77-99% for the consensus sequence). Analysis of SP-B mRNA in lung indicates that the abundance of the alternatively-spliced form is very low and varies among individuals. Although the relative abundance of the deletion form of SP-B mRNA remains constant among normal lungs, it is found with relatively higher abundance in the lungs of some individuals with diseases such as congenital alveolar proteinosis, respiratory distress syndrome, bronchopulmonary dysplasia, alveolar capillary dysplasia and hypophosphatasia. This observation points to the possibility that the alternative splicing is a potential regulatory mechanism of SP-B and may play a role in the pathogenesis of disease under certain circumstances.

Alternative Splicing↗
PubMed2006

Binding of the polypyrimidine tract-binding protein-associated splicing factor (PSF) to the hepatitis delta virus RNA.

The hepatitis delta virus (HDV) has a very limited protein coding capacity and must rely on host proteins for its replication. A ribonucleoprotein complex was detected following UV cross-linking between HeLa nuclear proteins and an RNA corresponding to the right terminal stem-loop domain of HDV genomic RNA. Mass spectrometric analysis of the complex revealed the polypyrimidine tract-binding protein-associated splicing factor (PSF) as a novel HDV RNA-interacting protein. Co-immunoprecipitation demonstrated the interaction between HDV RNA and PSF both in vitro in HeLa nuclear extract and in vivo within HeLa cells containing both polarities of the HDV genome. Analysis of the binding of various HDV-derived RNAs to purified, recombinant PSF further confirmed the specificity of the interaction and revealed that PSF directly binds to the terminal stem-loop domains of both polarities of HDV RNA. Our findings provide evidence of the involvement of a host mRNA processing protein in the HDV life cycle.

Amino Acid Sequence↗
PubMed1998

Cloning of a gene, YT521, for a novel RNA splicing-related protein induced by hypoxia/reoxygenation.

To elucidate the role of astrocytes in the stress response of the central nervous system to ischemia, early gene expression was examined in rat cultured astrocytes after the exposure to hypoxia/reoxygenation, and we have previously cloned a novel RNA binding protein, RA301, from the reoxygenated astrocytes. Furthermore, we have now cloned a new gene for RA301 binding protein, termed YT521, by a yeast two-hybrid screening technique to explore RA301 functions. The YT521 cDNA is about 3200 bp long with an open reading frame encoding 712 amino acids. This amino acid sequence contains arginine-aspartic acid-glutamic acid rich region and glutamic acid rich one, and has a low degree of homology with RNA binding proteins such as U1-70k. Northern blot analysis revealed that YT521 mRNA expression was up-regulated in reoxygenated astrocytes. Induction of YT521 mRNA was mediated by endogenously generated reactive oxygen species, as it was suppressed by treatment of the cells with diphenyl iodonium which blocks oxygen-free radical formation by astrocytes. These expression patterns resembled those of RA301 mRNA. Far Western blot analysis showed that YT521 protein was not only interacting with RA301 protein, but also with SC35 and SF2, both of which are splicing factors. These results suggest that YT521 is a novel candidate for RNA splicing-related protein.

Amino Acid Sequence↗
PubMed2006

Protein purification via temperature-dependent, intein-mediated cleavage from an immobilized metal affinity resin.

The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed in Escherichia coli and purified as an unspliced precursor. On in vitro incubation at 37 degrees Celsius or higher, the intein mediates efficient protein splicing. Mutations can be introduced into an intein fusion protein that prevent the second and third steps of protein splicing. As a result, the intein fusion protein can facilitate temperature-dependent formation of a thioester linkage between the N-extein and intein. This thioester is susceptible to in vitro hydrolysis or thiolysis at temperatures of 40 degrees Celsius or higher, and we have exploited this activity to generate a temperature-dependent protein purification scheme. Protein purification using this intein does not require the addition of exogenous thiols and is compatible with the use of immobilized metal affinity chromatography. The identity of the C-terminal residue of the N-extein has less influence on the cleavage reaction than in current purification systems in terms of premature in vivo cleavage and is complementary to current systems in terms of efficient in vitro cleavage.

Affinity Labels↗
PubMed1993

Topological switching of the COOH-terminal domain of peptidylglycine alpha-amidating monooxygenase by alternative RNA splicing.

Proteins encompassing the two catalytic domains (monooxygenase and lyase) and the COOH-terminal domain of rat peptidylglycine alpha-amidating monooxygenase (rPAM)3 were purified from recombinant Escherichia coli overexpressing each domain and used to raise domain-specific polyclonal antibodies. Four alternatively spliced forms of PAM RNA (PAM-1, -2, -3, and -4) were transcribed in vitro and used to synthesize PAM proteins in a cell-free translation system. The orientation of the proteins in microsomal membrane vesicles was analyzed using trypsin protection assays and immunoprecipitation with the domain-specific antibodies. Only one of the two potential N-glycosylation sites (Asn765-Phe-Ser) in PAM-1 was efficiently utilized by microsomal membranes. PAM-1 and PAM-2 were shown to be type Ia membrane proteins with their two catalytic domains residing within microsomal vesicles and their COOH-terminal domains exposed to the cytosol. In contrast, PAM-3 and PAM-4 were shown to be soluble proteins contained entirely within vesicles. Thus, the COOH-terminal domain underwent topological switching between the cytosolic (PAM-1 and -2) and luminal (PAM-3) compartments as a function of alternative splicing of exons Ba/Bb. Computer analyses of the PAM protein sequence correlated the exons encoding PAM-1 with a model for the structural and functional domains of the PAM protein. The dual topologies of the PAM proteins confer an important means of functional regulation to this secretory granule associated neuropeptide processing enzyme.

Amino Acid Sequence↗
PubMed2003

Differentiation-induced colocalization of the KH-type splicing regulatory protein with polypyrimidine tract binding protein and the c-src pre-mRNA.

We have examined the subcellular localization of the KH-type splicing regulatory protein (KSRP). KSRP is a multidomain RNA-binding protein implicated in a variety of cellular processes, including splicing in the nucleus and mRNA localization in the cytoplasm. We find that KSRP is primarily nuclear with a localization pattern that most closely resembles that of polypyrimidine tract binding protein (PTB). Colocalization experiments of KSRP with PTB in a mouse neuroblastoma cell line determined that both proteins are present in the perinucleolar compartment (PNC), as well as in other nuclear enrichments. In contrast, HeLa cells do not show prominent KSRP staining in the PNC, even though PTB labeling identified the PNC in these cells. Because both PTB and KSRP interact with the c-src transcript to affect N1 exon splicing, we examined the localization of the c-src pre-mRNA by fluorescence in situ hybridization. The src transcript is present in specific foci within the nucleus that are presumably sites of src transcription but are not generally perinucleolar. In normally cultured neuroblastoma cells, these src RNA foci contain PTB, but little KSRP. However, upon induced neuronal differentiation of these cells, KSRP occurs in the same foci with src RNA. PTB localization remains unaffected. This differentiation-induced localization of KSRP with src RNA correlates with an increase in src exon N1 inclusion. These results indicate that PTB and KSRP do indeed interact with the c-src transcript in vivo, and that these associations change with the differentiated state of the cell.

Animals↗
PubMed2000

Alternative splicing of protein 4.1R exon 16: ordered excision of flanking introns ensures proper splice site choice.

Alternative splicing plays a major role in regulating tissue-specific expression of cytoskeletal protein 4.1R isoforms. In particular, expression of the protein's functionally critical spectrin-actin binding domain, essential for maintenance of red cell membrane mechanical properties, is governed by a developmentally regulated splicing switch involving alternative exon 16. Using a model 3-exon 4.1R pre-messenger RNA (pre-mRNA), we explored the sequence requirements for excision of the introns flanking exon 16. These studies revealed that splicing of this alternative exon occurs preferentially in an ordered fashion. The first step is excision of the downstream intron to join exons 16 and 17, followed by excision of the upstream intron. Constructs designed to test the converse pathway were spliced less efficiently and with less fidelity, in part due to activation of a cryptic 5' splice site in exon 16. This downstream-first model for ordered splicing is consistent with the hypothesis that regulated alternative splicing requires cooperation between multiple exonic and/or intronic regulatory elements whose spatial organization is critical for recruitment of appropriate splicing factors. Our results predict that exon 16 splicing is regulated at the first step-excision of the downstream intron-and that cells unable to catalyze this step will exhibit exon 16 skipping. In cells that include exon 16, adherence to an ordered pathway is important for efficient and accurate production of mature 4.1R mRNA encoding an intact spectrin-actin binding domain. (Blood. 2000;95:692-699)

Alternative Splicing↗
PubMed2001

A comprehensive characterization of a group IB intron and its encoded maturase reveals that protein-assisted splicing requires an almost intact intron RNA.

The group I intron (AnCOB) of the mitochondrial apocytochrome b gene from Aspergillus nidulans encodes a bi-functional maturase protein that is also a DNA endonuclease. Although the AnCOB intron self-splices, the encoded maturase protein greatly facilitates splicing, in part, by stabilizing RNA tertiary structure. To determine their role in self-splicing and in protein-assisted splicing, several peripheral RNA sub-domains in the 313 nucleotide intron were deleted (P2, P9, P9.1) or truncated (P5ab, P6a). The sequence in two helices (P2 and P9) was also inverted. Except for P9, the deleted regions are not highly conserved among group I introns and are often dispensable for catalytic activity. Nevertheless, despite the very tight binding of AnCOB RNA to the maturase and the high activity of the bimolecular complex (the rate of 5' splice-site cleavage was >20 min(-1) with guanosine as the cofactor), the intron was surprisingly sensitive to these modifications. Several mutations inactivated splicing completely and virtually all impaired splicing to varying degrees. Mutants containing comparatively small deletions in various regions of the intron significantly decreased binding affinity (generally >10(4)-fold), indicating that none of the domains that remained constitutes the primary recognition site of the maturase. The data argue that tight binding requires tertiary interactions that can be maintained by only a relatively intact intron RNA, and that the binding mechanism of the maturase differs from those of two other well-characterized group I intron splicing factors, CYT-18 and Cpb2. A model is proposed in which the protein promotes widespread cooperative folding of an RNA lacking extensive initial tertiary structure.

Apoproteins↗
PubMed1995

Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein.

Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2',5'-phosphodiester bond (RNA branch) with the 5' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction.

Base Sequence↗
PubMed1997

A protein related to splicing factor U2AF35 that interacts with U2AF65 and SR proteins in splicing of pre-mRNA.

Recognition of a functional 3' splice site in pre-mRNA splicing requires a heterodimer of the proteins U2AF65/U2AF35. U2AF65 binds to RNA at the polypyrimidine tract, whereas U2AF35 is thought to interact through its arginine/serine-rich (RS) domain with other RS-domain-containing factors bound at the 5' splice site, assembled in splicing enhancer complexes, or associated with the U4/U6.U5 small nuclear ribonucleoprotein complex. It is unclear, however, how such network interactions can all be established through the small RS domain in U2AF. Here we describe the function of a U2AF35-related protein (Urp), which is the human homologue of a mouse imprinted gene. Nuclear extracts depleted of Urp are defective in splicing, but activity can be restored by addition of recombinant Urp. U2AF35 could not replace Urp in complementation, indicating that their functions do not overlap. Co-immunodepletion showed that Urp is associated with the U2AF65/U2AF35 heterodimer. Binding studies revealed that Urp specifically interacts with U2AF65 through a U2AF35-homologous region and with SR proteins (a large family of RS-domain-containing proteins) through its RS domain. Therefore, Urp and U2AF35 may independently position RS-domain-containing factors within spliceosomes.

Amino Acid Sequence↗
PubMed2004

Intein harbouring large tandem repeats in replicative DNA helicase of Trichodesmium erythraeum.

Protein splicing inteins can be small as approximately 130 aa or up to approximately 600 aa when harbouring an endonuclease domain. Here we report the identification and characterization of an unusually large intein, 1650 aa long and the largest of known inteins, encoded by the replicative DNA helicase gene dnaB of the oceanic N2-fixing cyanobacterium Trichodesmium erythraeum. This Ter DnaB-1 intein co-exists with a 177-aa mini-intein in the same host protein and harbours large tandem repeats in which an 84-aa sequence is repeated 16 times. Comparison between this tandem repeats and the recently reported tandem repeats of Ter DnaE-1 intein revealed differences and similarities. The two tandem repeats, residing in different inteins of different host proteins, differ by 50% in size and have little sequence similarity. Tandem repeats in the Ter DnaB-1 intein were required for the protein splicing activity when tested in Escherichia coli, in contrast to tandem repeats of the Ter DnaE-1 intein that inhibited protein splicing. On the other hand, tandem repeats of both inteins are located in the same corresponding region of the intein sequence and have the same number of repeating units. These suggest that the two tandem repeats could be related but have diverged greatly in size, sequence and effect on protein splicing. Alternatively, they could have independent origins but evolved certain similarities because of common constraints in structure and maintenance.

Amino Acid Sequence↗
PubMed1994

Cytostellin distributes to nuclear regions enriched with splicing factors.

Cytostellin, a approximately 240 kDa phosphoprotein found in all cells examined from human to yeast, is predominantly intranuclear in interphase mammalian cells and undergoes continuous redistribution during the cell cycle. Here, mammalian cytostellin is shown to localize to intranuclear regions enriched with multiple splicing proteins, including spliceosome assembly factor, SC-35. Cytostellin and the splicing proteins also co-localize to discrete foci (called 'dots'), which are distributed throughout the cell during mitosis and part of G1. The cytostellin that is localized to these dots resists extraction by Triton X-100, indicating that it is tightly associated with insoluble cell structures. All immunostainable cytostellin reappears in the nucleus before S-phase. Although cytostellin and the splicing proteins co-localize in interphase and dividing cells, cytostellin is not detected in purified spliceosomes, and it associates with six unidentified proteins, forming a macromolecular complex that is biochemically distinct from the proteins that comprise spliceosomes. This macromolecular complex is detected at constant levels throughout the cell cycle, and the level of cytostellin protein remains constant during the cell cycle. Nevertheless, intranuclear cytostellin immunostaining fluctuates markedly during the cell cycle. The monoclonal antibody (mAb) H5 epitope of cytostellin is 'masked' in serum-starved cells, but 60 minutes after serum stimulation intense cytostellin immunoreactivity appears in the nuclear speckles. This rapid induction of cytostellin immunoreactivity in subnuclear regions enriched with many splicing factors, as well as accumulations of RNA polymerase II (Pol II) transcripts, suggests that cytostellin may have a function related to mRNA biogenesis.

Animals↗
PubMed1997

smg mutants affect the expression of alternatively spliced SR protein mRNAs in Caenorhabditis elegans.

The expression of alternatively spliced mRNAs from genes is an ubiquitous phenomenon in metazoa. A screen for trans-acting factors that alter the expression of alternatively spliced mRNAs reveals that the smg genes of Caenorhabditis elegans participate in this process. smg genes have been proposed to function in degradation of nonsense mutant mRNAs. Here we show that smg genes affect normal gene expression by modulating the levels of alternatively spliced SRp20 and SRp30b mRNAs. These SR genes contain alternatively spliced exons that introduce upstream stop codons. The effect of smg genes on SR transcripts is specific, because the gene encoding the catalytic subunit of the cAMP-dependent protein kinase, which also contains an alternatively spliced exon that introduces upstream stop codon, is not effected in a smg background. These results suggest that the levels of alternatively spliced mRNAs may, in part, be regulated by alternative mRNA stability.

Alternative Splicing↗
PubMed2001

The adenovirus E4-ORF4 splicing enhancer protein interacts with a subset of phosphorylated SR proteins.

SR proteins purified from uninfected HeLa cells inhibit adenovirus IIIa pre-mRNA splicing by binding to the intronic IIIa repressor element (3RE). In contrast, SR proteins purified from late adenovirus-infected cells are functionally inactivated as splicing repressor proteins by a virus-induced dephosphorylation. We have shown that the adenovirus E4-ORF4 protein, which binds the cellular protein phos phatase 2A (PP2A) and activates IIIa splicing in vitro and in vivo, induces SR protein dephosphorylation. Here we show that E4-ORF4 interacts with only a subset of SR proteins present in HeLa cells. Thus, E4-ORF4 interacts efficiently with SF2/ASF and SRp30c, but not with other SR proteins. Interestingly, E4-ORF4 interacts with SF2/ASF through the latter's RNA recognition motifs. Furthermore, E4-ORF4 interacts preferentially with the hyperphosphorylated form of SR proteins found in uninfected HeLa cells. E4-ORF4 mutant proteins that fail to bind strongly to PP2A or SF2/ASF do not relieve the repressive effect of HeLa SR proteins on IIIa pre-mRNA splicing in transient transfection experiments, suggesting that an interaction between all three proteins is required for E4-ORF4-induced SR protein dephosphorylation.

Adenovirus E4 Proteins↗
PubMed2004

PDZ-containing proteins: alternative splicing as a source of functional diversity.

Scaffold proteins allow specific protein complexes to be assembled in particular regions of the cell at which they organize subcellular structures and signal transduction complexes. This characteristic is especially important for neurons, which are highly polarized cells. Among the domains contained by scaffold proteins, the PSD-95, Discs-large, ZO-1 (PDZ) domains are of particular relevance in signal transduction processes and maintenance of neuronal and epithelial polarity. These domains are specialized in the binding of the carboxyl termini of proteins allowing membrane proteins to be localized by the anchoring to the cytoskeleton mediated by PDZ-containing scaffold proteins. In vivo studies carried out in Drosophila have taught that the role of many scaffold proteins is not limited to a single process; thus, in many cases the same genes are expressed in different tissues and participate in apparently very diverse processes. In addition to the differential expression of interactors of scaffold proteins, the expression of variants of these molecular scaffolds as the result of the alternative processing of the genes that encode them is proving to be a very important source of variability and complexity on a main theme. Alternative splicing in the nervous system is well documented, where specific isoforms play roles in neurotransmission, ion channel function, neuronal cell recognition, and are developmentally regulated making it a major mechanism of functional diversity. Here we review the current state of knowledge about the diversity and the known function of PDZ-containing proteins in Drosophila with emphasis in the role played by alternatively processed forms in the diversity of functions attributed to this family of proteins.

Alternative Splicing↗
PubMed1995

Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains.

A subpopulation of the largest subunit of RNA polymerase II (Pol II LS) is located in 20-50 discrete subnuclear domains that are closely linked to speckle domains, which store splicing proteins. The speckle-associated fraction of Pol II LS is hyperphosphorylated on the COOH-terminal domain (CTD), and it is highly resistant to extraction by detergents. A diffuse nucleoplasmic fraction of Pol II LS is relatively hypophosphorylated on the CTD, and it is easily extracted by detergents. In transcriptionally active nuclei, speckle bound hyperphosphorylated Pol II LS molecules are distributed in irregularly shaped speckle domains, which appear to be interconnected via a reticular network. When transcription is inhibited, hyperphosphorylated Pol II LS and splicing protein SC35 accumulate in speckle domains, which are transformed into enlarged, dot-like structures lacking interconnections. When cells are released from transcriptional inhibition, Pol IIO and SC35 redistribute back to the interconnected speckle pattern of transcriptionally active cells. The redistribution of Pol II and SC35 is synchronous, reversible, and temperature dependent. It is concluded that: (a) hyperphosphorylation of Pol II LS's CTD is a better indicator of its tight association to discrete subnuclear domains than its transcriptional activity; (b) during states of transcriptional inhibition, hyperphosphorylated Pol II LS can be stored in enlarged speckle domains, which under the light microscope appear to coincide with the storage sites for splicing proteins; and (c) Pol II and splicing proteins redistribute simultaneously according to the overall transcriptional activity of the nucleus.

Amanitins↗
PubMed1996

The Cbp2 protein suppresses splice site mutations in a group I intron.

The Cbp2 protein facilitates the folding of a group I intron in the COB pre-mRNA of yeast mitochondria. Based on its ability to suppress mutations affecting the auto-catalytic reaction, the protein appears to play a role in the selection of splice sites. Adding Cbp2 did not overcome the effects of mutations in P1 whose primary effect was on the first step of splicing. In contrast, most mutations affecting the ligation of exons were suppressed in vitro by Cbp2. These included mutations in P1, P9.0 and P10. In fact, a mutant transcript lacking both P9.0 and P10 ligated efficiently in the presence of Cbp2. P9.0 and P10 mutations also reduced the rate of cleavage at the 5' splice junction, and this effect was only partially mitigated by adding Cbp2. A competitive secondary structure near the 3' splice junction blocked Cbp2-stimulated splicing, but this mutation could be suppressed by co-transcriptional splicing in the presence of Cbp2. Our data underscore the importance of the interaction between the 5' and 3' splice junctions in group I introns and suggest that nucleotide-nucleotide interactions that stabilize the structure of group I introns can be superceded by protein-RNA interactions.

Apoproteins↗