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PubMed1993

Polymorphic expression of CD46 protein isoforms due to tissue-specific RNA splicing.

CD46 is a member of the regulators of complement activation (RCA) family and serves to protect autologous cells from complement mediated lysis. The CD46 gene consists of 14 exons and extensive RNA splicing produces protein isoforms of different molecular weight. Predominant protein isoforms of 66 and 56 kDa arise from splicing in or out of exon 8 which encodes a region rich in serine, threonine and proline residues known to be heavily O-glycosylated. An inherited allelic polymorphism controls the relative expression of these isoforms in PBL and other tissues. This study has analysed an independent and overriding tissue specific regulation of CD46 splicing. Salivary gland and kidney produce RNA transcripts that preferentially include exon 8, giving rise to the 66 kDa protein species, while exon 8 is spliced out in brain tissue to give the 56 kDa protein. The cytoplasmic tail of CD46 is encoded by either exon 13 (CYT 1) or exon 14 (CYT 2). There is a preferential deletion of exon 13 from transcripts in salivary gland, kidney and brain to encode a protein containing cytoplasmic tail CYT 2. This preferential production of the CYT 2 tail is contrary to that seen on peripheral blood lymphocytes where equivalent expression of both CYT 1 and CYT 2 is observed. Our results suggest that while the splicing of exons within most cells is controlled by nucleotide sequences within or close to the CD46 gene (i.e. cis-regulation), splicing in tissues such as salivary gland, kidney and brain is regulated by trans-splicing factors encoded by another gene(s).

Alternative Splicing↗
PubMed1994

Alternatively spliced, germline J alpha 11-2-C alpha mRNAs are the predominant T cell receptor alpha transcripts in mouse kidney.

We recently reported the expression of a truncated T cell receptor (TCR) alpha mRNA in kidney and brain of normal mice. In the kidney, the truncated TCR alpha transcript was expressed by bone marrow-dependent, non-T large interstitial cells located predominantly in the medulla. Here, we report the molecular characterization of the truncated TCR alpha transcript from kidney. Using a modified anchored-PCR (A-PCR) technique and directional cloning, 37 cDNA clones extending 5' of the C alpha region were generated. cDNA sequencing showed that 29 of the clones (78%) originated in the J alpha 11-2 region. Of these clones, 17 started upstream or in the J alpha 11-2 exon and contained the entire J alpha 11-2 sequence correctly spliced to the first C alpha exon. Analysis of the sequence revealed the presence of multiple stop codons in all three reading frames. The other 12 clones originated further upstream of the J alpha 11-2 exon and did not include the J alpha 11-2 exon, but rather arose from the joining of a cryptic splice donor signal to the usual TCR alpha C splice acceptor. This alternatively spliced transcript contained an open reading frame extending from the upstream J alpha 11-2 region to 82 nucleotides downstream of the beginning of the TCR C alpha region, and potentially encoded a 36 amino acid polypeptide. The remaining eight clones all contained the J alpha TA61 region correctly spliced to C alpha with two of these extending upstream of the J alpha TA61 exon. The predominance of J alpha 11-2-C alpha containing clones was confirmed by RNase protection assay using total RNA from kidney and spleen of scid mice. The 3' region of the transcript contained a fully conserved, correctly spliced TCR alpha C region which was polyadenylated at the 3' end. The truncated TCR alpha mRNA could be detected in preparations of cytoplasmic RNA, indicating that this transcript follows a normal RNA processing pathway. Our results demonstrate that the truncated TCR alpha mRNA expressed in normal mouse kidney is a germline J-C transcript resulting from transcription initiated predominantly upstream of the J alpha 11-2 region. This germline transcript in the kidney is undergoing alternative splicing leading to the appearance of an open reading frame coding for a short polypeptide. These results suggest that the product of this transcript may be functionally relevant.

Alternative Splicing↗
PubMed1994

Defective splicing induced by 4NQO in the hamster hprt gene.

The molecular analysis of mutations affecting mRNA processing may contribute to a better understanding of the splicing mechanism through the identification of genomic sequences necessary for the recognition of splice sites. In this paper we report the sequence analysis of 14 splice mutants induced by 4-nitroquinoline 1-oxide (4NQO) at the hamster hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus. We show that mutations at the 3' acceptor splice site or at the first or fifth base of the 5' donor splice site are responsible for exon skipping. In addition, mutations in exon sequences also determine the skipping of one or more exons. Our data indicate that point mutations in intron regions at either side of an internal exon may induce the skipping of the same exon, supporting a model where the exon is the unit of early spliceosome assembly. Furthermore, they suggest that the splicing of hprt mRNA precursors may proceed through a clustering of exons 2, 3 and 4 which are then spliced in a concerted way.

4-Nitroquinoline-1-oxide↗
PubMed1995

Alternatively spliced isoforms of the NMDARI receptor subunit.

Molecularly diverse forms of the NMDA-receptor subunit NRI are formed by alternative RNA splicing. Differential splicing of three exons generates as many as eight NRI splice variants, seven of which have been identified in cDNA libraries. The alternatively spliced exons encode a 21 amino acid sequence in the N-terminus domain (termed NI), and adjacent sequences of 37 and 38 amino acids in the C-terminus domain (termed C1 and C2, respectively). Splicing out the exon segment that encodes the C2 cassette removes the first stop codon, resulting in a new open reading frame that encodes an unrelated sequence of 22 amino acids (C2') before a second stop codon is reached. Differential RNA splicing alters the structural, physiological and pharmacological properties of receptors that comprise NRI subunits. Diversity of NMDA receptors is also caused by differential association with members of the NR2 gene family. The finding of cell-specific expression and developmental regulation of NRI splice variants, and of the NR2 subunits, provides an explanation for the diversity of properties of NMDA receptors in different neuronal populations.

Amino Acid Sequence↗
PubMed1995

trans-splicing: an update.

5'-end maturation of messenger RNAs via acquisition of a trans-spliced leader sequence occurs in several primitive eukaryotes, some of which are parasitic. This type of trans-splicing proceeds though a two-step reaction pathway directly analogous to that of cis-splicing and like cis-splicing it requires multiple U snRNP cofactors. This minireview attempts to provide a brief synopsis of our current understanding of the evolution and biological significance of trans-splicing. Progress in deciphering the mechanism of trans-splicing, particularly as it relates to current models of cis-splicing, is also discussed.

Animals↗
PubMed1986

Splicing and polyadenylylation at cryptic sites in RNA transcribed from pSV2-neo.

Mapping the structures of RNAs transcribed from the chimeric plasmid pSV2-neo in transfected COS cells revealed discontinuities within the neo portion of the transcripts. Two cryptic 5' splice sites and three cryptic 3' splice sites were identified. The cryptic 5' splice sites matched 5 or 7 bases of the 5' consensus sequence. Each cryptic 3' splice site consisted of an AG that was preceded by a 15 nucleotide region which was 73% pyrimidine. They differed from the 3' consensus sequence mainly by the presence of a purine, rather than a pyrimidine, immediately adjacent to the AG at the splice site. Greater than 99% of the transcripts were spliced at the SV40 intron. Approx. 50% of these transcripts were spliced, in a complex pattern, at the additional sites within the neo region. Deletion of the SV40 early polyadenylylation signal from pSV2-neo revealed the presence of a cryptic polyadenylylation signal in a region of pBR322 sequence. It is located in the 5' portion of the beta-lactamase gene and occurs 6 +/- 2 nucleotides downstream from the sequence AATAATAATGAA, which contains three overlapping variant hexanucleotides. The cryptic signal appears to be approx. 10-fold less efficient than the SV40 polyadenylylation signal.

Animals↗
PubMed1989

Quantification analysis of 5'-splice signal sequences in mRNA precursors. Mutations in rabbit beta-globin gene.

Concerning the signals which direct excision of introns from mRNA precursors in genes of higher eukaryotes, a consensus sequence composed of nine nucleotides, CAAG/GTAGAGT, has been proposed for the 5'-splice site, but actual 5'-splice site sequences differ from it to a greater or lesser degree. In the present paper, the 5'-splice site sequence of the rabbit beta-globin gene was analyzed using a quantification method (categorical discriminant analysis). In this method, each 9-nucleotide sequence in the pre-mRNA was characterized by its sample score, which shows the extent to which the sequence contains the signal. This approach could explain not only the location of the 5'-splice site, but also the experimental results of various point mutations in the 5'-splice region, as reported by Aebi et al. Our method further predicted the positions of cryptic 5'-splice sites, which are activated when the authentic 5'-splice site is abolished.

Animals↗
PubMed1990

Splice junction mutations in a yeast tRNA gene which alter the rate and precision of processing.

We have introduced mutations into a tRNALeu3 gene which alter the intron boundaries and examined their effects on RNA splicing. Our results show that the 5'-proximal splice junction is not specified by the position of an adjacent base-paired stem present in all naturally occurring tRNA precursors. Also, efficient cleavage of 5'-splice junctions unique to these mutants, -CpU-, -UpA- and -UpG-, indicates the purine found at the 5'-side of this site in all natural precursors is dispensable. Some alterations of the sequence and structure at the 5'-proximal splice site reduce the rate of cleavage therein and result in accumulation of molecules composed of the 5'-half of the tRNA plus the intron. The precise position of the 5'-proximal cleavage site can vary +/- 1 base in these mutants. The 3'-proximal splice junction is rendered inactive by changing the prospective splice junction sequence from -ApC- to -CpC- and reducing the size of an unpaired loop at this site from six to two bases. Very small amounts of RNA composed of the 3'-half of the tRNA plus the intron accumulate from this precursor. We conclude that splice junction sequence and structure affect both the rate and precision of intervening sequence removal.

Base Sequence↗
PubMed1988

Molecular genetics of group I introns: RNA structures and protein factors required for splicing--a review.

In vivo and in vitro genetic techniques have been widely used to investigate the structure-function relationships and requirements for splicing of group-I introns. Analyses of group-I introns from extremely diverse genetic systems, including fungal mitochondria, protozoan nuclei, and bacteriophages, have yielded results which are complementary and highly consistent. In vivo genetic studies of fungal mitochondrial systems have served to identify cis-acting sequences within mitochondrial introns, and trans-acting protein products of mitochondrial and nuclear genes which are important for splicing, and to show that some mitochondrial introns are mobile genetic elements. In vitro genetic studies of the self-splicing intron within the Tetrahymena thermophila nuclear large ribosomal RNA precursor (Tetrahymena LSU intron) have been used to examine essential and nonessential RNA sequences and structures in RNA-catalyzed splicing. In vivo and in vitro genetic analysis of the intron within the bacteriophage T4 td gene has permitted the detailed examination of mutant phenotypes by analyzing splicing in vivo and self-splicing in vitro. The genetic studies combined with phylogenetic analysis of intron structure based on comparative nucleotide sequence data [Cech 73 (1988) 259-271] and with biochemical data obtained from in vitro splicing experiments have resulted in significant advances in understanding the biology and chemistry of group-I introns.

Introns↗
PubMed1995

Tissue-specific alternative splicing of hybrid Shaker/lacZ genes correlates with kinetic differences in Shaker K+ currents in vivo.

Alternative splicing of the Shaker (Sh) locus of Drosophila generates several transcripts with divergent 5' and 3' domains that produce kinetically distinct K+ currents in Xenopus oocytes. Although suggestive that alternative splicing may be involved in generating K+ channel diversity, clear tissue-specific differences in the distribution of particular Sh gene products have not been demonstrated. Using lacZ as a reporter gene for accurate splicing of variable Sh3' domains, we observe differences in beta-galactosidase expression patterns in transgenic animals that indicate both temporal and spatial regulation of 3' splice choice. The differences in 3'splice choice can account for variation in recovery kinetics of Sh-encoded K+ currents recorded in adult flies. The results indicate that tissue-specific expression of functionally distinct Sh K+ channels is regulated, in part, at the level of pre-mRNA splicing and implicate sequences in or around the 3' splice sites in regulating the choice of 3' domain.

Alternative Splicing↗
PubMed1995

Tissue-dependent expression of a novel splice variant of the human oestrogen receptor.

We have isolated a novel splice variant of ER mRNA from normal endometrial tissue using RT/PCR. The variant contains an unusual splice junction formed by splicing sequences within exons 4 and 7 together. The translated protein product would be predicted to lack part of exon 4, all of exons 5 and 6 and, due to a missense alignment at the new splice junction, the remaining sequence from exon 7 would be translated out of frame and terminate at the exon 7/8 splice junction. As a result, the protein would lack most of the hormone binding domain (HBD) and the major oestrogen-dependent transactivating region (AF-2), but still contain the DNA binding domain (DNA-BD) and N-terminal transactivating region (AF-1). In contrast to the exon 5 deleted variant of ER (delta 5), which was expressed in both normal endometrium and liver, this novel variant was present in endometrium but not in liver samples. These results confirm that some ER splice variants are expressed in normal, non-malignant oestrogen responsive tissues. In addition, they demonstrate the tissue specific expression of a novel and interesting splice variant of ER in these normal tissues.

Alternative Splicing↗
PubMed2005

The splicing factor U2AF65 is functionally conserved in the thermotolerant deep-sea worm Alvinella pompejana.

Due to their inherent stability, thermophilic bacteria and archaea serve as important resources for biochemical and biophysical analyses of many biological processes. Unfortunately, scientists characterizing eukaryote-specific processes, such as nuclear pre-mRNA splicing, are unable to take advantage of these sources of thermostable proteins. To identify and provide a source of thermostable eukaryotic proteins, we are characterizing splicing factors in the thermotolerant deep-sea vent polychaete, Alvinella pompejana. This worm, also known as the Pompeii worm, is found in the extreme environment of deep-sea hydrothermal vents, and is one of the most thermotolerant eukaryotic organisms known. We report on detailed analyses of U2AF65, the large subunit of the U2 small nuclear ribonucleoprotein auxiliary factor, an essential splicing factor important for intron definition and alternative splicing. The cloning and characterization of Pompeii U2AF65 show it is highly similar to human U2AF65 in sequence and function and is more thermostable than the human protein when bound to RNA in vitro. Notably, Pompeii U2AF65 can restore splicing in a human extract depleted of human U2AF. We also determine that the general splicing mechanisms and signal sequences are conserved in the Pompeii worm, an annelid which has previously been uncharacterized in terms of splicing factors and signals.

Amino Acid Sequence↗
PubMed2004

Expression of EDA+ and EDB+ fibronectin splice variants in bone.

The extracellular matrix component fibronectin (fn) has fundamental functions in cell attachment, differentiation, proliferation, and migration. Isoforms of cellular fibronectin, named EDA+ fibronectin or embryonal EDB+ fibronectin, are generated by alternative splicing of its mRNA precursors. Little is known about the expression of EDA+ and EDB+ fibronectin splice variants in human bone. The aim of this study was to investigate the expression pattern of fibronectin splice variants in bone cell lines and in different human bone tissue samples (mature bone, early stages of fracture healing, hypotrophic nonunion, osteosarcoma). Analysis was done by immunostaining with recombinant and monoclonal antibodies, qualitative RT-PCR and LightCycler-based real-time quantitative RT-PCR assay. In osteoblast and osteosarcoma cell lines, abundant expression of EDA+ and EDB+ fibronectin was found in immunocytochemistry. High transcription levels of both splice variants mRNA were seen in quantitative RT-PCR in osteosarcoma cell lines. In mature bone, EDA+ and EDB+ were not detectable in immunohistochemistry. Transcription of mRNA in both splice variants was absent in these samples. Early stages of fracture healing and osteosarcoma cell samples exhibited extensive staining for EDA+ and EDB+ fibronectin, and high mRNA levels were found. Both osteosarcoma and bone fracture healing tissue expressed high mRNA levels of the fibronectin splice variants independent of benign or malignant behavior. Low level of EDA+ fibronectin mRNA transcription and focal immunohistochemical staining of EDA+ fibronectin was found in hypotrophic nonunions, whereas EDB+ fibronectin was not detected by immunohistochemistry and qualitative or quantitative PCR. EDA+ fibronectin was found in granulation tissue-forming processes in bone independent from bone-forming activity. EDB+ fibronectin was seen only in high-turnover new osteoid-forming processes like early stages of fracture healing and osteosarcoma and was absent in low-turnover processes like mature bone and hypotrophic nonunion. Both EDA+ and EDB+ fibronectin mark active processes in bone without differentiation between malignant or benign activity. In conclusion, EDA+ and EDB+ fibronectin splice variants are strong markers for active fibrogenetic and osteoid-forming processes in human bones.

Adolescent↗
PubMed2006

Modulation of calcium signalling by dominant negative splice variant of ryanodine receptor subtype 3 in native smooth muscle cells.

The ryanodine receptor subtype 3 (RYR3) is expressed ubiquitously but its physiological function varies from cell to cell. Here, we investigated the role of a dominant negative RYR3 isoform in Ca2+ signalling in native smooth muscle cells. We used intranuclear injection of antisense oligonucleotides to specifically inhibit endogenous RYR3 isoform expression. In mouse duodenum myocytes expressing RYR2 subtype and both spliced and non-spliced RYR3 isoforms, RYR2 and non-spliced RYR3 were activated by caffeine whereas the spliced RYR3 was not. Only RYR2 was responsible for the Ca2+-induced Ca2+ release mechanism that amplified Ca2+ influx- or inositol 1,4,5-trisphosphate-induced Ca2+ signals. However, the spliced RYR3 negatively regulated RYR2 leading to the decrease of amplitude and upstroke velocity of Ca2+ signals. Immunostaining in injected cells showed that the spliced RYR3 was principally expressed near the plasma membrane whilst the non-spliced isoform was revealed around the nucleus. This study shows for the first time that the short isoform of RYR3 controls Ca2+ release through RYR2 in native smooth muscle cells.

Alternative Splicing↗
PubMed2005

Neuronal proteins custom designed by alternative splicing.

The evolution of alternative splicing in eukaryotes greatly expanded the number of functionally distinct proteins that could be produced from a finite gene pool. Extensive in the brains of higher organisms, alternative splicing might be the primary mechanism for generating the spectrum of protein activities that support complex brain functions. Alternative splicing is controlled at the level of individual neurons to custom design proteins for optimal performance. The expression profiles of splice isoforms are modified during development and as neuronal activity changes. Alternative splicing can lead to incremental, long lasting changes in ion channel and receptor activities, independent of changes in gene transcription. Recent studies of tissue-specific splicing factors are revealing how coordinated alterations in alternative splicing of RNA transcripts control synaptic function.

Alternative Splicing↗
PubMed2006

CD14-dependent modulation of NF-kappaB alternative splicing in the lung after burn injury.

Nuclear factor kappa-B (NF-kappaB), a key downstream player of the LPS signaling pathway, has been shown to undergo alternative splicing in in vitro studies. In this study, we examined the effect of injury and the role of CD14 on NF-kappaB alternative splicing using a murine burn model. CD14 knockout and respective wild-type mice were sacrificed after 18% total body surface area burn. RT-PCR and subsequent sequencing analysis revealed that injury induced multiple novel splicing variants of relA, relB, and NF-kappaB2 in the lungs of CD14 knockout but not wild-type mice. These novel variants resulted either from exon skipping, alternative usage of splicing signals, or intron retention. All but one variant resulted in a frameshift leading to premature termination of translation. These splicing variants encoded for proteins that lacked the domains essential for NF-kappaB transcription factor functions. Two NF-kappaB2 variants acquired only minor changes in their C-terminus that might affect their post-translational cleavage into active isoforms. These results suggest that alternative splicing may be one of the mechanisms by which NF-kappaB activity in the lungs can be regulated after injury. Furthermore, the CD14-mediated LPS signaling pathway may play a role in the regulation of NF-kappaB alternative splicing in the lungs after injury.

Alternative Splicing↗
PubMed2003

In-cell generation of antibody single-chain Fv transcripts by targeted RNA trans-splicing.

The humoral immune response propels the production of a diversified pool of antibodies with high affinity and selectivity for the eliciting antigen. Their isolation entails either B-cell cloning or the linking of authentic pairs of variable region genes encoding them. We hypothesized that targeted RNA trans-splicing (TS) inside the B-cell nucleus could be harnessed as a novel means to link both variable region genes and reconstitute genuine immune B-cell specificities. This could be accomplished by a special targeting gene harboring a peptide linker exon flanked by sequences capable of targeting both heavy (HC) and light chain (LC) transcripts. Following sequential trans-splicing reactions, the resulting RNA in each cell would encode the two variable regions, joined by the peptide linker. In this study, we examined genetic components and configurations required for the separate trans-splicing steps and for the combined two-step reactions. Using a model antibody, we show that in transiently transfected cells, we can target variable region exons through both their acceptor and donor splice sites, precisely joining an exon encoding a synthetic linker and the complementary variable region so as to form a single-chain Fv. We also demonstrate the accurate formation of single-chain Fv transcript as a result of trans-splicing of RNA synthesized from two chromosomal genes expressed by a stably transfected B-cell hybridoma. Our attempts to link the two variable region genes via a synthetic linker exon through sequential trans-splicing events were only successful with regard to both ends of the linker and to the 3' end of the light chain, but repeatedly resulted in a deletion at the 5' end of the joined heavy chain transcript. The implications of our findings on the potential application of trans-splicing for the isolation of useful antibodies are discussed.

Amino Acid Sequence↗
PubMed2006

Involvement of DEAD-box proteins in group I and group II intron splicing. Biochemical characterization of Mss116p, ATP hydrolysis-dependent and -independent mechanisms, and general RNA chaperone activity.

The RNA-catalyzed splicing of group I and group II introns is facilitated by proteins that stabilize the active RNA structure or act as RNA chaperones to disrupt stable inactive structures that are kinetic traps in RNA folding. In Neurospora crassa and Saccharomyces cerevisiae, the latter function is fulfilled by specific DEAD-box proteins, denoted CYT-19 and Mss116p, respectively. Previous studies showed that purified CYT-19 stimulates the in vitro splicing of structurally diverse group I and group II introns, and uses the energy of ATP binding or hydrolysis to resolve kinetic traps. Here, we purified Mss116p and show that it has RNA-dependent ATPase activity, unwinds RNA duplexes in a non-polar fashion, and promotes ATP-independent strand-annealing. Further, we show that Mss116p binds RNA non-specifically and promotes in vitro splicing of both group I and group II intron RNAs, as well as RNA cleavage by the aI5gamma-derived D135 ribozyme. However, Mss116p also has ATP hydrolysis-independent effects on some of these reactions, which are not shared by CYT-19 and may reflect differences in its RNA-binding properties. We also show that a non-mitochondrial DEAD-box protein, yeast Ded1p, can function almost as efficiently as CYT-19 and Mss116p in splicing the yeast aI5gamma group II intron and less efficiently in splicing the bI1 group II intron. Together, our results show that Mss116p, like CYT-19, can act broadly as an RNA chaperone to stimulate the splicing of diverse group I and group II introns, and that Ded1p also has an RNA chaperone activity that can be assayed by its effect on splicing mitochondrial introns. Nevertheless, these DEAD-box protein RNA chaperones are not completely interchangeable and appear to function in somewhat different ways, using biochemical activities that have likely been tuned by coevolution to function optimally on specific RNA substrates.

Adenosine Triphosphate↗