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PubMed2000

Review: lamina-associated polypeptide 2 isoforms and related proteins in cell cycle-dependent nuclear structure dynamics.

The lamina-associated polypeptide (LAP) 2 family comprises up to six alternatively spliced proteins in mammalian cells and three isoforms in Xenopus. LAP2beta is a type II integral protein of the inner nuclear membrane, which binds to lamin B and the chromosomal protein BAF, and may link the nuclear membrane to the underlying lamina and provide docking sites for chromatin. LAP2alpha shares only the N-terminus with the other isoforms and contains a unique C-terminus. It is a nonmembrane protein associated with the nucleoskeleton and may help to organize higher order chromatin structure by interacting with A-lamins and chromosomes. Recent studies using mutant proteins have just begun to unravel functions of LAP2 isoforms during postmitotic nuclear reassembly. LAP2alpha associates with chromosomes via an alpha-specific domain at early stages of assembly, possibly providing a structural framework for chromosome reorganization. The subsequent interaction of both LAP2alpha and LAP2beta with the chromosomal BAF may stabilize chromatin structure and target membranes to the chromosomes. At later stages LAP2 may regulate the assembly of lamins. LAP2 isoforms have been found to share a homologous approximately 40 amino acid long region, the LEM domain, with nuclear membrane proteins MAN1 and emerin, which has been implicated in Emery-Dreifuss muscular dystrophy.

Animals↗
PubMed1997

Cloning and characterisation of a thermostable alpha-DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. TY.

The gene for an extremely thermostable DNA polymerase has been cloned from chromosomal DNA of the recently characterised hyperthermophilic archaeon Thermococcus sp. TY by using degenerate primers derived from consensus sequences of known archaeal enzymes. The corresponding enzyme was overexpressed in Escherichia coli. Sequence comparison of the gene with related DNA polymerase genes revealed that it is interrupted by three regions showing high similarities to self-splicing protein elements, so-called "inteins". This is the first DNA polymerase containing such a large number of self-splicing elements. To ensure an efficient expression, these regions were deleted on the DNA level. The resulting protein showed DNA polymerase and 3'-5' exonuclease activity at high temperatures, being a promising candidate for use in the polymerase chain reaction (PCR).

Archaeal Proteins↗
PubMed2000

Human 100-kDa homologous DNA-pairing protein is the splicing factor PSF and promotes DNA strand invasion.

Proteins promoting homologous pairing could be involved in various fundamental biological processes. Previously we detected two mammalian nuclear proteins of 100 and 75 kDa able to promote homologous DNA pairing. Here we report isolation and characterisation of the human (h) 100-kDa DNA-pairing protein, hPOMp100, from HeLa nuclei. The peptide sequences of hPOMp100 revealed identity to the human splicing factor PSF and a DNA-binding subunit of p100/p52 heterodimer of unknown function. Bacterially expressed PSF promotes DNA pairing identical to that of hPOMp100. hPOMp100/PSF binds not only RNA but also both single-stranded (ss) and double-stranded (ds) DNA and facilitates the renaturation of complementary ssDNAs. More important, the protein promotes the incorporation of a ss oligonucleotide into a homologous superhelical dsDNA, D-loop formation. A D-loop is the first heteroduplex DNA intermediate generated between recombining DNA molecules. Moreover, this reaction could be implicated in re-establishing stalled replication forks. Consistent with this hypothesis, DNA-pairing activity of hPOMp100/PSF is associated with cellular proliferation. Significantly, phosphorylation of hPOMp100/PSF by protein kinase C inhibits its binding to RNA but stimulates its binding to DNA and D-loop formation and may represent a regulatory mechanism to direct this multifunctional protein to DNA metabolic pathways.

Base Pairing↗
PubMed2003

Semiquantitative mRNA measurements of osteoinductive growth factors in human iliac-crest bone: expression of LMP splice variants in human bone.

Although osteotropic growth factors are known to play an important role in bone metabolism, knowledge about their expression in relation to age, sex and smoking remains limited. In this study we report mRNA levels of the recently discovered Lim mineralization protein splice variants (LMP-1, LMP-2, LMP-3) and the established osteotropic growth factors BMP-2, BMP-6, BMP-7, TGF-beta, IGF-I, IGF-II and b-FGF in human iliac crest bone. Standardized bone biopsy specimens were obtained from the iliac crest during graft harvesting in 62 patients (38 males, 24 females, mean age 44.7 years, range 13-78 years) undergoing spinal surgery. Samples were immediately stored in liquid nitrogen for PCR analysis. Semi-quantitative RT-PCR was performed for TGF-beta, IGF-I, IGF-II, BMP2, BMP-6, BMP7, bFGF, LMP-1, LMP-2 and LMP-3 using beta-actin as internal standard. Triplicate measurements were made of each growth factor and beta-actin. mRNA for all examined growth factors was detected in 69% of the specimens. The lowest degree of detection was present for b-FGF and BMP-2, both of which were found in 85% of the specimens. LMP-1 was detected in 98% of the specimens. LMP-2 in 94% and LMP-3 in 27%, respectively. LMP-1 was generally expressed in higher amounts than LMP-2 and LMP-3. Nondetectable levels of the growth factors were more frequent in the >60-year-old males compared with >60-year-old females ( P < 0.05) and <60-year-old males ( P < 0.01). LMP-1 expression was more variable among young individuals, but mean values were similar between age groups. TGF-beta, BMP-2 and BMP-7 values did not differ between age groups, but generally a higher variation was found among older patients. IGF-I values were significantly higher ( P < 0.05) in males over 60 years, whereas the highest level of bFGF mRNA was present in males younger than 20 years ( P < 0.05). In addition, regression analysis revealed correlation between BMP-2 and BMP-7 (R2 = 0.74, P < 0.0005), LMP-2 and BMP-2 (R2 = 0.27, P < 0.0005) and LMP-2 and bFGF (R2 = 0.40, P < 0.0005). In conclusion, we have demonstrated expression of LMP-1 and LMP-2 in human bone. LMP-1 was expressed in higher amounts and showed a higher degree of variation among young individuals. LMP-2 was correlated to a number of other growth factors, suggesting that LMPs may also play a role in human bone metabolism. Higher variation in the expression of TGF-beta, BMP-2 and BMP-7 was found in the older age groups, but whether or not this can be correlated to age-related changes in bone turnover requires further studies.

Adolescent↗
PubMed1998

Expressed protein ligation, a novel method for studying protein-protein interactions in transcription.

Expressed protein ligation is a novel protein semi-synthesis method that permits the in vitro ligation of a chemically synthesized C-terminal segment of a protein to a recombinant N-terminal segment fused through its C terminus to an intein protein splicing element. In principle, the practical convenience of this method, combined with the expanded opportunities in protein engineering that it provides, makes it well suited for probing the molecular basis of complex processes such as transcription. Here we describe the successful application of expressed protein ligation to the approximately 600 amino acid sigma70 subunit of Escherichia coli RNA polymerase. The resulting semi-synthetic sigma70 constructs are shown to be fully functional and have been used to map the binding region of the bacteriophage T4 anti-sigma protein, AsiA, to within amino acids 567-600 of sigma70. The success of these semi-synthesis studies sets the stage for the future generation of semi-synthetic sigma70 molecules in which unnatural amino acids and biophysical probes are site-specifically incorporated in the RNA polymerase complex.

Bacterial Proteins↗
PubMed1990

Regulated expression of the small nuclear ribonucleoprotein particle protein SmN in embryonic stem cell differentiation.

The SmN protein is a component of small nuclear ribonucleoprotein particles and is closely related to the ubiquitous SmB and B' splicing proteins. It is expressed in a limited range of tissues and cell types, including several undifferentiated embryonal carcinoma cell lines and undifferentiated embryonic stem cells. The protein declines to undetectable levels when embryonal carcinoma or embryonic stem cells are induced to differentiate, producing primitive endoderm or parietal endoderm or yielding embryonal bodies. This decline is due to a corresponding decrease in the level of the SmN mRNA. The potential role of SmN in the regulation of alternative splicing in embryonic cell lines and early embryos is discussed.

Actins↗
PubMed1997

Transcription-dependent redistribution of nuclear protein 4.1 to SC35-enriched nuclear domains.

Protein 4.1, originally identified as a component of the membrane-skeleton of the red blood cell, has also been localized in the nucleus of mammalian cells. To learn more about nuclear 4.1 protein, we have analyzed the nature of its association with the nuclear structure in comparison with SC35 and snRNP antigens, splicing proteins of the nuclear speckle domains. When MDCK or HeLa cells were digested with DNase I and washed in the presence of high salt (2 M NaCl), snRNP antigens were extracted whereas protein 4.1 and SC35 remained colocalizing in nuclear speckles. In cells treated with RNase A or heat shocked, nuclear 4.1 distribution also resembled that of SC35. Experiments carried out in transcriptionally active nuclei showed that protein 4.1 distributed in irregularly shaped speckles which appeared to be interconnected. During transcriptional inhibition, protein 4.1 accumulated in rounded speckles lacking interconnections. When cells were released from transcriptional inhibition, protein 4.1 redistributed back to the interconnected speckle pattern of transcriptionally active cells, as it was also observed for SC35. Finally, coprecipitation of 4.1 and SC35 proteins from RNase A digested HeLa nuclei further indicates that these two proteins are associated, forming part of the nuclear speckle domains to which they attach more tightly than snRNP antigens.

Animals↗
PubMed2000

Molecular cloning and expression analysis of a putative nuclear protein, SR-25.

We cloned a full-length mouse cDNA and its human homologue encoding a novel protein designated as "SR-25." In Northern blot analysis, SR-25 mRNA was expressed in all organs tested, and relatively abundant in testis and thymus. Deduced amino acid sequences of mouse SR-25 and human SR-25 showed 77.7% identity. SR-25 has a serine-arginine repeat (SR repeat) and two types of amino acid clusters: a serine cluster and a highly basic cluster. Based on the presence of many nuclear localizing signals and a similarity to RNA splicing proteins, SR-25 is strongly suggested to be a nuclear protein and may contribute to RNA splicing.

Amino Acid Sequence↗
PubMed2005

ADAMTS proteinases: a multi-domain, multi-functional family with roles in extracellular matrix turnover and arthritis.

Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family are known to influence development, angiogenesis, coagulation and progression of arthritis. As proteinases their substrates include the von Willebrand factor precursor and extracellular matrix components such as procollagen, hyalectans (hyaluronan-binding proteoglycans including aggrecan), decorin, fibromodulin and cartilage oligomeric matrix protein. ADAMTS levels and activities are regulated at multiple levels through the control of gene expression, mRNA splicing, protein processing and inhibition by TIMP (tissue inhibitor of metalloproteinases). A recent screen of human cartilage has shown that multiple members of the ADAMTS family may be important in connective tissue homeostasis and pathology.

ADAM Proteins↗
PubMed1996

Zinc finger-mediated protein interactions modulate Ikaros activity, a molecular control of lymphocyte development.

The Ikaros gene, an essential regulator of lymphocyte differentiation, encodes, by means of differential splicing, protein isoforms with a distinct number of Kruppel-type zinc fingers organized in two domains. Deletion of the N-terminal zinc finger domain responsible for the sequence-specific DNA binding of the Ikaros proteins results in an early and complete arrest in lymphocyte development in homozygous mutant mice. In sharp contrast, heterozygotes reliably develop T cell leukemias and lymphomas. Here we show that the C-terminal zinc finger domain present in all of the Ikaros wild-type and mutant isoforms is responsible for their stable interactions off DNA and plays a pivotal role in determining their overall activity. Mutations in the C-terminal zinc fingers which ablate Ikaros protein interactions have a dramatic effect on the ability of these proteins to bind DNA and activate transcription. Therefore, interactions between Ikaros isoforms with an intact DNA binding domain are essential for their function. In contrast, interactions between isoforms with and without a DNA binding domain result in Ikaros complexes that do not bind DNA and, as a consequence, cannot activate transcription. Dominant-negative Ikaros isoforms are generated in smaller amounts by the wild-type Ikaros gene but are also produced exclusively by the N-terminally deleted Ikaros locus. Given these data, we propose that interactions between Ikaros isoforms are essential for normal progression through the lymphoid pathways. Mutations in the Ikaros gene that prevent Ikaros protein interactions or which change the relative ratio of DNA to non-DNA binding isoforms have profound effects in both lymphoid specification and homeostasis.

3T3 Cells↗
PubMed2004

Autoregulation of polypyrimidine tract binding protein by alternative splicing leading to nonsense-mediated decay.

Polypyrimdine tract binding protein (PTB) is a regulator of alternative splicing, mRNA 3' end formation, mRNA stability and localization, and IRES-mediated translation. Transient overexpression of PTB can influence alternative splicing, sometimes resulting in nonphysiological splicing patterns. Here, we show that alternative skipping of PTB exon 11 leads to an mRNA that is removed by NMD and that this pathway consumes at least 20% of the PTB mRNA in HeLa cells. We also show that exon 11 skipping is itself promoted by PTB in a negative feedback loop. This autoregulation may serve both to prevent disruptively high levels of PTB expression and to restore nuclear levels when PTB is mobilized to the cytoplasm. Our findings suggest that alternative splicing can act not only to generate protein isoform diversity but also to quantitatively control gene expression and complement recent bioinformatic analyses, indicating a high prevalence of human alternative splicing leading to NMD.

Alternative Splicing↗
PubMed2000

Dipeptide seryl-histidine and related oligopeptides cleave DNA, protein, and a carboxyl ester.

The amino acids histidine (His) and serine (Ser), or amino acids similar to Ser, function together as key catalytic amino acids in the active sites of such diverse enzymes as the serine- and thiol-proteases, lipases, and esterases. Ser and His are also conserved in the intein-extein junctions of the phylogenetically widespread self-splicing proteins and at the N- and C-termini of the homing endonucleases spliced from them. Here we show that the dipeptide seryl-histidine (Ser-His) and related oligopeptides can themselves cleave DNA, protein, and the ester p-nitrophenyl acetate (p-NPA) over wide ranges of pH and temperature. Denaturing polyacrylamide gel electrophoresis (PAGE) of 5'-end labeled DNA samples incubated with Ser-His reveals a pattern of two bands per nucleotide position, consistent with the generation of both 3'-hydroxyl and 3'-phosphate DNA cleavage fragments, as would be expected of phosphodiester hydrolysis by Ser-His. To the best of our knowledge, Ser-His is the shortest peptide ever reported to show cleavage activity with multiple categories of natural substrates. The amenability of the dipeptide to variation through addition of amino acid residues, either internally or to the C-terminus while retaining its multiple cleavage activities, combined with its reactivity over wide ranges of pH and temperature, demonstrates the evolutionary capacity of the Ser/His dyad and evokes many questions about possible roles it may have played in molecular evolution and its potential role as a core for selection of oligopeptides with enhanced cleavage activities and target specificity.

Amino Acid Sequence↗
PubMed1993

The cardiac form of the tissue-specific SmN protein is identical to the brain and embryonic forms of the protein.

The SmN protein is a component of small nuclear ribonucleoprotein particles and closely related to the ubiquitously expressed SmB and B' splicing proteins. However, SmN is only expressed in a limited range of tissues and cell types such as brain, heart and early embryonic cells. The isolation of cDNA clones derived from the mRNA encoding SmN in different cell types has indicated that the brain and embryonic forms of the protein are identical and are encoded by a distinct gene to that encoding SmB and B'. It has been suggested however, that the cardiac form of SmN is encoded by a distinct mRNA which is derived from a different gene from that encoding the brain and embryonic forms of SmN. By using the polymerase chain reaction as well as cDNA cloning we have shown that this is not the case and that the cardiac, brain and embryonic forms of the protein are identical and are translated from the same mRNA encoded by a single gene. The significance of this finding is discussed in terms of the complex expression pattern of this gene and the possible functional role of SmN.

Amino Acid Sequence↗
PubMed2003

Transcriptional analysis of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is the causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome. In this work, the RNAs of PCV2 synthesized during productive infection in porcine kidney cells were characterized. A total of nine RNAs were detected. They include the viral capsid protein RNA (CR), a cluster of five Rep-associated RNAs (designated Rep, Rep', Rep3a, Rep3b, and Rep3c), and three NS-associated RNA (designated NS515, NS672, and NS0). Members of the Rep-associated RNA cluster all share common 5' and 3' nucleotide sequences and they also share 200 common 3' nucleotides with the NS-associated RNAs. Rep, capable of coding for the replication-associated-protein (RepP), appears to be the primary transcript that gives rise to Rep', Rep3a, Rep3b, and Rep3c by alternate splicing. Protein sequence alignment showed that RepP and the Rep' protein of PCV2 are equivalent to those described for PCV type 1 (PCV1) (a nonpathogenic virus), which had been shown to be essential for viral DNA replication. The results also suggest that NS515, NS672, and NS0 are transcribed from three different promoters inside ORF1 downstream of the Rep promoter. To date, only three RNAs (CR, Rep, and Rep') have been reported for PCV1-infected porcine kidney cells. Therefore, it is important to apply similar strategies from this study to reexamine the transcription pattern of PCV1.

Amino Acid Sequence↗
PubMed1994

Incorporation of 5-fluorouracil into U2 and U6 snRNA inhibits mRNA precursor splicing.

The splicing activities of 5-fluorouracil (FUra)-substituted U2 and U6 small nuclear RNAs (snRNAs) were examined in an in vitro splicing system. Yeast splicing extracts were specifically depleted of endogenous U2 and U6 snRNAs by antisense oligonucleotide-directed RNase H hydrolysis. Splicing activity was recovered when the extracts were reconstituted with synthetic U2 and U6 snRNAs. However, U2 snRNA with all uracils substituted with FUra (FU2) did not restore any splicing activity. Nondenaturing gel electrophoresis showed that FU2 failed to promote the assembly of spliceosome complexes. The ability of U2 snRNA to restore splicing in U2-depleted extracts increased as FUra content decreased but was still only 60% of control activity at 25% substitution of uracils with FUra. Addition of FU2 to nondepleted extracts caused strong inhibition of splicing accompanied by increased degradation of the pre-mRNA, suggesting that FU2 forms an inactive complex with a protein splicing factor that normally binds to the pre-mRNA. FU6 restored full splicing activity to U6-depleted extracts, but at a 5-fold higher concentration than U6 snRNA. These results demonstrate that the incorporation of FUra can impair the functions of catalytic RNA molecules.

Base Sequence↗
PubMed2004

An endoplasmic reticulum trafficking signal prevents surface expression of a voltage- and Ca2+-activated K+ channel splice variant.

Protein delivery to restricted plasma membrane domains is exquisitely regulated at different stages of the cell trafficking machinery. Traffic control involves the recognition of export/retention/retrieval signals in the endoplasmic reticulum (ER)/Golgi complex that will determine protein fate. A splice variant (SV), SV1, of the voltage- and Ca(2+)-activated K(+) channel alpha-subunit accumulates the channel in the ER, preventing its surface expression. We show that SV1 insert contains a nonbasic, hydrophobic retention/retrieval motif, CVLF, that does not interfere with proper folding and tetramerization of SV1. Localization of proteins in the ER by CVLF is independent of its position; originally, on the first internal loop, SV1 insert or CVLF perform equally well if placed at the middle or end of the alpha-subunit intracellular carboxyl terminus. Also, CVLF is able to restrict the traffic of an independently expressed transmembrane protein, beta 1-subunit. CVLF is present in proteins across species and in lower organisms. Thus, CVLF may have evolved to serve as a regulator of cellular traffic.

Amino Acid Motifs↗
PubMed2002

Characterization of a putative murine mitochondrial transporter homology of hMRS3/4.

We report here a novel murine gene, Mrs3/4, encoding a putative mitochondrial transporter homologous to the yeast mitochondrial RNA splicing protein 3 and 4 (yMRS3&4) and its human counterpart hMRS3/4. By analyses of radiation hybrids, Mrs3/4 was mapped to mouse Chromosome (Chr) 19 between D19Mit66 and D19Mit24 with a distance of 22.4 cR to D19Mit66, a region that is syntenic to human Chromosome 10q24, where the human gene is located. Structural analysis shows that these proteins belong to the mitochondrial carrier family (MCF), which is characterized by three repeats with two transmembrane domains in each repeat. The murine Mrs3/4 gene has two splicing forms similar to the human homolog. The long form corresponds to the 341-amino acid (aa) protein with six transmembrane domains, and the short form corresponds to the 177-aa protein with three transmembrane domains. Both forms have well-conserved signature sequences of MCF. Targeting experiments showed that both forms have mitochondrial localization. Northern blot analyses showed that Mrs3/4 is ubiquitously expressed as a 1.8-kb transcript with a relative abundance in the heart, liver, kidney, and testis. In conclusion, the reported Mrs3/4 gene shows a strong conservation, mitochondrial protein localization, and a ubiquitous expression, which suggests that it has an important role in mammalian cells, most likely as an ion transporter across the mitochondrial inner membrane.

Amino Acid Sequence↗