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PubMed2000

Cellular distribution of the splice variants of the receptor for pituitary adenylate cyclase-activating polypeptide (PAC(1)-R) in the rat brain by in situ RT-PCR.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide and its specific receptor (the PAC(1) receptor) is widely distributed in the rat brain. It has been reported that alternative splicing of the region encoding the third intracellular loop of the PAC(1) receptor generates six isoforms which are differentially coupled to signal transduction pathways, but the precise distribution and localization of these splice isoforms in the brain remain to be determined. Using the initial specific primer pairs which correspond to the 'hip' or 'hop' types of receptors for the solution-phase reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated that the major splice variants of the PAC(1) receptor in various regions of the rat brain are the short splice isoform 'PAC(1)-R-s' which does not contain either the 'hip' or 'hop' cassette and the another splice isoform, 'PAC(1)-R-hop', which contains the 'hop' cassette. With an innovative molecular histochemical technique, in situ RT-PCR, we determined that these two splice isoforms are both intensely expressed in the mitral cells of the olfactory bulb, the Purkinje cells of the cerebellum, the pyramidal cells of the hippocampus and neocortex, and many neurons in the nuclei of hypothalamus and thalamus as well as other regions. The initial mapping of the cell type-specific expression of these two splice variants of the PAC(1) receptor provides the basis for a better understanding of the functional significance of the PAC(1)-R and its ligand PACAP in various brain regions.

Alternative Splicing↗
PubMed2001

Osteocalcin is incompletely spliced in non-osseous tissues.

Osteocalcin (OC) is known to be a bone tissue-specific protein, expression of which is believed to be controlled by the OC promoter. In this communication, we provided evidence to demonstrate that tissue-specific expression of OC was also regulated at the RNA splicing level. We identified incompletely spliced variants of human OC mRNA, which retain one or more introns during RNA splicing, existing dominantly in non-osseous organs. Northern blot analysis identified two OC RNA transcripts expressed in normal human tissues, but the expression level of the transcripts varied between the tissues. Most non-osseous tissues expressed transcripts with higher molecular weight, prominent in ovary, kidney, pancreas, spleen, thymus, prostate, and testis, than the expected size of OC mRNA as seen in bone marrow. RT-PCR analysis identified up to six OC transcripts in most tissues tested except bone marrow. Sequence analysis showed that four of five RNA variants contained intron 1 in common and the dominant one contained all three introns. MG63, an osteoblastic osteosarcoma cell, expressed only the completely-spliced form of OC, whereas incompletely spliced RNA was dominant in most prostate tumor cells. Combined study of in situ hybridization and immunohistochemistry revealed that OC RNA was highly expressed in prostate tumor epithelial cells while only very low levels of protein were detected, which confirms that there are OC RNA variants in non-osseous tissues. In conclusion, we demonstrated that OC mRNA is also expressed in several non-osseous tissues. However, only bone preferentially underwent the complete splicing event of all three introns. The function of other splicing variants of OC mRNA needs to be further investigated.

Alternative Splicing↗
PubMed2002

Expression of novel splice variants of the G protein subunit, Go alpha, is tissue-specific and age-dependent in the rat.

Heterotrimeric G proteins play an essential role in coupling numerous surface membrane receptors to intracellular signal transduction pathways. Relatively little is known about the splice variants of G proteins, including whether they undergo differential expression as a function of aging. We screened for splice variants of the alpha subunit of the dominant inhibitory G protein family member G(o) in a variety of tissues from rat and examined the expression of these splice variants during development. The splice variants were characterized using specific primers for G(o)alpha1 and G(o)alpha2 in conjunction with reverse transcription-polymerase chain reaction, and subsequently sequenced. G(o)alpha1 expression dominated over G(o)alpha2 in all neuronal tissues screened, including cerebral cortex, pituitary, spinal cord, colon myenteric plexus, dorsal root ganglion, and prenatal cortex. The sequence data of G(o)alpha1 supports the presence of three splice variants: G(o)alpha1a, G(o)alpha1b, G(o)alpha1c. The G(o)alpha1a variant was reported previously [J. Biol. Chem. 262 (1987) 14241], whereas G(o)alpha1b and G(o)alpha1c represent novel variants. The G(o)alpha1b splice variant demonstrates a 94 bp deletion using a cryptic donor site in exon 10. The G(o)alpha1c variant demonstrates a complete deletion of exon 10. A protein product with a molecular weight of approximately 34 kDa consistent with that expected for G(o)alpha1c was identified using Western blot analysis and two-dimensional gel electrophoresis. The expression of G(o)alpha1a decreased postnatally, supporting a potential physiological role during fetal development, whereas G(o)alpha1c expression increased postnatally. The age-dependent and tissue-specific expression of the G(o)alpha1 splice variants presage a broader functional role than has been observed historically with G(o).

Aging↗
PubMed1998

The human P2X4 receptor gene is alternatively spliced.

ATP acts as a fast excitatory neurotransmitter by binding to a large family of membrane proteins, P2X receptors, that have been shown to be ligand-gated, non-selective cation channels. We report the cloning of a full-length and alternatively spliced form of the human P2X4 gene. Clones were identified from a human stomach cDNA library using a rat P2X4 probe. Nucleotide sequence analysis of positive clones identified the full-length human P2X4 cDNA, which codes for a 388-residue protein that is highly homologous (82%) to the rat gene, and an alternatively spliced cDNA. In the alternatively spliced cDNA, the 5'-untranslated region and the first 90 amino acids in the coding region of full-length human P2X4 are replaced by a 35 amino acid coding sequence that is highly homologous with a region of chaperonin proteins in the hsp-90 family. The open reading frames of the full-length and splice variant clones were confirmed by in vitro translation. Northern analysis indicated expression of the full-length P2X4 message in numerous human tissues including smooth muscle, heart, and skeletal muscles. Alternatively spliced RNAs were identified in smooth muscle and brain by RT-PCR and confirmed by RNAse protection assays using a 710 bp anti-sense RNA probe that spanned the alternatively spliced and native P2X4 regions. Injection of full-length, but not alternatively spliced, cRNA into Xenopus oocytes resulted in the expression of ATP gated non-selective cation currents.

Adenosine Triphosphate↗
PubMed1992

Cellular expression of tropoelastin mRNA splice variants.

The primary transcript of tropoelastin is alternatively spliced into multiple mRNAs. The pattern and frequency of exon splicing is developmentally regulated, but the cellular profile of isoform expression within and among elastic tissues is not known. We used splice-variant specific antisense oligomeric deoxyribonucleotide probes in an in situ hybridization assay to assess the distribution of cells undergoing specific alternative splicing of tropoelastin pre-mRNA in developing bovine elastic tissues. Antisense oligomers were synthesized to exon sequences that are not alternatively spliced (exon 36) and to sequences that become abutted after high frequency (exon 33) and low frequency (exons 13 and 14) alternative splicing. The specificity of these probes for tropoelastin splice variants was verified by Southern hybridization to tropoelastin cDNAs with known exon deletions, and their specificity for tropoelastin mRNA was demonstrated by Northern hybridization. In situ hybridization with [35S]-labeled oligomers on sections of bovine lobar pulmonary artery and other elastic tissues showed that all elastogenic cells produce multiple forms of tropoelastin mRNA. These observations suggest that the production of tropoelastin isoforms is common to all cells within an elastin tissue and that this multiplicity may not be involved in regional differences in elastic tissue architecture.

Animals↗
PubMed1997

Regulation of pre-mRNA splicing in metazoa.

Recent studies have identified additional cis-acting sequence elements that can either positively or negatively regulate pre-mRNA splicing. Trans-acting protein factors involved in tissue-specific splicing regulation have been characterized. The functions of splicing factors have been explored in vivo and certain splicing factors have been demonstrated to be essential for proper development and cell viability. Kinases that can specifically phosphorylate splicing factors have been identified and phosphorylation has been shown to influence the activity of splicing factors, consistent with a role for phosphorylation in splicing regulation.

Animals↗
PubMed1996

Solution structure of the donor site of a trans-splicing RNA.

BACKGROUND: RNA splicing is both ubiquitous and essential for the maturation of precursor mRNA molecules in eukaryotes. The process of trans-splicing involves the transfer of a short spliced leader (SL) RNA sequence to a consensus acceptor site on a separate pre-mRNA transcript. In Caenorhabditis elegans, a majority of pre-mRNA transcripts receive the 22-nucleotide SL from the SL1 RNA. Very little is known about the various roles that RNA structures play in the complex conformational rearrangements and reactions involved in premRNA splicing. RESULTS: We have determined the solution structure of a domain of the first stem loop of the SL1 RNA of C. elegans, using homonuclear and heteronuclear NMR techniques; this domain contains the splice-donor site and a nine-nucleotide hairpin loop. In solution, the SL1 RNA fragment adopts a stem-loop structure: nucleotides in the stem region form a classical A-type helix while nucleotides in the hairpin loop specify a novel conformation that includes a helix, that extends for the first three residues; a syn guanosine nucleotide at the turn region; and an extrahelical adenine that defines a pocket with nucleotides at the base of the loop. CONCLUSION: The proximity of this pocket to the splice donor site, combined with the observation that the nucleotides in this motif are conserved among all nematode SL RNAs, suggests that this pocket may provide a recognition site for a protein or RNA molecule in the trans-splicing process.

Animals↗
PubMed1998

The function of multisite splicing enhancers.

Splicing enhancers are RNA sequences consisting of one or more binding sites (enhancer elements) for specific serine/arginine (SR)-rich proteins. When associated with these elements, SR proteins activate splicing by recruiting the splicing machinery to the adjacent intron through protein-protein interactions. Here, we show that the rate and efficiency of splicing increases linearly, rather than synergistically, as the number of identical or nonidentical enhancer elements present on pre-mRNA is increased. We conclude that only one splicing enhancer complex at a time is capable of interacting with the constitutive splicing machinery. Thus, the function of multisite enhancer elements to increase the probability of an interaction between the enhancer complex and the splicing machinery rather than to promote functional synergy.

Animals↗
PubMed1999

An RNA switch at the 5' splice site requires ATP and the DEAD box protein Prp28p.

Pre-mRNA splicing requires dramatic RNA rearrangements hypothesized to be catalyzed by ATP-dependent RNA unwindases of the DExD/H box family. In a rearrangement critical for the fidelity of 5' splice site recognition, a base-pairing interaction between the 5' splice site and U1 snRNA must be switched for a mutually exclusive interaction between the 5' splice site and U6 snRNA. By lengthening the U1:5' splice site duplex, we impeded this switch in a temperature-dependent manner and prevented formation of the spliceosome's catalytic core. Using genetics, we identified the DExD/H box protein Prp28p as a potential mediator of the switch. In vitro, the switch requires both Prp28p and ATP. We propose that Prp28p directs isomerization of RNA at the 5' splice site and promotes fidelity in splicing.

Actins↗
PubMed2000

Multisite RNA binding and release of polypyrimidine tract binding protein during the regulation of c-src neural-specific splicing.

We studied the role of polypyrimidine tract binding protein in repressing splicing of the c-src neuron-specific N1 exon. Immunodepletion/add-back experiments demonstrate that PTB is essential for splicing repression in HeLa extract. When splicing is repressed, PTB cross-links to intronic CUCUCU elements flanking the N1 exon. Mutation of the downstream CU elements causes dissociation of PTB from the intact upstream CU elements and allows splicing. Thus, PTB molecules bound to multiple elements cooperate to repress splicing. Interestingly, in neuronal WERI-1 cell extract where N1 is spliced, PTB also binds to the upstream CU elements but is dissociated in the presence of ATP. We conclude that splicing repression by PTB is modulated in different cells by a combination of cooperative binding and ATP-dependent dissociation.

Adenosine Triphosphate↗
PubMed1999

Accurate quantification of the mRNA of NMDAR1 splice variants measured by competitive RT-PCR.

N-methyl-D-aspartate receptors (NMDAR) belong to the subclass of ionotropic glutamate receptors and are widely distributed in the vertebrate brain. Molecular cloning has revealed the existence of six NMDAR subunits: one NMDAR1 (NR1), four different NMDAR2 (NR2A-D) and one NMDAR3A (NR3A). Alternative splicing of the single NR1 gene generates eight isoforms with distinct functional properties [M. Hollmann, J. Boulter, C. Maron, L. Beasley, J. Sullivan, G. Pecht, S. Heinemann, Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor, Neuron 10 (1993) 943-954 [8]; R.S. Zukin, M.V.L. Bennett, Alternatively spliced isoforms of the NMDAR1 receptor subunit, TiNS 18 (1995) 306-313 [20]]. Despite the progress made in the functional analysis of NMDARs the molecular architecture of this receptor remains to be elucidated. In situ hybridization studies have already indicated that splicing of the NR1 gene is regionally regulated in the rodent brain, which may contribute to functional diversity of NMDARs in distinct brain areas [D.J. Laurie, P.H. Seeburg, Regional and developmental heterogeneity in splicing of the rat brain NMDAR1 mRNA, J. Neurosci. 14 (1994) 3180-3194 [10]; D.G. Standaert, C.M. Testa, A.B. Young, J.B. Penney Jr., Organization of N-methyl-D-aspartate glutamate receptor gene expression in the basal ganglia of the rat, J. Comp. Neurology 343 (1994) 1-16 [18]; M. Hollmann, S. Heinemann, Cloned glutamate receptors, Ann. Rev. Neurosci. 17 (1994) 31-108 [9]]. Since in situ hybridization techniques do not allow accurate quantification of distinct NR1 splice variants and are also very time-consuming, an accurate and sensitive competitive RT-PCR assay was developed. This method was then used to study the distribution of three NR1 splice variants in the rat brain, and the results are compared with former in situ hybridization studies.

Alternative Splicing↗
PubMed2000

Bimolecular exon ligation by the human spliceosome bypasses early 3' splice site AG recognition and requires NTP hydrolysis.

Here we report further characterization of an in vitro assay system for exon ligation by the human spliceosome in which the 3' splice site AG is supplied by a different RNA molecule than that containing the 5' splice and branch sites. By varying the time during splicing reactions when the 3' splice site AG is made available to the splicing machinery, we show that AG recognition need not occur until after lariat formation. Thus an early AG recognition event required for spliceosome formation and lariat formation on some mammalian introns is not required for exon ligation. Depletion/add-back studies and cold competitor challenge experiments reveal that commitment of a 3' splice site AG to exon ligation requires NTP hydrolysis. Because it both physically and kinetically uncouples exon ligation from spliceosome assembly and lariat formation, the bimolecular system will be a valuable tool for further mechanistic analysis of the second step of splicing.

Adenosine Triphosphate↗
PubMed2000

Pentamidine inhibits mitochondrial intron splicing and translation in Saccharomyces cerevisiae.

Pentamidine inhibits in vitro splicing of nuclear group I introns from rRNA genes of some pathogenic fungi and is known to inhibit mitochondrial function in yeast. Here we report that pentamidine inhibits the self-splicing of three group I and two group II introns of yeast mitochondria. Comparison of yeast strains with different configurations of mitochondrial introns (12, 5, 4, or 0 introns) revealed that strains with the most introns were the most sensitive to growth inhibition by pentamidine on glycerol medium. Analysis of blots of RNA from yeast strains grown in raffinose medium in the presence or absence of pentamidine revealed that the splicing of seven group I and two group II introns that have intron reading frames was inhibited by the drug to varying extents. Three introns without reading frames were unaffected by the drug in vivo, and two of these were inhibited in vitro, implying that the drug affects splicing by acting directly on RNA in vitro, but on another target in vivo. Because the most sensitive introns in vivo are the ones whose splicing depends on a maturase encoded by the intron reading frames, we tested pentamidine for effects on mitochondrial translation. We found that the drug inhibits mitochondrial but not cytoplasmic translation in cells at concentrations that inhibit mitochondrial intron splicing. Therefore, pentamidine is a potent and specific inhibitor of mitochondrial translation, and this effect explains most or all of its effects on respiratory growth and on in vivo splicing of mitochondrial introns.

Antifungal Agents↗
PubMed2000

The SRm160/300 splicing coactivator subunits.

The SRm160/300 splicing coactivator, which consists of the serine/arginine (SR)-related nuclear matrix protein of 160 kDa and a 300-kDa nuclear matrix antigen, functions in splicing by promoting critical interactions between splicing factors bound to pre-mRNA, including snRNPs and SR family proteins. In this article we report the isolation of a cDNA encoding the 300-kDa antigen and investigate the activity of it and SRm160 in splicing. Like SRm160, the 300-kDa antigen contains domains rich in alternating S and R residues but lacks an RNA recognition motif; the protein is accordingly named "SRm300." SRm300 also contains a novel and highly conserved N-terminal domain, several unique repeated motifs rich in S, R, and proline residues, and two very long polyserine tracts. Surprisingly, specific depletion of SRm300 does not prevent the splicing of pre-mRNAs shown previously to require SRm160/300. Addition of recombinant SRm160 alone to SRm160/300-depleted reactions specifically activates splicing. The results indicate that SRm160 may be the more critical component of the SRm160/300 coactivator in the splicing of SRm160/300-dependent pre-mRNAs.

Amino Acid Sequence↗
PubMed2001

Activation of a cryptic 5' splice site by U1 snRNA.

In the course of analyzing 5' splice site mutations in the second intron of Schizosaccharomyces pombe cdc2, we identified a cryptic 5' junction containing a nonconsensus nucleotide at position +2. An even more unusual feature of this cryptic 5' junction was its pattern of activation. By analyzing the profile of splicing products for an extensive series of cdc2 mutants in the presence and absence of compensatory U1 alleles, we have obtained evidence that the natural 5' splice site participates in activation of the cryptic 5' splice site, and that it does so via base pairing to U1 snRNA. Furthermore, the results of follow-up experiments strongly suggest that base pairing between U1 snRNA and the cryptic 5' junction itself plays a dominant role in its activation. Most remarkably, a mutant U1 can activate the cryptic 5' splice site even in the presence of a wild-type sequence at the natural 5' junction, providing unambiguous evidence that this snRNA redirects splicing via base pairing. Although previous work has demonstrated that U5 and U6 snRNAs can activate cryptic 5' splice sites through base pairing interactions, this is the first example in which U1 snRNA has been implicated in the final selection of a cryptic 5' junction.

Base Pairing↗
PubMed2002

Mutations in RRM4 uncouple the splicing repression and RNA-binding activities of polypyrimidine tract binding protein.

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequence-specific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays.

Alternative Splicing↗
PubMed2002

Region-specific alternative splicing in the nervous system: implications for regulation by the RNA-binding protein NAPOR.

Alternative RNA splicing generates extensive proteomic diversity in the nervous system, yet few neural-specific RNA binding proteins have been implicated in splicing control. Here we show that the biochemical properties and spatial expression of mouse neuroblastoma apoptosis-related RNA-binding protein (NAPOR; also called NAPOR-1) are consistent with its roles in the regulation of the exon 5 and exon 21 splicing events of the N-methyl-D-aspartate (NMDA) receptor R1 transcript. NAPOR, which is closely related to CUG binding protein 2 (CUG-BP2), promotes exon 21 and represses exon 5 splicing in functional coexpression assays. These NMDA mRNA isoforms are distributed, in vivo, in a region-specific manner in rat brain, such that high levels of exon 21 selection and exon 5 skipping coincide with high NAPOR mRNA expression in the forebrain. Within the forebrain, this spatial correspondence is most striking in the visual cortex. In contrast, low NAPOR expression coincides with the reciprocal pattern of alternative splicing in the hindbrain. Complementary experiments demonstrate a tissue-specific distribution of NAPOR, CUG-BP, and other highly related proteins within the nervous system as assayed by probing forebrain and hindbrain nuclear extracts with monoclonal antibody, mAb 3B1. Thus, NAPOR may be one of a group of closely related proteins involved in splicing regulation within the brain. An intronic RNA element responsible for the silencing of exon 21 splicing is identified by mutational analysis and shown to bind directly to recombinant NAPOR protein, suggesting a model in which exon 21 selection is positively regulated by an antirepression mechanism of action.

Alternative Splicing↗
PubMed1998

The two steps of group II intron self-splicing are mechanistically distinguishable.

The two transesterification reactions catalyzed by self-splicing group II introns take place in either two active sites or two conformations of a single active site involving rearrangements of the positions of the reacting groups. We have investigated the effects on the rates of the chemical steps of the two reactions due to sulfur substitution of nonbridging oxygens at both the 5' and 3' splice sites as well as the deoxyribose substitution of the ribose 2' hydroxyl group at the 5' splice site. The data suggest that the two active sites differ in their interactions with several of these groups. Specifically, sulfur substitution of the pro-Sp nonbridging oxygen at the 5' splice site reduces the chemical rate of the step one branching reaction by at least 250-fold, whereas substitution of the pro-Sp oxygen at the 3' splice site has only a 4.5-fold effect on the chemical rate of step two. Previous work demonstrated that the Rp phosphorothioate substitutions at both the 5' and 3' splice sites reduced the rate of both steps of splicing to an undetectable level. These results suggest that either two distinct active sites catalyze the two steps or that more significant alterations must be made in a single bifunctional active site to accommodate the two different reactions.

Binding Sites↗