A protein splice-junction motif in hedgehog family proteins.
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Amino Acid Sequence↗
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Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein.
Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, we characterized the binding of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. We also identified a new 60-kDa tissue-specific protein that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins.
Amino Acid Sequence↗
Genomic structure of the locus encoding protein 4.1. Structural basis for complex combinational patterns of tissue-specific alternative RNA splicing.
Protein 4.1 (P4.1) is a multifunctional protein with heterogeneity in molecular weight, intracellular localization, tissue- and development-specific expression patterns. We have analyzed the genomic structure of the locus encoding mouse P4.1 and have systematically analyzed diverse P4.1 mRNA isoforms expressed in erythroid and nonerythroid tissues. Our results indicate that the mouse protein 4.1 gene, over 90 kilobases long, comprises at least 23 exons (13 constitutive exons, 10 alternative exons) interrupted by 22 introns. The donor and acceptor splice site sequences match the consensus sequences for the exon-intron boundaries of most eukaryotic genes. No significant sequence difference was observed between splice junctions of alternative and constitutive exons. Apparently, most alternative exon-encoded peptides are located within particular functional domains of the P4.1 protein: two peptides encoded by alternative exons 4 and 5 are located near or within the glycophorin/calmodulin binding domain, whereas three other alternative exon-encoded peptides (19-amino acid encoded by exon 14, 14-amino acid encoded by exon 15, and 21-amino acid encoded by exon 16) are located near or within the spectrin-actin binding domain. Selective use of exon 2', which carries an upstream translation initiation codon (AUG), may produce an elongated P4.1 isoform (135 kDa) that is predominantly expressed in nonerythroid tissues. Combinatorial splicing of these exons may generate different isoforms that exhibit complicated tissue-specific expression patterns.
3T3 Cells↗
A large deletion within the protein 4.1 gene associated with a stable truncated mRNA and an unaltered tissue-specific alternative splicing.
Protein 4.1 is a major protein of the red blood cell skeleton. It binds to the membrane through its 30-kD N-terminal domain and to the spectrin-actin lattice through its 10-kD domain. We describe here the molecular basis of a heterozygous hereditary elliptocytosis (HE) associated with protein 4.1 partial deficiency. The responsible allele displayed a greater than 70-kb genomic deletion, beginning within intron 1 and ending within a 1.3-kb region upstream from exon 13. This deletion encompassed both erythroid and nonerythroid translation initiation sites. It accounts for the largest deletion known in genes encoding proteins of the red blood cell membrane. The corresponding mRNA was shortened by 1727 bases, due to the absence of exons 2 to 12. Nevertheless, this mRNA was stable. It showed a similar pattern in lymphoblastoid cells as in reticulocytes. Differential splicing of exons within the undeleted region remained regulated in a tissue-specific manner. Exons 14, 15, and 17a were absent from both reticulocyte and lymphocyte mRNAs, whereas exon 16 was present in reticulocytes but absent from lymphocytes. Thus, differential splicing on a local scale was not dependent on the overall structure of protein 4.1 mRNA in this particular instance.
Alleles↗
Intein-mediated reporter gene assay for detecting protein-protein interactions in living mammalian cells.
For nondestructive analysis of chemical processes in living mammalian cells, here we developed a new reporter gene assay for detecting cytosolic protein-protein interactions based on protein splicing of transcription factors with DnaE inteins. The protein splicing induces connection of a DNA-binding protein (modified LexA; mLexA) with a transcription activation domain of a herpes simplex virus protein (VP16AD). We thereby circumvented the limitation of earlier methods for monitoring protein-protein interactions, including the two-hybrid systems, protein complementation systems (PCS), and protein reconstitution systems, and rather combined their advantages. To test the applicability of this method, we monitored epidermal growth factor (EGF)-induced interactions on cell membranes of a known partner, an oncogenic product Ras and its target Raf-1. Ras was connected with N-terminal DnaE and mLexA, while Raf-1 was connected with C-terminal DnaE and VP16AD. Upon stimulation with EGF, the interaction between Ras and Raf-1 triggered folding of the DnaEs, thereby inducing protein splicing to form mLexA-VP16AD fusion protein, and transcription of a reporter gene, firefly luciferase. The extent of Ras-Raf-1 interaction was quantified by measuring the luciferase activity. The interaction was not able to be monitored by two-hybrid systems nor by PCS of split firefly luciferases; however, by using the protein splicing elements and the reporter gene, we obtained the bioluminescence signals sufficient for evaluation of the interactions close to cell membranes.
Animals↗
Identification and characterization of a novel serine-arginine-rich splicing regulatory protein.
We have identified an 86-kDa protein containing a single amino-terminal RNA recognition motif and two carboxy-terminal domains rich in serine-arginine (SR) dipeptides. Despite structural similarity to members of the SR protein family, p86 is clearly unique. It is not found in standard SR protein preparations, does not precipitate in the presence of high magnesium concentrations, is not recognized by antibodies specific for SR proteins, and cannot complement splicing-defective S100 extracts. However, we have found that p86 can inhibit the ability of purified SR proteins to activate splicing in S100 extracts and can even inhibit the in vitro and in vivo activation of specific splice sites by a subset of SR proteins, including ASF/SF2, SC35, and SRp55. In contrast, p86 activates splicing in the presence of SRp20. Thus, it appears that pairwise combination of p86 with specific SR proteins leads to altered splicing efficiency and differential splice site selection. In all cases, such regulation requires the presence of the two RS domains and a unique intervening EK-rich region, which appear to mediate direct protein-protein contact between these family members. Full-length p86, but not a mutant lacking the RS-EK-RS domains, was found to preferentially interact with itself, SRp20, ASF/SF2, SRp55, and, to a slightly lesser extent, SC35. Because of the primary sequence and unique properties of p86, we have named this protein SRrp86 for SR-related protein of 86 kDa.
Amino Acid Sequence↗
Thyroid hormones regulate beta-amyloid gene splicing and protein secretion in neuroblastoma cells.
The beta-amyloid protein (Abeta), the major component of the senile plaques found in Alzheimer brains, derives from a larger beta-amyloid precursor protein (APP). Alternative splicing of the APP gene yields three major APP messenger RNAs (mRNAs), which, in turn, give rise to the APP770, APP751, and APP695 protein isoforms. In this study we examined the effects of thyroid hormone on APP expression in N2a-beta neuroblastoma cells. T3 caused a significant increase in the APP770 mRNA band, in detriment of the APP695 mRNA, which was proportionately reduced. In agreement with these results, T3 markedly altered the relative ratio of intracellular APP isoforms, increasing the amount of APP770 and causing an equivalent reduction of the immature APP695 isoform. In accordance with these results, the soluble APP695-derived form was specifically reduced in the culture medium obtained from T3-treated cells. In contrast, the increase in intracellular APP770 was not followed by an enhanced release of soluble derivatives of this isoform. These results suggest that T3 regulates not only APP gene splicing, but also the processing and secretion of the APP peptides. According to our results, thyroid hormone might play a role in the development of Alzheimer's disease by modulating the intracellular and extracellular contents of APP isoforms.
Alternative Splicing↗
Putative tumor suppressor protein 4.1B is differentially expressed in kidney and brain via alternative promoters and 5' alternative splicing.
Protein 4.1B has been reported as a tumor suppressor in brain, but not in kidney, despite high expression in both tissues. Here we demonstrate that N-terminal variability in kidney and brain 4.1B isoforms arises through an unusual coupling of RNA processing events in the 5' region of the gene. We describe two transcriptional promoters at far upstream alternative exons 1A and 1B, and show that their respective transcripts splice differentially to exon 2'/2 in a manner that determines mRNA coding capacity. The consequence of this unique processing is that exon 1B transcripts initiate translation at AUG1 (in exon 2') and encode larger 4.1B isoforms with an N-terminal extension; exon 1A transcripts initiate translation at AUG2 (in exon 4) and encode smaller 4.1B isoforms. Tissue-specific differences in promoter utilization may thus explain the abundance of larger 4.1B isoforms in brain but not in kidney. In cell studies, differentiation of PC12 cells was accompanied by translocation of large protein 4.1B isoforms into the nucleus. We propose that first exon specification is coupled to downstream splicing events, generating 4.1B isoforms with diverse roles in kidney and brain physiology, and potentially unique functions in cell proliferation and tumor suppression.
Alternative Splicing↗
Inteins as targets for potential antimycobacterial drugs.
Protein splicing is a self-catalyzed process mediated by inteins. The observation that inteins occur only in microorganisms and that they often interrupt genes that play an essential role in nucleic acid metabolism makes them attractive as potential antibacterial targets. Because mycobacteria are the only intein-containing bacteria associated with human hosts, inteins would represent highly specific antimycobacterial targets. In Mycobacterium tuberculosis, two important proteins of DNA repair and replication, RecA and DnaB, respectively, are interrupted by inteins that must be excised by protein splicing before these proteins can function. This review describes the screening systems for the detection of mutations or inhibitors that interfere with proteins splicing that have been developed and published to date. In three of these experimental system, inteins have been inserted into proteins that are toxic under certain conditions. Protein splicing therefore leads to conditional growth inhibition or cell death and its inhibition can be monitored in terms of bacterial growth. A fourth assay for protein splicing and its inhibition is based on purified proteins and measures the formation of Green Fluorescent Protein or its inhibition. The advantages of inteins as antimycobacterial targets are discussed.
Animals↗
Degeneration of a homing endonuclease and its target sequence in a wild yeast strain.
Mobile introns and inteins self-propagate by 'homing', a gene conversion process initiated by site-specific homing endonucleases. The VMA intein, which encodes the PI-SceI endonuclease in Saccharomyces cerevisiae, is present in several different yeast strains. Surprisingly, a wild wine yeast (DH1-1A) contains not only the intein(+) allele, but also an inteinless allele that has not undergone gene conversion. To elucidate how these two alleles co-exist, we characterized the endonuclease encoded by the DH1-1A intein(+) allele and the target site in the intein(-) allele. Sequence analysis reveals seven mutations in the 31 bp recognition sequence, none of which occurs at positions that are individually critical for activity. However, binding and cleavage of the sequence by PI-SceI is reduced 10-fold compared to the S.cerevisiae target. The PI-SceI analog encoded by the DH1-1A intein(+) allele contains 11 mutations at residues in the endonuclease and protein splicing domains. None affects protein splicing, but one, a R417Q substitution, accounts for most of the decrease in DNA cleavage and DNA binding activity of the DH1-1A protein. Loss of activity in the DH1-1A endonuclease and target site provides one explanation for co-existence of the intein(+) and intein(-) alleles.
Alleles↗
Two novel arginine/serine (SR) proteins in maize are differentially spliced and utilize non-canonical splice sites.
The serine-arginine (SR)-rich splicing proteins are highly conserved RNA binding nuclear phosphor-proteins that play important roles in both regular and alternative splicing. Here we describe two novel putative SR genes from maize, designated zmRSp31A and zmRSp31B. Both genes contain characteristic RNA binding motifs RNP-1 and RNP-2, a serine/arginine-rich (RS) domain and share significant sequence similarity to the Arabidopsis atRSp31 family of SR proteins. Both zmRSp31A and zmRSp31B produce multiple transcripts by alternative splicing, of which majority of the alternatively spliced transcripts utilize non-canonical splice sites. zmRSp31A and zmRSp31B produce at least six and four transcripts, respectively, of which only one corresponds to the wild type proteins for each gene. All the alternatively spliced transcripts of both the genes, with one exception, are predicted to encode small truncated proteins containing only the RNP-2 domain of their first RNA recognition motif and completely lack the carboxyl terminal RS domain. We provide evidence that some of the alternatively spliced transcripts of both genes are associated with polysomes and interact with the translational machinery.
Alternative Splicing↗
Distribution and function of new bacterial intein-like protein domains.
Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein-splicing of their host-protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N-terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein-like (BIL) domains. Bacterial intein-like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C-terminal flank and could also protein-splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.
Amino Acid Sequence↗
Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs.
Surfactant protein C (SP-C), a hydrophobic protein of pulmonary surfactant is essential for surfactant function. Toward elucidating molecular mechanisms that mediate regulation of SP-C gene expression in rabbit lung, we isolated and characterized cDNAs encoding rabbit SP-C and studied the regulation of SP-C gene expression during fetal lung development and by adenosine 3',5'-cyclic monophosphate (cAMP) and dexamethasone in fetal lung tissues in vitro. We found that rabbit SP-C is highly homologous to SP-C of other species and is encoded by two mRNAs that differ by an insertion of 31 nucleotides in the 3' untranslated regions. SP-C mRNAs were classified into two types based on the nucleotide sequence; type I represents RNA without the 31 nucleotide insert and comprises approximately 80-90% of total SP-C mRNA content, whereas type II represents RNA containing the insert and comprises approximately 10-20% of total SP-C mRNA content. SP-C mRNAs were induced in a coordinate manner during fetal lung development and by cAMP and dexamethasone in fetal lung tissues in vitro. Southern hybridization analysis of genomic DNA suggested that SP-C mRNAs are encoded by a single gene. Polymerase [corrected] chain reaction-amplification of genomic DNA with oligonucleotide primers flanking the insertional sequence and sequence analysis of amplified DNA showed that SP-C mRNAs are produced by alternative use of 3' splice sites of intron 5 of SP-C gene.
Amino Acid Sequence↗
Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.
We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.
Alternative Splicing↗
Inhibition by SR proteins of splicing of a regulated adenovirus pre-mRNA.
The adenovirus L1 unit represents an example of an alternatively spliced precursor messenger (pre-mRNA) where on 5' splice can be jointed to one of two alternative 3' splice sites, producing the 52,55K or the IIIa mRNAs (Fig. 1a). Efficient usage of the distal IIIa 3' splice site requires late viral protein synthesis and is therefore confined to the late phase of virus infection. Here we show that, in extracts from uninfected cells, the classical SR proteins, which are essential splicing factors, inhibit IIIa pre-mRNA splicing by binding to an intronic repressor element and preventing recruitment of the U2 small nuclear ribonucleoprotein particle to the spliceosome. We further show that the viral repressor element has splicing-enhancer activity when appropriately placed in the pre-mRNA. Together, our results demonstrate that SR proteins function as activators or repressors of splicing depending on where on the pre-mRNA they bind.
Adenoviridae↗
Modular organization of inteins and C-terminal autocatalytic domains.
Analysis of the conserved sequence features of inteins (protein "introns") reveals that they are composed of three distinct modular domains. The N-terminal (N) and C-terminal (C) domains are predicted to perform different parts of the autocatalytic protein splicing reaction. An optional endonuclease domain (EN) is shown to correspond to different types of homing endonucleases in different inteins. The N domain contains motifs predicted to catalyze the first steps of protein splicing, leading to the cleavage of the intein N terminus from its protein host. Intein N domain motifs are also found in C-terminal autocatalytic domains (CADs) present in hedgehog and other protein families. Specific residues in the N domain of intein and CADs are proposed to form a charge relay system involved in cleaving their N-termini. The intein C domain is apparently unique to inteins and contains motifs that catalyze the final protein splicing steps: ligation of the intein flanks and cleavage of its C terminus to release the free intein and spliced host protein. All intein EN domains known thus far have dodecapeptide (DOD, LAGLI-DADG) type homing endonuclease motifs. This work identifies an EN domain with an HNH homing-endonuclease motif and two new small inteins with no EN domains. One of these small inteins might be inactive or a "pseudo intein." The results suggest a modular architecture for inteins, clarify their origin and relationship to other protein families, and extend recent experimental findings on the functional roles of intein N, C, and EN motifs.
Amino Acid Sequence↗
The splicing regulatory protein p18SRP is down-regulated in Alzheimer's disease brain.
Alzheimer's disease (AD) is the most common neurodegenerative disorder of aging, accounting for an estimated two-thirds of all cases of senile dementia. Using bioinformatics, the yeast two-hybrid-system, reverse transcription polymerase chain reaction, and fluorescence microscopy analysis, we demonstrate here that the new putative splicing regulatory protein p18SRP is a lysine-rich zinc finger domain-containing protein that interacts with the serine-arginine (SR)-rich splicing regulatory protein SRrp86. The additional finding of its down-regulation in the brain of AD subjects points to a possible pivotal role of p18SRP in the control of cellular survival.
Aged↗
A nuclear-encoded intein in the fungal pathogen Cryptococcus neoformans.
We have used comparative sequence analysis to identify an intein-like sequence (protein splicing element) present in Cryptococcus neoformans, a fungal pathogen of humans. The sequence encoding this element is present in the C. neoformans PRP8 gene, as an in-frame insertion relative to the PRP8 genes of other organisms. It contains sequences similar to those of the protein-splicing domains of two previously described yeast inteins (in Saccharomyces cerevisiae and Candida tropicalis), although it lacks any recognizable internal endonuclease domain. The Cryptococcus neoformans intein (Cne PRP8) is only the second to be found in a eukaryote nuclear genome; the previously described yeast inteins occur at the same site in the VMA gene homologues of S. cerevisiae and C. tropicalis. The host gene of the Cryptococcus intein, PRP8, encodes a highly conserved mRNA splicing protein found as part of the spliceosome. The Cne PRP8 intein may be a useful drug target in addressing the cryptococcal infections so prevalent in AIDS patients.
Amino Acid Sequence↗