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PubMed2026

The structured mRNA element 45ABC mediates auto- and cross-regulation of RBP45 genes via alternative splicing.

Alternative splicing (AS) is a common gene regulatory mechanism involving distinct interactions between trans-acting factors and cis-regulatory elements on the precursor messenger RNA (pre-mRNA). In this study, we have functionally characterized the structured motif 45ABC, which is located in the pre-mRNAs of RNA-binding protein (RBP) 45 genes in many plant species. Our data revealed that this element mediates a negative auto- and cross-regulatory feedback loop via AS of the three 45ABC-containing RBP45 genes in Arabidopsis thaliana. We identified a G-rich stretch within the first stem as a potential RBP45 binding site and observed increased RBP45-dependent AS upon structural weakening of this pairing element. The second stem includes the alternative 5' splice site being activated in the presence of RBP45. Based on the known interaction between RBP45 homologs and U1 snRNP components required for 5' splice site recognition, we propose that RBP45 recruitment to stem I of 45ABC may induce usage of the alternative 5' splice site in stem II. The resulting splicing variant is unproductive, thereby diminishing RBP45 expression. Analysing the splicing-regulatory impact of the three At-RBP45 genes in auto- and cross-regulation and a transcriptome-wide manner revealed unequal genetic redundancy with a major role of RBP45B. Furthermore, phenotypical analysis of single- and higher-order rbp45 mutants pointed at these genes' functions in controlling primary root growth and flowering time. Taken together, we demonstrated that both sequence and structural features of 45ABC are critical for proper splicing control, balancing RBP45 expression and functions in plants via a conserved mRNA motif.

Alternative Splicing↗

PubMed2004

Extensive search for discriminative features of alternative splicing.

Alternative pre-mRNA splicing events can be classified into various types, including cassette, mutually exclusive, alternative 3' splice site, alternative 5' splice site, retained intron. The detection of features of a particular type of alternative splicing events is an important and challenging problem in understanding the mechanism of alternative splicing. In this paper, we consider the problem of finding regulatory sequence patterns, which are specific to a particular type of alternative splicing events, on alternative exons and their flanking introns. For this problem, we have designed various pattern features and evaluated them on the alternative splicing data compiled in Lee's ASAP (Alternative Splicing Annotation Project) database. Through our work, we have succeeded in finding features with practically high accuracies.

Alternative Splicing↗

PubMed2024

Shaping human brain development and vulnerability through alternative splicing.

Alternative splicing contributes to shaping lineage-specific gene expression and phenotypes. In this issue of Cell Genomics, Recinos, Bao, Wang, et al.1 report that the balance between splicing isoforms of the microtubule-associated protein Tau in the brain is differentially regulated among primates by the RNA-binding protein MBNL2, with consequences for protein aggregation and neurodegeneration in humans.

Humans↗

PubMed2000

Test of intron predictions reveals novel splice sites, alternatively spliced mRNAs and new introns in meiotically regulated genes of yeast.

Correct identification of all introns is necessary to discern the protein-coding potential of a eukaryotic genome. The existence of most of the spliceosomal introns predicted in the genome of Saccharomyces cerevisiae remains unsupported by molecular evidence. We tested the intron predictions for 87 introns predicted to be present in non-ribosomal protein genes, more than a third of all known or suspected introns in the yeast genome. Evidence supporting 61 of these predictions was obtained, 20 predicted intron sequences were not spliced and six predictions identified an intron-containing region but failed to specify the correct splice sites, yielding a successful prediction rate of <80%. Alternative splicing has not been previously described for this organism, and we identified two genes (YKL186C/ MTR2 and YML034W) which encode alternatively spliced mRNAs; YKL186C/ MTR2 produces at least five different spliced mRNAs. One gene (YGR225W/ SPO70 ) has an intron whose removal is activated during meiosis under control of the MER1 gene. We found eight new introns, suggesting that numerous introns still remain to be discovered. The results show that correct prediction of introns remains a significant barrier to understanding the structure, function and coding capacity of eukaryotic genomes, even in a supposedly simple system like yeast.

Alternative Splicing↗

PubMed1986

Alternative splicing and alternative initiation of translation explain the four forms of the Ia antigen-associated invariant chain.

The Ia antigen-associated invariant chain (In) exists in humans as four related polypeptides, p33, p35, p41 and p43, all associated with HLA-class II antigens. As described previously, two of these forms of In chain, p33 and p35, result from the use of two in-phase initiation AUG codons on the unique In p33 mRNA. In addition to cDNA clones derived from In p33 mRNA, we have isolated a new cDNA clone, called p41-1, which differs from p33-1 by an additional segment in the coding region. The DNA sequence encoding the segment unique to p41-1 was identified in the genomic sequence in the intron between exon 6 and 7, and we refer to it as exon 6b. Cells transfected with a full length p41 cDNA clone in an expression vector synthesize the two larger forms of the In chain, p41 and p43. We propose that the larger mRNA, encoding p41, results from alternative splicing of exon 6b, and that p41 and p43 result from the use of the two functional initiation AUG codons identified in p33 mRNA. Alternative splicing, together with alternative initiation of translation, allows therefore the synthesis of four related In chain polypeptides from a single gene.

B-Lymphocytes↗

PubMed1987

Four Ia invariant chain forms derive from a single gene by alternate splicing and alternate initiation of transcription/translation.

We determined the structural basis for the presence of electrophoretically-distinct, antigenically-related forms of invariant chains in Ia oligomers, and established the mechanisms by which they can be expressed from a single gene. S1 nuclease protection assays indicated that, in B cells, transcription of this gene initiates at a minimum of three sites. Thus, unlike previously thought, invariant chain mRNAs have heterogeneous 5' untranslated segments that may differentially affect initiation of translation. Further, restriction mapping and nucleotide sequencing of cDNAs revealed two kinds of invariant chain mRNAs differing by an internal coding segment of 192 bp. This segment represents an alternatively spliced exon, as demonstrated by nucleotide sequencing of corresponding genomic regions. The exon (exon X) encodes a cysteine-rich stretch of 64 amino acids near the COOH terminus that displays a striking and surprising homology to an internal amino acid repeat of thyroglobulin, suggesting an evolutionary mechanism of exon shuffling. Transient expression of cDNAs indicated that both types of alternatively spliced mRNAs contain two in-frame AUGs functioning as alternate start sites for translation. Thus, transfections with exon X-lacking cDNAs resulted in the expression of Mr 33,000 and 31,000 proteins, detected by immunoprecipitation with anti-invariant chain antisera, and identical by two-dimensional gel (2-D) analyses to the B cell invariant-chain forms gamma 1 (Mr 31,000), gamma 2, and gamma 3 (Mr 33,000). Similarly, exon X-containing cDNAs expressed Mr 43,000 and 41,000 proteins, also identical by 2-D migration to Ia-associated proteins. Thus, human Ia molecules contain four forms of invariant chain of closely related but nonidentical primary structure that are generated from a single gene by a complex pattern of alternate transcriptional start, exon splicing, and translational start.

Base Sequence↗

PubMed2006

AltTrans: transcript pattern variants annotated for both alternative splicing and alternative polyadenylation.

BACKGROUND: The three major mechanisms that regulate transcript formation involve the selection of alternative sites for transcription start (TS), splicing, and polyadenylation. Currently there are efforts that collect data & annotation individually for each of these variants. It is important to take an integrated view of these data sets and to derive a data set of alternate transcripts along with consolidated annotation. We have been developing in the past computational pipelines that generate value-added data at genome-scale on individual variant types; these include AltSplice on splicing and AltPAS on polyadenylation. We now extend these pipelines and integrate the resultant data sets to facilitate an integrated view of the contributions from splicing and polyadenylation in the formation of transcript variants. DESCRIPTION: The AltSplice pipeline examines gene-transcript alignments and delineates alternative splice events and splice patterns; this pipeline is extended as AltTrans to delineate isoform transcript patterns for each of which both introns/exons and 'terminating' polyA site are delineated; EST/mRNA sequences that qualify the transcript pattern confirm both the underlying splicing and polyadenylation. The AltPAS pipeline examines gene-transcript alignments and delineates all potential polyA sites irrespective of underlying splicing patterns. Resultant polyA sites from both AltTrans and AltPAS are merged. The generated database reports data on alternative splicing, alternative polyadenylation and the resultant alternate transcript patterns; the basal data is annotated for various biological features. The data (named as integrated AltTrans data) generated for both the organisms of human and mouse is made available through the Alternate Transcript Diversity web site at http://www.ebi.ac.uk/atd/. CONCLUSION: The reported data set presents alternate transcript patterns that are annotated for both alternative splicing and alternative polyadenylation. Results based on current transcriptome data indicate that the contribution of alternative splicing is larger than that of alternative polyadenylation.

Alternative Splicing↗

PubMed1988

Splice site selection, rate of splicing, and alternative splicing on nascent transcripts.

Based on ultrastructural analysis of actively transcribing genes seen in electron micrographs, we present evidence that pre-mRNA splicing occurs with a reasonable frequency on the nascent transcripts of early Drosophila embryo genes and that splice site selection may generally precede polyadenylation. The details of the process observed are in agreement with results from in vitro splicing systems but differ in the more rapid completion of in vivo splicing. For those introns that are removed cotranscriptionally, a series of events is initiated following 3' splice site synthesis, beginning with ribonucleoprotein (RNP) particle formation at the 3' splice site within 48 sec, intron loop formation within 2 min, and splicing within 3 min. The initiation of the process is correlated with 3' splice site synthesis but is independent of 5' splice site synthesis, the position of the intron within the transcript, and the age or length of the transcript. In some cases, introns are removed from the 5' end of a transcript before introns are synthesized at the 3' end, supporting a possible role for the order of transcription in splice site pairing. In general, our observations are consistent with the 'first-come-first-served' principle of splice site selection, although an observed example of exon skipping indicates that alternative splicing possibilities can be accommodated within this general framework.

Animals↗

PubMed2005

Differential recruitment of pre-mRNA splicing factors to alternatively spliced transcripts in vivo.

Alternative splicing in mammalian cells has been suggested to be largely controlled by combinatorial binding of basal splicing factors to pre-mRNA templates. This model predicts that distinct sets of pre-mRNA splicing factors are associated with alternatively spliced transcripts. However, no experimental evidence for differential recruitment of splicing factors to transcripts with distinct splicing fates is available. Here we have used quantitative single-cell imaging to test this key prediction in vivo. We show that distinct combinations of splicing factors are recruited to sites of alternatively spliced transcripts in intact cells. While a subset of serine/arginine protein splicing factors, including SF2/ASF, SC35, and SRp20, is efficiently recruited to the tau gene when exon 10 is included, these factors are less frequently associated with tau transcription sites when exon 10 is excluded. In contrast, the frequency of recruitment of several other splicing factors is independent of splicing outcome. Mutation analysis of SF2/ASF shows that both protein-protein as well as protein-RNA interactions are required for differential recruitment. The differential behavior of the various splicing factors provides the basis for combinatorial occupancy at pre-mRNAs. These observations represent the first in vivo evidence for differential association of pre-mRNA splicing factors with alternatively spliced transcripts. They confirm a key prediction of a stochastic model of alternative splicing, in which distinct combinatorial sets of generic pre-mRNA splicing factors contribute to splicing outcome.

Alternative Splicing↗

PubMed1994

Alternative splicing generates secretory isoforms of human CD1.

Human CD1 genes are a family of five non-polymorphic genes that, although homologous to both class I and II major histocompatibility complex genes, map to chromosome 1. Only three of the antigens, CD1a, -b, and -c, have been clustered with monoclonal antibodies. They are noncovalently associated with beta 2-microglobulin and may function as nonclassical antigen-presenting molecules. Here we analyze their expression in mouse myeloma transfectants and human thymocytes and find mRNA splicing complexity. This manifests itself as incomplete splicing, alternative splicing, utilization of cryptic splice sites, and the generation of alternative reading frames. In the case of CD1A transfectants, we demonstrate that the major protein product is secreted and show by amino acid sequence analysis that this is derived from an unspliced transcript. A second major CD1a component appears to be retained intracellularly. The production of alternatively spliced transcripts in the thymus is not a feature of all CD1 genes. Although in the case of CD1A only the transcript encoding the cell surface CD1a isoform is found, CD1C and -E produce complex intrathymic splicing patterns. The CD1C transcripts predict the expression of a secreted CD1c isoform in the human thymus, which we detect in CD1C transfectant culture supernatants. CD1 gene expression is thus characterized by considerable splicing complexity, and the difference between the splicing patterns found in different environments suggests that this is tissue specific.

Alternative Splicing↗

PubMed1993

Novel variants of CD44 arising from alternative splicing: changes in the CD44 alternative splicing pattern of MCF-7 breast carcinoma cells treated with hyaluronidase.

CD44 is a cell-surface glycoprotein postulated to play a role in a variety of biological processes, including lymphocyte homing and tumor-cell metastasis. Several isoforms of CD44 have been identified in human cells, and the genesis of some of these isoforms has been attributed to alternative splicing. In the study presented here we amplified three novel transcript variants of CD44 from human cell lines using a reverse transcriptase-polymerase chain reaction strategy. Two of the novel isoforms differed from previously described CD44 isoforms as a result of alternative splicing that occurred at previously reported splice junctions. The third novel CD44 isoform was generated from a previously unreported alternative splice junction near the 5' end of the open reading frame. Southern blot analysis of genomic DNA revealed that these novel isoforms and all of the previously described CD44 isoforms arose from alternative splicing. The capability of cells to modify their CD44 alternative splicing pattern was demonstrated in MCF-7 cells, which altered their CD44-isoform expression pattern in response to treatment with hyaluronidase. A better understanding of mechanisms regulating CD44 alternative splicing may provide insights into diverse processes, including tumor-cell metastasis and lymphocyte homing.

Alternative Splicing↗

PubMed1999

Stage-specific changes in SR splicing factors and alternative splicing in mammary tumorigenesis.

Using a mouse model of mammary gland development and tumorigenesis we examined changes in both alternative splicing and splicing factors in multiple stages of mammary cancer. The emphasis was on the SR family of splicing factors known to influence alternative splicing in a wide variety of genes, and on alternative splicing of the pre-mRNA encoding CD44, for which alternative splicing has been implicated as important in a number of human cancers, including breast cancer. We observed step-wise increases in expression of individual SR proteins and alternative splicing of CD44 mRNA during mammary gland tumorigenesis. Individual preneoplasias differed as to their expression patterns for SR proteins, often expressing only a sub-set of the family. In contrast, tumors demonstrated a complex pattern of SR expression. Little difference was observed between neoplasias and their metastases. Alternative splicing of CD44 also changed through the disease paradigm such that tumors produced RNA containing a mixture of variable exons, whereas preneoplasias exhibited a more restricted exon inclusion pattern. In contrast, other standard splicing factors changed little in either concentration or splicing pattern in the same cells. These data suggest alterations in relative concentrations of specific splicing factors during early preneoplasia that become more pronounced during tumor formation. Given the ability of SR proteins to affect alternative processing decisions, our results suggest that a number of pre-mRNAs may undergo changes in alternative splicing during the early and intermediate stages of mammary cancer.

Alternative Splicing↗

PubMed1993

Discrete splicing alternatives in mRNA encoding the extracellular domain of the testis FSH receptor in the normal and hypogonadal (hpg) mouse.

The structure of RNA encoding the mouse testis FSH receptor was studied using reverse transcription and the polymerase chain reaction. Four major bands were observed by ethidium bromide staining and by hybridization to an FSH-receptor cDNA probe. The largest of these bands was the expected size (779 bp) while the other bands were spaced approximately 70 bp apart. Using alternative primers, each of the products was shown to contain exons 1, 9 and 10. Exons 2-8 in the FSH receptor gene are between 68 and 77 bp in size, suggesting that these multiple products arise by alternate splicing of the region encoding the extracellular domain of the receptor. A similar pattern of splicing was observed in cDNA from the testes of hypogonadal mice, showing that this alternative splicing pattern is not gonadotrophin-dependent.

Alternative Splicing↗

PubMed2006

EASI--enrichment of alternatively spliced isoforms.

Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This directly purifies the single-stranded regions of heteroduplexes between alternative splices formed in the PCR, enabling direct sequencing of all the rare alternative splice forms of any gene. As a proof of principle the alternative transcripts of three tumour suppressor genes, TP53, MLH1 and MSH2, were isolated from testis cDNA. These contain missing exons, cryptic splice sites or include completely novel exons. EASI beads are stable for months in the fridge and can be easily combined with standard protocols to speed the cloning of novel transcripts.

Alternative Splicing↗

PubMed2005

Protein modularity of alternatively spliced exons is associated with tissue-specific regulation of alternative splicing.

Recent comparative genomic analysis of alternative splicing has shown that protein modularity is an important criterion for functional alternative splicing events. Exons that are alternatively spliced in multiple organisms are much more likely to be an exact multiple of 3 nt in length, representing a class of "modular" exons that can be inserted or removed from the transcripts without affecting the rest of the protein. To understand the precise roles of these modular exons, in this paper we have analyzed microarray data for 3,126 alternatively spliced exons across ten mouse tissues generated by Pan and coworkers. We show that modular exons are strongly associated with tissue-specific regulation of alternative splicing. Exons that are alternatively spliced at uniformly high transcript inclusion levels or uniformly low levels show no preference for protein modularity. In contrast, alternatively spliced exons with dramatic changes of inclusion levels across mouse tissues (referred to as "tissue-switched" exons) are both strikingly biased to be modular and are strongly conserved between human and mouse. The analysis of different subsets of tissue-switched exons shows that the increased protein modularity cannot be explained by the overall exon inclusion level, but is specifically associated with tissue-switched alternative splicing.

Alternative Splicing↗

PubMed2004

How did alternative splicing evolve?

Alternative splicing creates transcriptome diversification, possibly leading to speciation. A large fraction of the protein-coding genes of multicellular organisms are alternatively spliced, although no regulated splicing has been detected in unicellular eukaryotes such as yeasts. A comparative analysis of unicellular and multicellular eukaryotic 5' splice sites has revealed important differences - the plasticity of the 5' splice sites of multicellular eukaryotes means that these sites can be used in both constitutive and alternative splicing, and for the regulation of the inclusion/skipping ratio in alternative splicing. So, alternative splicing might have originated as a result of relaxation of the 5' splice site recognition in organisms that originally could support only constitutive splicing.

Alternative Splicing↗

PubMed2004

ASD: the Alternative Splicing Database.

Alternative splicing is widespread in mammalian gene expression, and variant splice patterns are often specific to different stages of development, particular tissues or a disease state. There is a need to systematically collect data on alternatively spliced exons, introns and splice isoforms, and to annotate this data. The Alternative Splicing Database consortium has been addressing this need, and is committed to maintaining and developing a value-added database of alternative splice events, and of experimentally verified regulatory mechanisms that mediate splice variants. In this paper we present two of the products from this project: namely, a database of computationally delineated alternative splice events as seen in alignments of EST/cDNA sequences with genome sequences, and a database of alternatively spliced exons collected from literature. The reported splice events are from nine different organisms and are annotated for various biological features including expression states and cross-species conservation. The data are presented on our ASD web pages (http://www.ebi.ac.uk/asd).

Alternative Splicing↗

PubMed2000

Binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral mRNAs.

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.

Alternative Splicing↗