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PubMed2003

Analysis of differential gene expression in Echinococcus multilocularis larval stages by means of spliced leader differential display.

All parasitic helminths process a large subset of their mRNA molecules via spliced leader trans-splicing. Here we describe a rapid and efficient method, 'spliced leader differential display', to generate comprehensive expression fingerprints of trans-spliced factors on silver-stained acrylamide gels. The method allows systematic screening of the entire trans-spliced transcriptome of parasitic organisms using only minute amounts of starting material. Spliced leader differential display selectively displays 5' end-fragments of trans-spliced messages thus facilitating the identification of associated proteins and the full-length characterisation of corresponding mRNAs. We employed spliced leader differential display to analyse gene expression patterns of in vitro cultivated protoscoleces and metacestode vesicles from Echinococcus multilocularis. These analyses revealed that the majority of trans-spliced mRNAs are expressed at equal amounts in both larval stages whereas about 25% of the identified transcripts displayed stage-specific differential expression. Full-length transcripts for several of the obtained fragments were isolated and their constitutive or differential expression was verified by competitive PCR and Virtual Northern blotting. Spliced leader differential display was further used to identify genes which are induced in the E. multilocularis metacestode stage under growth promoting conditions in the presence of host hepatocytes. One mRNA, encoding a putative member of the epidermal growth factor family of mitogens, was shown to be 10-fold induced upon co-incubation of metacestode vesicles with host cells which indicates a possible involvement of parasite-determined, epidermal growth factor-like signal transduction systems in metacestode proliferation and development. The successful application of spliced leader differential display to gene expression analyses of echinococcal larvae, combined with easy handling and a low rate of false positives, recommends this method not only for studies on the host-parasite interplay during alveolar echinococcosis, but also for investigations on other parasitic organisms.

Adolescent↗
PubMed1998

Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein and of a trans-splicing system involving two intein fragments.

Most protein-splicing elements (inteins) function both as catalysts of protein splicing and as homing endonucleases. In order to identify the domains of inteins that are essential for protein splicing, the intein sequence embedded in the recA gene of Mycobacterium tuberculosis was genetically dissected. The effect of various modifications of the intein on the ability to mediate splicing was studied in Escherichia coli transformed with plasmids in which the coding sequence for the RecA intein was inserted in-frame between coding regions for the E. coli maltose-binding protein and a polypeptide containing a hexahistidine sequence as the N- and C-exteins, respectively. One type of genetic alteration of the RecA intein involved deletion of the central region encoding 229 amino acids (aa), representing the entire homing endonuclease homology domain. The residual intein (211 aa plus an undecapeptide spacer) was able to promote protein splicing as efficiently as the wild-type intein, indicating that the homing endonuclease domain plays no role in the protein-splicing process and that the protein-splicing active center is confined to the N- and C-terminal segments of the intein, less than 110 aa each. Another type of alteration involved the introduction of overlapping translation termination and initiation codons in-frame into the intein coding region. The modified RecA intein, although synthesized as two separate components, could nevertheless mediate protein splicing, indicating that the N- and C-terminal protein-splicing domains can interact with sufficient affinity and specificity to allow protein-splicing to occur in trans. The efficiency of trans-splicing was much enhanced when the homing endonuclease domain was entirely deleted so that the length of the interacting N- and C-terminal intein fragments was only about 110 aa each.

Bacillus↗
PubMed2001

Cis-acting elements regulate alternative splicing of exons 6A, 6B and 8 of the alpha1(XI) collagen gene and contribute to the regional diversification of collagen XI matrices.

Consecutive exons 6A, 6B, 7 and 8 that encode the variable region of the amino-terminal domain (NTD) of the col11a1 gene product undergo a complex pattern of alternative splicing that is both tissue-dependent and developmentally regulated. Expression of col11a1 is predominantly associated with cartilage where it plays a critical role in skeletal development. At least five splice-forms (6B-7-8, 6A-7-8, 7-8, 6B-7 and 7) are found in cartilage. Splice-forms containing exon 6B or 8 have distinct distributions in the long bone during development, while in non-cartilage tissues, splice-form 6A-7-8 is typically expressed. In order to study this complex and tissue-specific alternative splicing, a mini-gene that contains mouse genomic sequence from exon 5 to 11, flanking the variable region of alpha1(XI)-NTD, was constructed. The minigene was transfected into chondrocytic (RCS) and non-chondrocytic (A204) cell lines that endogenously express alpha1(XI), as well as 293 cells which do not express alpha1(XI). Alternative splicing in RCS and A204 cells reflected the appropriate cartilage and non-cartilage patterns while 293 cells produced only 6A-7-8. This suggests that 6A-7-8 is the default splicing pathway and that cell or tissue-specific trans-acting factors are required to obtain pattern of the alternative splicing of alpha1(XI) pre-mRNA observed in chondrocytes. Deletional analysis was used to identify cis-acting regions important for regulating splicing. The presence of the intact exon 7 was required to generate the full complex chondrocytic pattern of splicing. Furthermore, deletional mapping of exon 6B identified sequences required for expression of exon 6B in RCS cells and these may correspond to purine-rich (ESE) and AC-rich (ACE) exonic splicing enhancers.

Alternative Splicing↗
PubMed1998

Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum.

The human HPRT gene contains spans approximately 42,000 base pairs in genomic DNA, has a mRNA of approximately 900 bases and a protein coding sequence of 657 bases (initiation codon AUG to termination codon UAA). This coding sequence is distributed into 9 exons ranging from 18 (exon 5) to 184 (exon 3) base pairs. Intron sizes range from 170 (intron 7) to 13,075 (intron 1) base pairs. In a database of human HPRT mutations, 277 of 2224 (12.5%) mutations result in alterations in splicing of the mRNA as analyzed by both reverse transcriptase mediated production of a cDNA followed by PCR amplification and cDNA sequencing and by genomic DNA PCR amplification and sequencing. Mutations have been found in all eight 5' (donor) and 3' (acceptor) splice sequences. Mutations in the 5' splice sequences of introns 1 and 5 result in intron inclusion in the cDNA due to the use of cryptic donor splice sequences within the introns; mutations in the other six 5' sites result in simple exon exclusion. Mutations in the 3' splice sequences of introns 1, 3, 7 and 8 result in partial exon exclusion due to the use of cryptic acceptor splice sequences within the exons; mutations in the other four 3' sites result in simple exon exclusion. A base substitution in exon 3 (209G-->T) creates a new 5' (donor) splice site which results in the exclusion of 110 bases of exon 3 from the cDNA. Two base substitutions in intron 8 (IVS8-16G-->A and IVS8-3T-->G) result in the inclusion of intron 8 sequences in the cDNA due to the creation of new 3' (acceptor) splice sites. Base substitution within exons 1, 3, 4, 6 and 8 also result in splice alterations in cDNA. Those in exons 1 and 6 are at the 3' end of the exon and may directly affect splicing. Those within exons 3 and 4 may be the result of the creation of nonsense codons, while those in exon 8 cannot be explained by this mechanism. Lastly, many mutations that affect splicing of the HPRT mRNA have pleiotropic effects in that multiple cDNA products are found.

Animals↗
PubMed2002

Defining a 5' splice site by functional selection in the presence and absence of U1 snRNA 5' end.

Pre-mRNA splicing in metazoans is mainly specified by sequences at the termini of introns. We have selected functional 5' splice sites from randomized intron sequences through repetitive rounds of in vitro splicing in HeLa cell nuclear extract. The consensus sequence obtained after one round of selection in normal extract closely resembled the consensus of natural occurring 5' splice sites, suggesting that the selection pressures in vitro and in vivo are similar. After three rounds of selection under competitive splicing conditions, the base pairing potential to the U1 snRNA increased, yielding a G100%U100%R94%A67%G89%U76%R83% intronic consensus sequence. Surprisingly, a nearly identical consensus sequence was obtained when the selection was performed in nuclear extract containing U1 snRNA with a deleted 5' end, suggesting that other factors than the U1 snRNA are involved in 5' splice site recognition. The importance of a consecutive complementarity between the 5' splice site and the U1 snRNA was analyzed systematically in the natural range for in vitro splicing efficiency and complex formation. Extended complementarity was inhibitory to splicing at a late step in spliceosome assembly when pre-mRNA substrates were incubated in normal extract, but favorable for splicing under competitive splicing conditions or in the presence of truncated U1 snRNA where transition from complex A to complex B occurred more rapidly. This suggests that stable U1 snRNA binding is advantageous for assembly of commitment complexes, but inhibitory for the entry of the U4/U6.U5 tri-snRNP, probably due to a delayed release of the U1 snRNP.

3' Untranslated Regions↗
PubMed1998

Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo.

The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA-protein interactions in HIV both in vivo and in vitro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td) intron in vivo. All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro. Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA. In the absence of translation, two interactions between sequences in the upstream exon and the 5' and 3' splice sites trap the pre-mRNA in splicing-incompetent conformations. Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B. Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA. Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation. This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E. coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing is caused by an alternative structural element containing a cryptic 5' splice site, which serves as a substrate for the ribozyme. Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.

Animals↗
PubMed1999

Spliceosome-mediated RNA trans-splicing as a tool for gene therapy.

We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-mRNA encoding the beta-subunit of human chorionic gonadotropin gene 6 and pre-trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis- and trans-splicing demonstrated that cis-splicing can be inhibited by trans-splicing. RNA repair in a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significant beta-galactosidase activity. These observations suggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy.

Animals↗
PubMed2000

dull: rice mutants with tissue-specific effects on the splicing of the waxy pre-mRNA.

In the endosperm of japonica rice, du-1 and du-2 mutations cause the reduction of amylose contents. It was previously shown that the Wx(b) allele of rice, which is predominantly distributed in japonica rice, has a mutation in the 5' splice site of intron 1 resulting in the creation of two weak 5' splice sites within exon 1. In du-1 and du-2 mutants, spliced Wx(b) transcripts were highly reduced, whereas the processing of transcripts derived from three other genes highly expressed in endosperm was not apparently influenced. Results of competitive RT-PCR analysis indicate that transcripts spliced at the two newly created 5' splice sites were equally affected in these two mutants. Genetic and molecular analyses of the effects of du-1 and du-2 on Wx(a) pre-mRNA with normal splice sites indicate that these two mutations do not affect the processing of Wx(a) pre-mRNA after splicing, suggesting that du-1 and du-2 are mutations of genes required for the efficient splicing of mutated Wx(b) pre-mRNA. Furthermore, du-1 and du-2 showed differential effects in endosperm and pollen. Although both mutations caused similar effects on the splicing of Wx(a) transcripts in endosperm, du-1 caused higher reduction of Wx(b) mRNA in pollen than in endosperm, while du-2 had a lesser effect in pollen than in endosperm. Based on these results, we propose that the du-1 and du-2 loci of rice encode tissue-specifically regulated splicing factors that are involved in alternative splicing of pre-mRNA in rice.

Alternative Splicing↗
PubMed2004

A variable dinucleotide repeat in the CFTR gene contributes to phenotype diversity by forming RNA secondary structures that alter splicing.

Dinucleotide repeats are ubiquitous features of eukaryotic genomes that are not generally considered to have functional roles in gene expression. However, the highly variable nature of dinucleotide repeats makes them particularly interesting candidates for modifiers of RNA splicing when they are found near splicing signals. An example of a variable dinucleotide repeat that affects splicing is a TG repeat located in the splice acceptor of exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Higher repeat numbers result in reduced exon 9 splicing efficiency and, in some instances, the reduction in full-length transcript is sufficient to cause male infertility due to congenital bilateral absence of the vas deferens or nonclassic cystic fibrosis. Using a CFTR minigene system, we studied TG tract variation and observed the same correlation between dinucleotide repeat number and exon 9 splicing efficiency seen in vivo. Replacement of the TG dinucleotide tract in the minigene with random sequence abolished splicing of exon 9. Replacements of the TG tract with sequences that can self-base-pair suggested that the formation of an RNA secondary structure was associated with efficient splicing. However, splicing efficiency was inversely correlated with the predicted thermodynamic stability of such structures, demonstrating that intermediate stability was optimal. Finally, substitution with TA repeats of differing length confirmed that stability of the RNA secondary structure, not sequence content, correlated with splicing efficiency. Taken together, these data indicate that dinucleotide repeats can form secondary structures that have variable effects on RNA splicing efficiency and clinical phenotype.

Base Sequence↗
PubMed2004

Variation in sequence and organization of splicing regulatory elements in vertebrate genes.

Although core mechanisms and machinery of premRNA splicing are conserved from yeast to human, the details of intron recognition often differ, even between closely related organisms. For example, genes from the pufferfish Fugu rubripes generally contain one or more introns that are not properly spliced in mouse cells. Exploiting available genome sequence data, a battery of sequence analysis techniques was used to reach several conclusions about the organization and evolution of splicing regulatory elements in vertebrate genes. The classical splice site and putative branch site signals are completely conserved across the vertebrates studied (human, mouse, pufferfish, and zebrafish), and exonic splicing enhancers also appear broadly conserved in vertebrates. However, another class of splicing regulatory elements, the intronic splicing enhancers, appears to differ substantially between mammals and fish, with G triples (GGG) very abundant in mammalian introns but comparatively rare in fish. Conversely, short repeats of AC and GT are predicted to function as intronic splicing enhancers in fish but are not enriched in mammalian introns. Consistent with this pattern, exonic splicing enhancer-binding SR proteins are highly conserved across all vertebrates, whereas heterogeneous nuclear ribonucleoproteins, which bind many intronic sequences, vary in domain structure and even presence/absence between mammals and fish. Exploiting differences in intronic sequence composition, a statistical model was developed to predict the splicing phenotype of Fugu introns in mammalian systems and was used to engineer the spliceability of a Fugu intron in human cells by insertion of specific sequences, thereby rescuing splicing in human cells.

Animals↗
PubMed2003

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts.

The multiplicity of proteins compared with genes in mammals owes much to alternative splicing. Splicing signals are so subtle and complex that small perturbations may allow the production of new mRNA variants. However, the flexibility of splicing can also be a liability, and several genetic diseases result from single-base changes that cause exons to be skipped during splicing. Conventional oligonucleotide strategies can block reactions but cannot restore splicing. We describe here a method by which the use of a defective exon was restored. Spinal muscular atrophy (SMA) results from mutations of the Survival Motor Neuron (SMN) gene. Mutations of SMN1 cause SMA, whereas SMN2 acts as a modifying gene. The two genes undergo alternative splicing with SMN1, producing an abundance of full-length mRNA transcripts, whereas SMN2 predominantly produces exon 7-deleted transcripts. This discrepancy is because of a single nucleotide difference in SMN2 exon 7, which disrupts an exonic splicing enhancer containing an SF2ASF binding site. We have designed oligoribonucleotides that are complementary to exon 7 and contain exonic splicing enhancer motifs to provide trans-acting enhancers. These tailed oligoribonucleotides increased SMN2 exon 7 splicing in vitro and rescued the incorporation of SMN2 exon 7 in SMA patient fibroblasts. This treatment also resulted in the partial restoration of gems, intranuclear structures containing SMN protein that are severely reduced in patients with SMA. The use of tailed antisense oligonucleotides to recruit positively acting factors to stimulate a splicing reaction may have therapeutic applications for genetic disorders, such as SMA, in which splicing patterns are altered.

Alternative Splicing↗
PubMed1985

mRNA precursor splicing in vivo: sequence requirements determined by deletion analysis of an intervening sequence.

To define the extent of intervening sequence required for splicing higher eukaryotic mRNA precursors in vivo, we constructed deletions within the second intervening sequence of the human G gamma-globin gene that progressively approach the donor or acceptor splice sites. Most of the intervening sequence can be deleted with no effect on splicing. At the donor splice site, 6 bases of intervening sequence are sufficient for accurate and efficient splicing. At the acceptor splice site, 20 bases are sufficient for accurate and efficient splicing, and 16 bases are sufficient for accurate splicing but at a reduced level. However, 15 bases are insufficient for splicing at a significant level. The effect of deletions ending near the acceptor splice site is independent of whether an A-G dinucleotide is introduced into the acceptor splice site region by the deletion.

Base Sequence↗
PubMed1995

Modulation of 5' splice site choice in pre-messenger RNA by two distinct steps.

Ser/Arg-rich proteins (SR proteins) are essential splicing factors that commit pre-messenger RNAs to splicing and also modulate 5' splice site choice in the presence or absence of functional U1 small nuclear ribonucleoproteins (snRNPs). Here, we perturbed the U1 snRNP in HeLa cell nuclear extract by detaching the U1-specific A protein using a 2'-O-methyl oligonucleotide (L2) complementary to its binding site in U1 RNA. In this extract, the standard adenovirus substrate is spliced normally, but excess amounts of SR proteins do not exclusively switch splicing from the normal 5' splice site to a proximal site (site 125 within the adenovirus intron), suggesting that modulation of 5' splice site choice exerted by SR proteins requires integrity of the U1 snRNP. The observation that splicing does not necessarily follow U1 binding indicates that interactions between the U1 snRNP and components assembled on the 3' splice site via SR proteins may also be critical for 5' splice site selection. Accordingly, we found that SR proteins promote the binding of the U2 snRNP to the branch site and stabilize the complex formed on a 3'-half substrate in the presence or absence of functional U1 snRNPs. A novel U2/U6/3'-half substrate crosslink was also detected and promoted by SR proteins. Our results suggest that SR proteins in collaboration with the U1 snRNP function in two distinct steps to modulate 5' splice site selection.

Blotting, Western↗
PubMed1998

Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein.

Protein splicing involves the self-catalyzed excision of protein splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. To study the enzymology of this interesting process we have expressed and purified N- and C-terminal segments of the Mycobacterium tuberculosis RecA intein, each approximately 100 amino acids long, fused to appropriate exteins. These fragments were reconstituted into a functional protein splicing element by renaturation from 6 M urea. When renaturation was carried out in the absence of thiols, the reconstituted splicing element accumulated as an inactive disulfide-linked complex of the two intein fragments, which could be induced to undergo protein splicing by reduction of the disulfide bond. This provided a useful tool for separately investigating the requirements for the reconstitution of the intein fragments to yield a functional protein splicing element and for the protein splicing process per se. For example, the pH dependence of these processes was quite different, with reconstitution being most efficient at pH 8.5 and splicing most rapid at pH 7.0. The availability of such an in vitro protein splicing system opens the way for the exploration of intein structure and the unusual enzymology of protein splicing. In addition, this trans-splicing system is a potential protein ligase that can link any two polypeptides fused to the N- and C-terminal intein segments.

Bacterial Proteins↗
PubMed2003

Splicing error in E1alpha pyruvate dehydrogenase mRNA caused by novel intronic mutation responsible for lactic acidosis and mental retardation.

An intronic point mutation was identified in the E1alpha PDH gene from a boy with delayed development and lactic acidosis, an X-linked disorder associated with a partial defect in pyruvate dehydrogenase (PDH) activity. Protein analysis demonstrated a corresponding decrease in immunoreactivity of the alpha and beta subunits of the PDH complex. In addition to the normal spliced mRNA product of the E1alpha PDH gene, patient samples contained significant levels of an aberrantly spliced mRNA with the first 45 nucleotides of intron 7 inserted in-frame between exons 7 and 8. The genomic DNA analysis found no mutation in the coding regions but revealed a hemizygous intronic G to A substitution 26 nucleotides downstream from the normal exon 7 5'-splice site. Splicing experiments in COS-7 cells demonstrated that this point mutation at intron 7 position 26 is responsible for the aberrant splicing phenotype, which involves a switch from the use of the normal 5'-splice site (intron 7 position 1) to the cryptic 5'-splice site downstream of the mutation (intron 7 position 45). The intronic mutation is unusual in that it generates a consensus binding motif for the splicing factor, SC35, which normally binds to exonic enhancer elements resulting in increased exon inclusion. Thus, the aberrant splicing phenotype is most likely explained by the generation of a de novo splicing enhancer motif, which activates the downstream cryptic 5'-splice site. The mutation documented here is a novel case of intron retention responsible for a human genetic disease.

Acidosis, Lactic↗
PubMed2004

An intronic polypyrimidine-rich element downstream of the donor site modulates cystic fibrosis transmembrane conductance regulator exon 9 alternative splicing.

Two intronic elements, a polymorphic TGmTn locus at the end of intron 8 and an intronic splicing silencer in intron 9, regulate aberrant splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Previous studies (Pagani, F., Buratti, E., Stuani, C., Romano, M., Zuccato, E., Niksic, M., Giglio, L., Faraguna, D., and Baralle, F. E. (2000) J. Biol. Chem. 275, 21041-21047 and Buratti, E., Dork, T., Zuccato, E., Pagani, F., Romano, M., and Baralle, F. E. (2001) Embo J. 20, 1774-1784) have demonstrated that trans-acting factors that bind to these sequences, TDP43 and Ser/Arg-rich proteins, respectively, mediate splicing inhibition. Here, we report the identification of two polypyrimidine-binding proteins, TIA-1 and polypyrimidine tract-binding protein (PTB), as novel players in the regulation of CFTR exon 9 splicing. In hybrid minigene experiments, TIA-1 induced exon inclusion, whereas PTB induced exon skipping. TIA-1 bound specifically to a polypyrimidine-rich controlling element (PCE) located between the weak 5'-splice site (ss) and the intronic splicing silencer. Mutants of the PCE polypyrimidine motifs did not bind TIA-1 and, in a splicing assay, did not respond to TIA-1 splicing enhancement. PTB antagonized in vitro TIA-1 binding to the PCE, but its splicing inhibition was independent of its binding to the PCE. Recruitment of U1 small nuclear RNA to the weak 5'-ss by complementarity also induced exon 9 inclusion, consistent with the facilitating role of TIA-1 in weak 5'-ss recognition by U1 small nuclear ribonucleoprotein. Interestingly, in the presence of a high number of TG repeats and a low number of T repeats in the TGmTn locus, TIA-1 activated a cryptic exonic 3'-ss. This effect was independent of both TIA-1 binding to the PCE and U1 small nuclear RNA recruitment to the 5'-ss. Moreover, it was abolished by deletion of either the TG or T sequence. These data indicate that, in CFTR exon 9, TIA-1 binding to the PCE recruits U1 small nuclear ribonucleoprotein to the weak 5'-ss and induces exon inclusion. The TIA-1-mediated alternative usage of the 3'-splice sites, which depends on the composition of the unusual TGmTn element, represents a new mechanism of splicing regulation by TIA-1.

Alternative Splicing↗
PubMed2004

Novel down-regulatory mechanism of the surface expression of the vasopressin V2 receptor by an alternative splice receptor variant.

In rat kidney, two alternatively spliced transcripts are generated from the V2 vasopressin receptor gene. The large transcript (1.2 kb) encodes the canonical V2 receptor, whereas the small transcript encodes a splice variant displaying a distinct sequence corresponding to the putative seventh transmembrane domain and the intracellular C terminus of the V2 receptor. This work showed that the small spliced transcript is translated in the rat kidney collecting tubules. However, the protein encoded by the small transcript (here called the V2b splice variant) is retained inside the cell, in contrast to the preferential surface distribution of the V2 receptor (here called the V2a receptor). Cells expressing the V2b splice variant do not exhibit binding to 3H-labeled vasopressin. Interestingly, we found that expression of the splice variant V2b down-regulates the surface expression of the V2a receptor, most likely via the formation of V2a.V2b heterodimers as demonstrated by co-immunoprecipitation and fluorescence resonance energy transfer experiments between the V2a receptor and the V2b splice variant. The V2b splice variant would then be acting as a dominant negative. The effect of the V2b splice variant is specific, as it does not affect the surface expression of the G protein-coupled interleukin-8 receptor (CXCR1). Furthermore, the sequence encompassing residues 242-339, corresponding to the C-terminal domain of the V2b splice variant, also down-regulates the surface expression of the V2a receptor. We suggest that some forms of nephrogenic diabetes insipidus are due to overexpression of the splice variant V2b, which could retain the wild-type V2a receptor inside the cell via the formation of V2a.V2b heterodimers.

Alternative Splicing↗
PubMed2005

Functionally diverse complement of large conductance calcium- and voltage-activated potassium channel (BK) alpha-subunits generated from a single site of splicing.

The pore-forming alpha-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are encoded by a single gene that undergoes extensive alternative pre-mRNA splicing. However, the extent to which differential exon usage at a single site of splicing may confer functionally distinct properties on BK channels is largely unknown. Here we demonstrated that alternative splicing at site of splicing C2 in the mouse BK channel C terminus generates five distinct splice variants: ZERO, e20, e21(STREX), e22, and a novel variant deltae23. Splice variants display distinct patterns of tissue distribution with e21(STREX) expressed at the highest levels in adult endocrine tissues and e22 at embryonic stages of mouse development. deltae23 is not functionally expressed at the cell surface and acts as a dominant negative of cell surface expression by trapping other BK channel splice variant alpha-subunits in the endoplasmic reticulum and perinuclear compartments. Splice variants display a range of biophysical properties. e21(STREX) and e22 variants display a significant left shift (>20 mV at 1 microM [Ca2+]i) in half-maximal voltage of activation compared with ZERO and e20 as well as considerably slower rates of deactivation. Splice variants are differentially sensitive to phosphorylation by endogenous cAMP-dependent protein kinase; ZERO, e20, and e22 variants are all activated, whereas e21 (STREX) is the only variant that is inhibited. Thus alternative pre-mRNA splicing from a single site of splicing provides a mechanism to generate a physiologically diverse complement of BK channel alpha-subunits that differ dramatically in their tissue distribution, trafficking, and regulation.

Alternative Splicing↗