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C Gooding

Publications and source records attributed to C Gooding.

14 recordsLinked to original sources
PubMed2005

Regulation of alternative splicing by PTB and associated factors.

PTB (polypyrimidine tract-binding protein) is a repressive regulator of alternative splicing. We have investigated the role of PTB in three model alternative splicing systems. In the alpha-actinin gene, PTB represses the SM (smooth muscle) exon by binding to key sites in the polypyrimidine tract. Repressive binding to these sites is assisted by co-operative binding to additional downstream sites. SM exon splicing can be activated by CELF proteins, which also bind co-operatively to interspersed sites and displace PTB from the pyrimidine tract. Exon 11 of PTB pre-mRNA is repressed by PTB in an autoregulatory feedback loop. Exon 11-skipped RNA gets degraded through nonsense-mediated decay. Less than 1% of steady-state PTB mRNA is represented by this isoform, but inhibition of nonsense-mediated decay by RNA interference against Upf1 shows that at least 20% of PTB RNA is consumed by this pathway. This represents a widespread but under-appreciated role of alternative splicing in the quantitative regulation of gene expression, an important addition to its role as a generator of protein isoform diversity. Repression of alpha-tropomyosin exon 3 is an exceptional example of PTB regulation, because repression only occurs at high levels in SM cells, despite the fact that PTB is widely expressed. In this case, a PTB-interacting cofactor, raver1, appears to play an important role. By the use of 'tethering' assays, we have identified discrete domains within both PTB and raver1 that mediate their repressive activities on this splicing event.

Actinin↗
PubMed2001

Differential alternative splicing activity of isoforms of polypyrimidine tract binding protein (PTB).

Polypyrimidine tract binding protein (PTB) is an RNA-binding protein that regulates splicing by repressing specific splicing events. It also has roles in 3'-end processing, internal initiation of translation, and RNA localization. PTB exists in three alternatively spliced isoforms, PTB1, PTB2, and PTB4, which differ by the insertion of 19 or 26 amino acids, respectively, between the second and third RNA recognition motif domains. Here we show that the PTB isoforms have distinct activities upon alpha-tropomyosin (TM) alternative splicing. PTB1 reduced the repression of TM exon 3 in transfected smooth muscle cells, whereas PTB4 enhanced TM exon 3 skipping in vivo and in vitro. PTB2 had an intermediate effect. The PTB4 > PTB2 > PTB1 repressive hierarchy was observed in all in vivo and in vitro assays with TM, but the isoforms were equally active in inducing skipping of alpha-actinin exons and showed the opposite hierarchy of activity when tested for activation of IRES-driven translation. These findings establish that the ratio of PTB isoforms could form part of a cellular code that in turn controls the splicing of various other pre-mRNAs.

Alternative Splicing↗
PubMed1999

Polypyrimidine tract binding protein functions as a repressor to regulate alternative splicing of alpha-actinin mutally exclusive exons.

The smooth muscle (SM) and nonmuscle (NM) isoforms of alpha-actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat alpha-actinin genomic clone encompassing the mutually exclusive exons was isolated and sequenced. The SM exon was found to utilize two branch points located 382 and 386 nucleotides (nt) upstream of the 3' splice site, while the NM exon used a single branch point 191 nt upstream. Mutually exclusive splicing arises from the proximity of the SM branch points to the NM 5' splice site, and this steric repression could be relieved in part by the insertion of spacer elements. In addition, the SM exon is repressed in non-SM cells and extracts. In vitro splicing of spacer-containing transcripts could be activated by (i) truncation of the transcript between the SM polypyrimidine tract and exon, (ii) addition of competitor RNAs containing the 3' end of the actinin intron or regulatory sequences from alpha-tropomyosin (TM), and (iii) depletion of the splicing extract by using biotinylated alpha-TM RNAs. A number of lines of evidence point to polypyrimidine tract binding protein (PTB) as the trans-acting factor responsible for repression. PTB was the only nuclear protein observed to cross-link to the actinin RNA, and the ability of various competitor RNAs to activate splicing correlated with their ability to bind PTB. Furthermore, repression of alpha-actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA could be specifically restored by the addition of recombinant PTB. Thus, alpha-actinin mutually exclusive splicing is enforced by the unusual location of the SM branch point, while constitutive repression of the SM exon is conferred by regulatory elements between the branch point and 3' splice site and by PTB.

Actinin↗
PubMed1998

Co-transcriptional commitment to alternative splice site selection.

Production of mRNA in eukaryotic cells involves not only transcription but also various processing reactions such as splicing. Recent experiments have indicated that there are direct physical connections between components of the transcription and processing machinery, supporting previous suggestions that pre-mRNA splicing occurs co-transcriptionally. Here we have used a novel functional approach to demonstrate co-transcriptional regulation of alternative splicing. Exon 3 of the alpha-tropomyosin gene is specifically repressed in smooth muscle cells. By delaying synthesis of an essential downstream inhibitory element, we show that the decision to splice or repress exon 3 occurs during a limited window of opportunity following transcription, indicating that splice site selection proceeds rapidly after transcription.

Alternative Splicing↗
PubMed1998

Role of an inhibitory pyrimidine element and polypyrimidine tract binding protein in repression of a regulated alpha-tropomyosin exon.

Splicing of exons 2 and 3 of a-tropomyosin (TM) involves mutually exclusive selection of either exon 3, which occurs in most cells, or of exon 2 in smooth muscle (SM) cells. The SM-specific selection of exon 2 results from the inhibition of exon 3. At least two essential cis-acting elements are required for exon 3 inhibition, the upstream and downstream regulatory elements (URE and DRE). These elements are essential for repression of TM exon 3 in SM cells, and also mediate a low level of repression of exon 3 in an in vitro 5' splice site competition assay in HeLa extracts. Here, we show that the DRE consists of at least two discrete components, a short region containing a number of UGC motifs, and an essential pyrimidine-rich tract (DY). We show that the specific sequence of the DY element is important and that DY is able to bind to factors in HeLa nuclear extracts that mediate a low background level of exon 3 skipping. Deletion of a sequence within DY identified as an optimal binding site for PTB impairs (1) regulation of splicing in vivo, (2) skipping of exon 3 in an in vitro 5' splice site competition, (3) the ability of DY competitors to affect the 5' splice site competition in vitro, and (4) binding of PTB to DY. Addition of recombinant PTB to in vitro splicing reactions is able to partially reverse the effects of the DY competitor RNA. The data are consistent with a model for regulation of TM splicing that involves the participation of both tissue-specific and general inhibitory factors and in which PTB plays a role in repressing both splice sites of exon 3.

Base Sequence↗
PubMed1996

Smooth muscle alternative splicing induced in fibroblasts by heterologous expression of a regulatory gene.

Alternative splicing is a common mechanism for regulating gene expression in different cell types. In order to understand this important process, the trans-acting factors that enforce the choice of particular splicing pathways in different environments must be identified. We have used the rat alpha-tropomyosin gene as a model system of tissue-specific alternative splicing. Exon 3 of alpha-tropomyosin is specifically inhibited in smooth muscle cells allowing the alternative inclusion of exon 2. We have used a novel gene transfer and selection strategy to detect a gene whose expression in fibroblasts is sufficient to switch them to smooth muscle-specific splicing of alpha-tropomyosin and also alpha-actinin. Extracts from the regulating fibroblasts contain an apparently novel 55 kDa protein which binds to RNA elements required for regulation of tropomyosin splicing. This protein is not detected in extracts of non-regulating cells and is therefore a strong candidate cell-specific splicing regulator. These experiments advance our understanding of smooth muscle splicing regulation as well as establishing a means for direct cloning of tissue-specific splicing regulators which have so far been refractory to biochemical analysis.

Actinin↗
PubMed1994

Smooth muscle-specific switching of alpha-tropomyosin mutually exclusive exon selection by specific inhibition of the strong default exon.

Exons 2 and 3 of alpha-tropomyosin are spliced in a strict mutually exclusive manner. Exon 3 is a default choice, being selected in almost all cell types where the gene is expressed. The default selection arises from a competition between the two exons, in which the stronger branch point/pyrimidine tract elements of exon 3 win. Exon 2 is selected predominantly or exclusively only in smooth muscle cells. We show here that the basis for the smooth muscle-specific switching of exon selection is inhibition of exon 3. Exon 3 is still skipped with smooth muscle specificity, even in the absence of exon 2. We have defined two conserved sequence elements, one in each of the introns flanking exon 3, that are essential for this regulation. Mutation of either element severely impairs regulated suppression of exon 3. No other exon or intron sequences appear to be necessary for regulation. We have also demonstrated skipping of exon 3 that is dependent upon both regulatory elements in an in vitro splicing assay. We further show that both splice sites of exon 3 must be inhibited in a concerted fashion to switch to selection of exon 2. This may relate to the requirement for negative elements on both sides of the exon.

Alternative Splicing↗
PubMed1992

LCR/MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells.

We have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin promoter and the second intron of the human beta-globin gene, and this expression cassette is then placed downstream of the LCR and transfected into MEL cells. The cDNAs are expressed at levels similar to those of the murine beta-globin in the induced MEL cells. Heterologous genomic sequences can also be expressed at similar levels when linked to to the LCR and beta-globin promoter. In addition we demonstrate that, after induction of differentiation, MEL cells are capable of secreting heterologous proteins over a prolonged time period, making this system suitable for use in continuous production systems such as hollow fibre bioreactors. The utility of the LCR/MEL cell system is demonstrated by the expression of growth hormone at high levels (greater than 100 mg/l) 7 days after induction. Since the expression levels seen do not depend upon gene amplification and are independent of the integration position of the expression cassette, it is possible to obtain clones with stable high-level expression within 3-4 weeks after transfection.

Animals↗
PubMed1988

Human hTM alpha gene: expression in muscle and nonmuscle tissue.

We have isolated a cDNA clone from a human skeletal muscle library which contains the complete protein-coding sequence of a skeletal muscle alpha-tropomyosin. This cDNA sequence defines a fourth human tropomyosin gene, the hTM alpha gene, which is distinct from the hTMnm gene encoding a closely related isoform of skeletal muscle alpha-tropomyosin. In cultured human fibroblasts, the hTM alpha gene encodes both skeletal-muscle- and smooth-muscle-type alpha-tropomyosins by using an alternative mRNA-splicing mechanism.

Amino Acid Sequence↗
PubMed1980

In vivo imaging of the rat anatomy with nuclear magnetic resonance.

Live rats were imaged by nuclear magnetic resonance (NMR). These images demonstrated fine detail and high object contrast. Motion artifacts are not apparent in 4-minute images, and major blood vessels are demonstrated as regions of low signal intensity because of blood flow. Selective contrast enhancement is possible by varying NMR imager accumulation parameters.

Animals↗