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PubMed2004

A bioinformatics analysis of alternative exon usage in human genes coding for extracellular matrix proteins.

Alternative splicing increases protein diversity through the generation of different mRNA molecules from the same gene. Although alternative splicing seems to be a widespread phenomenon in the human transcriptome, it is possible that different subgroups of genes present different patterns, related to their biological roles. Analysis of a subgroup may enhance common features of its members that would otherwise disappear amidst a heterogeneous population. Extracellular matrix (ECM) proteins are a good set for such analyses since they are structurally and functionally related. This family of proteins is involved in a large variety of functions, probably achieved by the combinatorial use of protein domains through exon shuffling events. To determine if ECM genes have a different pattern of alternative splicing, we compared clusters of expressed sequences of ECM to all other genes regarding features related to the most frequent type of alternative splicing, alternative exon usage (AEU), such as: the number of alternative exon-intron structures per cluster, the number of AEU events per exon-intron structure, the number of exons per event, among others. Although we did not find many differences between the two sets, we observed a higher frequency of AEU events involving entire protein domains in the ECM set, a feature that could be associated with their multi-domain nature. As other subgroups or even the ECM set in different tissues could present distinct patterns of AEU, it may be premature to conclude that alternative splicing is homogeneous among groups of related genes.

Alternative Splicing↗
PubMed1999

An element in the 5' common exon of the NCAM alternative splicing unit interacts with SR proteins and modulates 5' splice site selection.

The neural cell adhesion molecule (NCAM) gene contains an 801 nt exon that is included preferentially in neuronal cells. We have set up an in vitro splicing system that mimics the neuro-specific alternative splicing profile of NCAM exon 18. Splicing regulation is observed using model pre-mRNAs that contain competing 5' or 3' splice sites, suggesting that distinct pathways regulate NCAM 5' and 3' splice site selection. While inclusion of exon 18 is the predom-inant choice in neuronal cells, an element in the 5' common exon 17 improves exon 17/exon 19 splicing in a neuronal cell line. A similar behavior is observed in vitro as the element can stimulate the 5' splice site of exon 17 or a heterologous 5' splice site. The minimal 32 nt sequence of the exon 17 enhancer consists of purine stretches and A/C motifs. Mutations in the purine stretches compromise the binding of SR proteins and decreases splicing stimulation in vitro. Mutations in the A/C motifs do not affect SR protein binding but reduce enhancing activity. Our results suggest that the assembly of an enhancer complex containing SR proteins in a 5' common exon ensures that NCAM mRNAs lacking exon 18 are made in neuronal cells.

Alternative Splicing↗
PubMed1994

Evidence of selection for protein introns in the recAs of pathogenic mycobacteria.

Protein introns are recently discovered genetic elements whose intervening sequences are removed from a precursor protein by an unusual protein splicing reaction. This involves the excision of a central spacer molecule, the protein intron, and the religation of the amino- and carboxy-terminal fragments of the precursor. The recA gene of Mycobacterium tuberculosis contains one such element and we now show that the other major mycobacterial pathogen, Mycobacterium leprae, also possesses a protein intron in its recA, although other mycobacterial recA genes do not. However, these two protein introns are different in size, sequence and location of insertion of their coding sequences into the recAs of M. tuberculosis and M. leprae, indicating that acquisition of the protein introns has occurred independently in the two species, and thus suggesting that there has been selection for splicing in the maturation of RecA in the pathogenic mycobacteria. The M. leprae protein intron provides an example of conditional protein splicing, splicing occurring in M. leprae itself but not when expressed in Escherichia coli, unlike most previously described protein introns. These observations suggest that protein introns may perform a function for their host, rather than being just selfish elements.

Amino Acid Sequence↗
PubMed1996

Open reading frame P--a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA.

The open reading frame P (ORF P) is located in the domain and on the DNA strand of the herpes simplex virus 1 transcribed during latent infection. ORF P is not expressed in productively infected cells as a consequence of repression by the binding of the major viral regulatory protein to its high-affinity binding site. In cells infected with a mutant virus carrying a derepressed gene, ORF P protein is extensively posttranslationally processed. We report that ORF P interacts with a component of the splicing factor SF2/ASF, pulls down a component of the SM antigens, and colocalizes with splicing factors in nuclei of infected cells. The hypothesis that ORF P protein may act to regulate viral gene expression, particularly in situations such as latently infected sensory neurons in which the major regulatory protein is not expressed, is supported by the evidence that in cells infected with a mutant in which the ORF P gene was derepressed, the products of the regulatory genes alpha 0 and alpha 22 are reduced in amounts early in infection but recover late in infection. The proteins encoded by these genes are made from spliced mRNAs, and the extent of recovery of these proteins late in infection correlates with the extent of accumulation of post-translationally processed forms of ORF P protein.

Carcinoma, Hepatocellular↗
PubMed2006

Maps, codes, and sequence elements: can we predict the protein output from an alternatively spliced locus?

Alternative splicing choices are governed by splicing regulatory protein interactions with splicing silencer and enhancer elements present in the pre-mRNA. However, the prediction of these choices from genomic sequence is difficult, in part because the regulators can act as either enhancers or silencers. A recent study describes how for a particular neuronal splicing regulatory protein, Nova, the location of its binding sites is highly predictive of the protein's effect on an exon's splicing.

Alternative Splicing↗
PubMed2004

Muscleblind proteins regulate alternative splicing.

Although the muscleblind (MBNL) protein family has been implicated in myotonic dystrophy (DM), a specific function for these proteins has not been reported. A key feature of the RNA-mediated pathogenesis model for DM is the disrupted splicing of specific pre-mRNA targets. Here we demonstrate that MBNL proteins regulate alternative splicing of two pre-mRNAs that are misregulated in DM, cardiac troponin T (cTNT) and insulin receptor (IR). Alternative cTNT and IR exons are also regulated by CELF proteins, which were previously implicated in DM pathogenesis. MBNL proteins promote opposite splicing patterns for cTNT and IR alternative exons, both of which are antagonized by CELF proteins. CELF- and MBNL-binding sites are distinct and regulation by MBNL does not require the CELF-binding site. The results are consistent with a mechanism for DM pathogenesis in which expanded repeats cause a loss of MBNL and/or gain of CELF activities, leading to misregulation of alternative splicing of specific pre-mRNA targets.

Alternative Splicing↗
PubMedDate not supplied

Assessing the impact of alternative splicing on domain interactions in the human proteome.

We have constructed a database of alternatively spliced protein forms (ASP), consisting of 13,384 protein isoform sequences of 4422 human genes (www.bioinformatics.ucla.edu/ASP). We identified fifty protein domain types that were selectively removed by alternative splicing at much higher frequencies than average (p-value < 0.01). These include many well-known protein-interaction domains (e.g., KRAB; ankyrin repeats; Kelch) including some that have been previously shown to be regulated functionally by alternative splicing (e.g., collagen domain). We present a number of novel examples (Kruppel transcription factors; Pbx2; Enc1) from the ASP database, illustrating how this pattern of alternative splicing changes the structure of a biological pathway, by redirecting protein interaction networks at key switch points. Our bioinformatics analysis indicates that a major impact of alternative splicing is removal of protein-protein interaction domains that mediate key linkages in protein interaction networks. ASP expands the available dataset of human alternatively spliced protein forms from 1989 human genes (SwissProt release 42) to 5413 (nonredundant set, ASP + SwissProt), a nearly 3-fold increase. ASP will enhance the existing pool of protein sequences that are searched by mass spectroscopy software during the identification of peptide fragments.

Alternative Splicing↗
PubMed1993

Nova, the paraneoplastic Ri antigen, is homologous to an RNA-binding protein and is specifically expressed in the developing motor system.

Paraneoplastic opsoclonus-ataxia, a disorder of motor control, develops in breast or lung cancer patients who harbor an antibody (Ri) that recognizes their tumors and a nuclear neuronal protein of 55 kd. We have characterized a gene, Nova, encoding an antigen recognized by the Ri antibody. Nova encodes a novel, highly conserved protein, homologous to the RNA-binding protein hnRNP K, the yeast splicing protein MER1, and a motif in several retroviral proteases. Northern blot analysis detects Nova transcripts only in brain, and several alternatively spliced forms are present in brain and tumor cells. Nova expression is restricted to the ventral brain stem and spinal cord in E18 mice. Since Nova encodes a target antigen in the motor disorder paraneoplastic opsoclonus-ataxia that is expressed in the developing subcortical motor system, it is a likely participant in both the pathogenesis of paraneoplastic opsoclonus-ataxia and the developmental biology of the motor system. The homology between Nova and hnRNP K suggests that Nova regulates RNA splicing or metabolism in a specific subset of developing neurons.

Amino Acid Sequence↗
PubMed1989

Mitochondrial splicing requires a protein from a novel helicase family.

Proteins involved in mitochondrial splicing but encoded by nuclear genes have been characterized in Saccharomyces and Neurospora. The role in splicing of these proteins is largely unknown. Here we report that mutations in the nuclear gene MSS116 directly affect the splicing of several introns of the cytochrome b (cob) and cytochrome c oxidase subunit I (cox1) primary transcripts. This implies that the MSS116 protein (pMSS116) is an important component of the mitochondrial splicing machinery. The sequence of the cloned MSS116 gene shows that its protein product is homologous to the translation eIF-4A factor and the human nuclear protein p68. We show further that these proteins share several conserved amino-acid blocks with DNA helicases and related proteins. This suggests that pMSS116 has an RNA helicase activity. RNA helicases may be involved in many different processes including translation and splicing.

Amino Acid Sequence↗
PubMed2000

Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803.

A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been shown to mediate efficient in vivo and in vitro trans-splicing in a foreign protein context. A cis-splicing Ssp DnaE intein construct displayed splicing activity similar to the trans-splicing form, which suggests that the N- and C-terminal intein fragments have a high affinity interaction. An in vitro trans-splicing system was developed that used a bacterially expressed N-terminal fragment of the Ssp DnaE intein and either a bacterially expressed or chemically synthesized intein C-terminal fragment. Unlike artificially split inteins, the Ssp DnaE intein fragments could be reconstituted in vitro under native conditions to mediate splicing as well as peptide bond cleavage. This property allowed the development of an on-column trans-splicing system that permitted the facile separation of reactants and products. Furthermore, the trans-splicing activity of the Ssp DnaE intein was successfully applied to the cyclization of proteins in vivo. Also, the isolation of the unspliced precursor on chitin resin allowed the cyclization reaction to proceed in vitro. The Ssp DnaE intein thus represents a potentially important protein for in vivo and in vitro protein manipulation.

Base Sequence↗
PubMed2005

Single-step affinity purification of toxic and non-toxic proteins on a fluidics platform.

Single-step fusion-based affinity purification of proteins with pH-controllable linkers was carried out in a fluidic device. The linkers were previously derived from self-splicing protein elements called inteins. Two different linkers were generated to solve two distinct separation problems: one for rapid single-step affinity purification of a wide range of proteins, and the other specifically for the purification of cytotoxic proteins. Scale-down factors of 185 resulted in separations in a 27 microl bed-volume. A rotating CD format was chosen because of its simplicity in effecting fluid movement through centrifugal force without the complications associated with electro-osmosis and other pumping methods. The design and fabrication of the fluidic device and the protein purification process are described. This work, which demonstrates the purification of active proteins by two distinct fluidic separations, is widely applicable to small-scale massively parallel proteomic separations.

Chromatography, Affinity↗
PubMed2000

Mitochondrial gene therapy: an arena for the biomedical use of inteins.

Mitochondrial DNA (mtDNA) mutations underlie many rare diseases and might also contribute to human ageing. Gene therapy is a tempting future possibility for intervening in mitochondriopathies. Expression of the 13 mtDNA-encoded proteins from nuclear transgenes (allotopic expression) might be the most effective gene-therapy strategy. Its only confirmed difficulty is the extreme hydrophobicity of these proteins, which prevents their import into mitochondria from the cytosol. Inteins (self-splicing 'protein introns') might offer a solution to this problem: their insertion into such transgenes could greatly reduce the encoded proteins' hydrophobicity, enabling import, with post-import excision restoring the natural amino acid sequence.

Animals↗
PubMed2000

Developmental expression of Y-box protein 1 mRNA and alternatively spliced Y-box protein 3 mRNAs in spermatogenic cells in mice.

Y-box proteins bind DNA and RNA and are characterized by a cold shock domain and a carboxyl-terminus containing clusters of aromatic and basic residues that alternate with clusters of acidic residues. Y-box proteins 1 and 3 in mouse testis were cloned here by 3' rapid amplification of cDNA ends (RACE) using a degenerate primer. Northern blots and reverse transcription-polymerase chain reaction (RT-PCR) established that the levels of Y-box protein 1 and 3 mRNAs are regulated individually: (i) Y-box protein 1 mRNA is strongly expressed in kidney, whereas Y-box protein 3 mRNA is strongly expressed in heart and muscle; (ii) Y-box protein 1 and 3 mRNAs are weakly expressed in early prepubertal testis and strongly expressed in pachytene spermatocytes, round spermatids, and elongated spermatids; and (iii) prepubertal testes and meiotic and haploid spermatogenic cells express two alternatively spliced Y-box protein 3 mRNAs encoding isoforms with different carboxyl termini, whereas somatic tissues primarily express one form. Sucrose gradients reveal that approximately 27% of both Y-box protein 3 mRNAs are translationally active in adult testis. In conclusion, spermatogenic cells in mice express five isoforms of Y-box proteins including Y-box protein 1, and two isoforms each of Y-box proteins 2 and 3. This multiplicity is intriguing because Y-box proteins are thought to activate transcription and repress translation in spermatogenic cells.

Alternative Splicing↗
PubMed2001

Expression of Golli mRNA during development in primary immune lymphoid organs of the rat.

The gene-of-the-oligodendrocyte lineage (Golli)-MBP transcription unit contains three Golli-specific exons together with eight exons of the "classical" myelin basic protein (MBP) gene, yielding alternatively spliced proteins which share amino acid sequence with MBP. Unlike MBP, a late antigen expressed only in the nervous system, Golli gene products are expressed pre- and post-natally at many sites. In this study, we determined the sequence of Golli in rat by RT-PCR and 5' RACE and showed that Golli sequences are expressed in primary lymphoid organs as early as e16.5, which could explain the anergic rat T cell response we previously observed in Golli-induced meningitis.

Amino Acid Sequence↗
PubMed2000

Crystallographic study of intein homing endonuclease II encoded in the archaeal DNA polymerase gene.

Intein homing endonucleases are proteins spliced out from a precursor protein and site-specific enzymes that make double-strand breaks in inteinless alleles. Crystals of intein homing endonuclease II from the hyperthermophilic archaeon Pyrococcus kodakaraensis strain KOD1 (PI-PkoII) have been grown at room temperature using ammonium sulfate as a precipitant. The diffraction pattern of the crystal extends to 3.0 A resolution at room temperature upon exposure to synchrotron X-rays at KEK-PF, Japan. The crystals have symmetry consistent with space group C222(1), with unit-cell parameters a = 107.6, b = 150.5, c = 146.8 A. A full set of X-ray diffraction data were collected to 3.0 A Bragg spacing from a native crystal with an overall R(merge) of 4.8% and a completeness of 96.6%.

Archaeal Proteins↗
PubMed2004

Novel lipid modifications of secreted protein signals.

Secreted signaling proteins function in a diverse array of essential patterning events during metazoan development, ranging from embryonic segmentation in insects to neural tube differentiation in vertebrates. These proteins generally are expressed in a localized manner, and they may elicit distinct concentration-dependent responses in the cells of surrounding tissues and structures, thus functioning as morphogens that specify the pattern of cellular responses by their tissue distribution. Given the importance of signal distribution, it is notable that the Hedgehog (Hh) and Wnt proteins, two of the most important families of such signals, are known to be covalently modified by lipid moieties, the membrane-anchoring properties of which are not consistent with passive models of protein mobilization within tissues. This review focuses on the mechanisms underlying biogenesis of the mature Hh proteins, which are dually modified by cholesteryl and palmitoyl adducts, as well as on the relationship between Hh proteins and the self-splicing proteins (i.e., proteins containing inteins) and the Hh-like proteins of nematodes. We further discuss the cellular mechanisms that have evolved to handle lipidated Hh proteins in the spatial deployment of the signal in developing tissues and the more recent findings that implicate palmitate modification as an important feature of Wnt signaling proteins.

Amino Acid Sequence↗
PubMed1996

Nuclear morphogenesis and the onset of transcriptional activity in early hamster embryos.

Coiled bodies and interchromatin granules are distinct subnuclear domains that contain splicing small nuclear ribonucleoproteins (snRNPs) and protein-splicing factors. Here we have studied the morphogenesis of coiled bodies and clusters of interchromatin granules in relation to the onset of transcriptional activity in early hamster embryos. The results indicate that major embryonic transcription by RNA polymerase II is first detected during the early two-cell stage (15-20 h post-fertilization), whereas RNA polymerase I activity and nucleologenesis are only observed in late two-cell embryos (30-40 h postfertilization). Splicing snRNPs and heterogeneous nuclear RNP (hnRNP) proteins are shown to be imported into the pronuclei following fertilization, and prominent clusters of interchromatin granules containing the splicing factor SC-35 are already observed in both maternal and paternal pronuclei of one-cell embryos. Interestingly, these large clusters of interchromatin granules do not appear to concentrate splicing snRNPs. In contrast, coiled bodies are first detected during the two-cell stage after the onset of transcription, and they are clearly enriched in snRNPs. Taken together with results previously obtained in mouse embryos, these data suggest that the assembly of coiled bodies and clusters of interchromatin granules is independent from the onset of embryonic transcriptional activity, and that coiled bodies represent the major snRNP-enriched subnuclear domain in the early mammalian embryo.

Animals↗
PubMed1999

Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation.

A method is described to express and purify human DNA (cytosine-5) methyltransferase (human DNMT1) using a protein splicing (intein) fusion partner in a baculovirus expression vector. The system produces approximately 1 mg of intact recombinant enzyme >95% pure per 1.5 x 10(9) insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two C-terminal amino acids, proline and glycine, that were substituted for lysine and aspartic acid for optimal cleavage from the intein affinity tag. Human DNMT1 was used for steady-state kinetic analysis with poly(dI-dC).poly(dI-dC) and unmethylated and hemimethylated 36- and 75-mer oligonucleotides. The turnover number (k(cat)) was 131-237 h(-1) on poly(dI-dC).poly(dI-dC), 1.2-2.3 h(-1) on unmethylated DNA, and 8.3-49 h(-1) on hemimethylated DNA. The Michaelis constants for DNA (K(m)(CG)) and S-adenosyl-L-methionine (AdoMet) (K(m)(AdoMet)) ranged from 0.33-1.32 and 2.6-7.2 microM, respectively, whereas the ratio of k(cat)/K(m)(CG) ranged from 3.9 to 44 (237-336 for poly(dI-dC).poly(dI-dC)) x 10(6) M(-1) h(-1). The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7-21-fold. The values of k(cat) on hemimethylated DNAs showed a 2-3-fold difference, depending upon which strand was pre-methylated. Furthermore, human DNMT1 formed covalent complexes with substrates containing 5-fluoro-CNG, indicating that substrate specificity extended beyond the canonical CG dinucleotide. These results show that, in addition to maintenance methylation, human DNMT1 may also carry out de novo and non-CG methyltransferase activities in vivo.

Animals↗