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T A Cooper

Publications and source records attributed to T A Cooper.

At least 19 recordsLinked to original sources
PubMed2001

Highlights of alternative splicing regulation session: yes, no, maybe--a history of paradigm shifts.

Highlights from the Sixth Annual Meeting of the RNA Society, Banff, Alberta, Canada, 29 May to 3 June 2001. Cooper summarizes the discussions and presentations from the session entitled "Control of Splice Site Selection" held at the Sixth Annual Meeting of the RNA Society. Paradigms are shifting as experiments show that some of the proteins involved in regulating splicing can act as splicing enhancers or repressors, depending on the cellular context. The complex interactions among the ribonucleoproteins (RNPs) and proteins, and the role of cis elements, in controlling cell-specific splicing are highlighted. The importance of properly regulated splicing is emphasized by examples of disease pathologies in which alternative splicing is aberrant.

Alternative Splicing↗
PubMed2001

The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4.

Exon enhancers are accessory pre-mRNA splicing signals that stimulate exon splicing. One class of proteins, the serine-arginine-rich (SR) proteins, have been demonstrated to bind enhancers and activate splicing. Here we report that A/C-rich exon enhancers (ACE elements) are recognized by the human YB-1 protein, a non-SR protein. Sequence-specific binding of YB-1 was observed both to an ACE derived from an in vivo iterative selection protocol and to ACE elements in an alternative exon (v4) from the human CD44 gene. The ACE element that was the predominant YB-1 binding site in CD44 exon v4 was required for maximal in vivo splicing and in vitro spliceosome assembly. Expression of wild-type YB-1 increased inclusion of exon v4, whereas a truncated form of YB-1 did not. Stimulation of exon v4 inclusion by wild-type YB-1 required the ACE necessary for YB-1 binding in vitro, suggesting that YB-1 stimulated exon inclusion in vivo by binding to an exonic ACE element. These observations identify a protein in addition to SR proteins that participates in the recognition of exon enhancers.

Alternative Splicing↗
PubMed2001

Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide expansion in the 3' untranslated region of the DM protein kinase gene. People with DM1 have an unusual form of insulin resistance caused by a defect in skeletal muscle. Here we demonstrate that alternative splicing of the insulin receptor (IR) pre-mRNA is aberrantly regulated in DM1 skeletal muscle tissue, resulting in predominant expression of the lower-signaling nonmuscle isoform (IR-A). IR-A also predominates in DM1 skeletal muscle cultures, which exhibit a decreased metabolic response to insulin relative to cultures from normal controls. Steady-state levels of CUG-BP, a regulator of pre-mRNA splicing proposed to mediate some aspects of DM1 pathogenesis, are increased in DM1 skeletal muscle; overexpression of CUG-BP in normal cells induces a switch to IR-A. The CUG-BP protein mediates this switch through an intronic element located upstream of the alternatively spliced exon 11, and specifically binds within this element in vitro. These results support a model in which increased expression of a splicing regulator contributes to insulin resistance in DM1 by affecting IR alternative splicing.

Alternative Splicing↗
PubMed2001

The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing.

Alternative splicing of cardiac troponin T (cTNT) exon 5 undergoes a developmentally regulated switch such that exon inclusion predominates in embryonic, but not adult, striated muscle. We previously described four muscle-specific splicing enhancers (MSEs) within introns flanking exon 5 in chicken cTNT that are both necessary and sufficient for exon inclusion in embryonic muscle. We also demonstrated that CUG-binding protein (CUG-BP) binds a conserved CUG motif within a human cTNT MSE and positively regulates MSE-dependent exon inclusion. Here we report that CUG-BP is one of a novel family of developmentally regulated RNA binding proteins that includes embryonically lethal abnormal vision-type RNA binding protein 3 (ETR-3). This family, which we call CELF proteins for CUG-BP- and ETR-3-like factors, specifically bound MSE-containing RNAs in vitro and activated MSE-dependent exon inclusion of cTNT minigenes in vivo. The expression of two CELF proteins is highly restricted to brain. CUG-BP, ETR-3, and CELF4 are more broadly expressed, and expression is developmentally regulated in striated muscle and brain. Changes in the level of expression and isoforms of ETR-3 in two different developmental systems correlated with regulated changes in cTNT splicing. A switch from cTNT exon skipping to inclusion tightly correlated with induction of ETR-3 protein expression during differentiation of C2C12 myoblasts. During heart development, the switch in cTNT splicing correlated with a transition in ETR-3 protein isoforms. We propose that ETR-3 is a major regulator of cTNT alternative splicing and that the CELF family plays an important regulatory role in cell-specific alternative splicing during normal development and disease.

Adult↗
PubMed2000

Binding of PurH to a muscle-specific splicing enhancer functionally correlates with exon inclusion in vivo.

Regulated alternative splicing of avian cardiac troponin T (cTNT) pre-mRNA requires multiple intronic elements called muscle-specific splicing enhancers (MSEs) that flank the alternative exon 5 and promote muscle-specific exon inclusion. To understand the function of the MSEs in muscle-specific splicing, we sought to identify trans-acting factors that bind to these elements. MSE3, which is located 66-81 nucleotides downstream of exon 5, assembles a complex that is both sequence- and muscle-specific. Purification and characterization of the MSE3 complex identified one component as 5-aminoimidazole-4-carboxamide ribonucleotideformyltransferase/IMP cyclohydrolase (PurH), an enzyme involved in de novo purine synthesis. Recombinant human PurH protein directly binds MSE3 RNA and PurH is the primary determinant of sequence-specific binding in the native complex. Furthermore, we show a direct correlation between the in vitro binding affinity of both the MSE3 complex and recombinant PurH with functional activation of exon inclusion in vivo. Together, these results strongly suggest that PurH performs a second function as a component of a complex that regulates MSE3-dependent exon inclusion.

Alternative Splicing↗
PubMed2000

RNA processing and human disease.

Gene expression involves multiple regulated steps leading from gene to active protein. Many of these steps involve some aspect of RNA processing. Diseases caused by mutations that directly affect RNA processing are relatively rare compared with mutations that disrupt protein function. The vast majority of diseases of RNA processing result from loss of function of a single gene due to mutations in cis-acting elements required for pre-messenger RNA (mRNA) splicing. However, a few diseases are caused by alterations in the trans-acting factors required for RNA processing and in the vast majority of cases it is the pre-mRNA splicing machinery that is affected. Clearly, alterations that disrupt splicing of pre-mRNAs from large numbers of genes would be lethal at the cellular level. A common theme among these diseases is that only subsets of genes are affected. This is consistent with an emerging view that different subsets of exons require different sets of cis-acting elements and trans-acting factors.

Alternative Splicing↗
PubMed1999

The relative strengths of SR protein-mediated associations of alternative and constitutive exons can influence alternative splicing.

We have characterized the functional role of SR protein-mediated exon/exon associations in the alternative splicing of exon 5 of chicken cardiac troponin T (cTnT). We have previously shown that SR proteins can promote the association of the alternative exon 5 with the flanking constitutive exon 6 of this pre-mRNA. In this study, we have shown that when exons 2, 3, and 4 of the cTnT pre-mRNA are spliced together, the composite exon 2/3/4 contains an additional SR protein binding site. Furthermore, we have found that SR proteins can also promote interactions between the pairs of exons 2/3/4-5 and 2/3/4-6. We then asked whether the SR protein binding sites in these exons play a role in cTnT alternative splicing in vivo. We found that the SR protein binding sites in exons 2/3/4 and 6 promote exon 5 skipping, and it has previously been shown that the SR protein binding site in exon 5 promotes exon 5 inclusion. Consistent with these results, we find that the SR protein-mediated association of exon 2/3/4 with 6 is preferred over associations involving exon 5, in that exons 2/3/4 and 6 are more efficient than exon 5 in competing an SR protein-mediated exon/exon association. We suggest that the relative strengths of SR protein-mediated associations of alternative and constitutive exons play a role in determining alternative splicing patterns.

Alternative Splicing↗
PubMed1999

Alternative splicing determines the intracellular localization of the novel nuclear protein Nop30 and its interaction with the splicing factor SRp30c.

We report on the molecular cloning of a novel human cDNA by its interaction with the splicing factor SRp30c in a yeast two-hybrid screen. This cDNA is predominantly expressed in muscle and encodes a protein that is present in the nucleoplasm and concentrated in nucleoli. It was therefore termed Nop30 (nucleolar protein of 30 kDa). We have also identified a related cDNA with a different carboxyl terminus. Sequencing of the NOP gene demonstrated that both cDNAs are generated by alternative 5' splice site usage from a single gene that consists of four exons, spans at least 1800 nucleotides, and is located on chromosome 16q21-q23. The alternative 5' splice site usage introduces a frameshift creating two different carboxyl termini. The carboxyl terminus of Nop30 is rich in serines and arginines and has been found to target the protein into the nucleus, whereas its isoform is characterized by proline/glutamic acid dipeptides in its carboxyl terminus and is predominantly found in the cytosol. Interaction studies in yeast, in vitro protein interaction assays, and co-immunoprecipitations demonstrated that Nop30 multimerizes and binds to the RS domain of SRp30c but not to other splicing factors tested. Overexpression of Nop30 changes alternative exon usage in preprotachykinin and SRp20 reporter genes, suggesting that Nop30 influences alternative splice site selection in vivo.

Alternative Splicing↗
PubMed1998

Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy.

Myotonic dystrophy (DM) is caused by a CTG expansion in the 3' untranslated region of the DM gene. One model of DM pathogenesis suggests that RNAs from the expanded allele create a gain-of-function mutation by the inappropriate binding of proteins to the CUG repeats. Data presented here indicate that the conserved heterogeneous nuclear ribonucleoprotein, CUG-binding protein (CUG-BP), may mediate the trans-dominant effect of the RNA. CUG-BP was found to bind to the human cardiac troponin T (cTNT) pre-messenger RNA and regulate its alternative splicing. Splicing of cTNT was disrupted in DM striated muscle and in normal cells expressing transcripts that contain CUG repeats. Altered expression of genes regulated posttranscriptionally by CUG-BP therefore may contribute to DM pathogenesis.

Alternative Splicing↗
PubMed1998

A short sequence within two purine-rich enhancers determines 5' splice site specificity.

Purine-rich enhancers are exon sequences that promote inclusion of alternative exons, usually via activation of weak upstream 3' splice sites. A recently described purine-rich enhancer from the caldesmon gene has an additional activity by which it directs selection of competing 5' splice sites within an alternative exon. In this study, we have compared the caldesmon enhancer with another purine-rich enhancer from the chicken cardiac troponin T (cTNT) gene for the ability to regulate flanking splice sites. Although similar in sequence and length, the two enhancers demonstrated strikingly different specificities towards 5' splice site choice when placed between competing 5' splice sites in an internal exon. The 32-nucleotide caldesmon enhancer caused effective usage of the exon-internal 5' splice site, whereas the 30-nucleotide cTNT enhancer caused effective usage of the exon-terminal 5' splice site. Both enhancer-mediated splicing pathways represented modulation of the default pathway in which both 5' splice sites were utilized. Each enhancer is multipartite, consisting of two purine-rich sequences of a simple (GAR)n repeat interdigitated with two enhancer-specific sequences. The entire enhancer was necessary for maximal splice site selectivity; however, a 5- to 7-nucleotide region from the 3' end of each enhancer dictated splice site selectivity. Mutations that interchanged this short region of the two enhancers switched specificity. The portion of the cTNT enhancer determinative for 5' splice site selectivity was different than that shown to be maximally important for activation of a 3' splice site, suggesting that enhancer environment can have a major impact on activity. These results are the first indication that individual purine-rich enhancers can differentiate between flanking splice sites. Furthermore, localization of the specificity of splice site choice to a short region within both enhancers indicates that subtle differences in enhancer sequence can have profound effects on the splicing pathway.

Animals↗
PubMed1998

Muscle-specific splicing of a heterologous exon mediated by a single muscle-specific splicing enhancer from the cardiac troponin T gene.

The chicken cardiac troponin T (cTNT) gene contains a single 30-nucleotide alternative exon that is included in embryonic striated muscle and skipped in the adult. Transient-transfection analysis of cTNT minigenes in muscle and fibroblast cell cultures previously identified four muscle-specific splicing enhancers (MSEs) that promote exon inclusion specifically in embryonic striated muscle cultures. Three MSEs located in the intron downstream from the alternative exon were sufficient for muscle-specific exon inclusion. In the present study, the boundaries of these MSEs were defined by scanning mutagenesis, allowing analysis of individual elements in gain-of-function experiments. Concatamers of MSE2 were necessary and sufficient to promote muscle-specific inclusion of a heterologous exon, indicating that it is a target for muscle-specific regulation. Sequences present in MSE2 are also found in MSE4, suggesting that these two MSEs act in a similar manner. MSE3 appears to be different from MSE2 and MSE4 yet is able to functionally replace both of these elements, demonstrating functional redundancy of elements that are likely to bind different factors. MSE2 and MSE4 each contain a novel sequence motif that is found adjacent to a number of alternative exons that undergo regulated splicing in striated muscle, suggesting a common role for this element in muscle-specific regulation.

Animals↗
PubMed1997

Identification of a new class of exonic splicing enhancers by in vivo selection.

In vitro selection strategies have typically been used to identify a preferred ligand, usually an RNA, for an identified protein. Ideally, one would like to know RNA consensus sequences preferred in vivo for as-yet-unidentified factors. The ability to select RNA-processing signals would be particularly beneficial in the analysis of exon enhancer sequences that function in exon recognition during pre-mRNA splicing. Exon enhancers represent a class of potentially ubiquitous RNA-processing signals whose actual prevalence is unknown. To establish an approach for in vivo selection, we developed an iterative scheme to select for exon sequences that enhance exon inclusion. This approach is modeled on the in vitro SELEX procedure and uses transient transfection in an iterative procedure to enrich RNA-processing signals in cultured vertebrate cells. Two predominant sequence motifs were enriched after three rounds of selection: a purine-rich motif that resembles previously identified splicing enhancers and a class of A/C-rich splicing enhancers (ACEs). Individual selected ACEs enhanced splicing in vivo and in vitro. ACE splicing activity was competed by RNAs containing the purine-rich splicing enhancer from cardiac troponin T exon 5. Thus, ACE activity is likely to require a subset of the SR splicing factors previously shown to mediate activity of this purine-rich enhancer. ACE motifs are found in two vertebrate exons previously demonstrated to contain splicing enhancer activity as well as in the well-characterized Drosophila doublesex (dsx) splicing enhancer. We demonstrate that one copy of the dsx repeat enhances splicing of a vertebrate exon in vertebrate cells and that this enhancer activity requires the ACE motif. We suggest the possibility that the dsx enhancer is a member of a previously unrecognized family of ACEs.

Animals↗
PubMed1997

Effects of parathyroidectomy on induced renal failure in dogs.

OBJECTIVE: To determine the effects of parathyroid hormone (PTH) depletion on dogs with induced chronic renal failure. ANIMALS: 2 groups of 26 mixed-breed dogs of both sexes (13 were parathyroidectomized [PTX] and 13 had sham surgery). PROCEDURE: After surgical reduction of renal mass and PTX, dogs were selected for a 24-month period of study and monitored for clinical, hematologic, blood biochemical, and organ function status. On development of uremia or after 24 months, dogs were euthanatized, and tissues were examined. RESULTS: Higher survival rate and smaller decrement in renal function (glomerular filtration rate) were observed in PTX dogs, compared with those that had sham surgery, but values did not reach statistical significance. The PTX dogs remained hypocalcemic during the study and had lower serum Ca2+ X P product values. Regardless of parathyroid state, survivors and fatalities could be separated on the basis of serum Ca2+ X P product values. Parathyroidectomy did not prevent renal deposition of calcium, and renal lesions were poorly correlated with renal cortical calcium concentration. Abnormalities reported in dogs with renal failure, which were attributed to PTH (glucose intolerance, pulmonary hypertension), were not observed in PTX dogs or those that had sham surgery. CONCLUSIONS AND CLINICAL RELEVANCE: PTX had beneficial effects, but these were mediated via changes in mineral homeostasis rather than via direct effects of PTH. Results attributable to PTX were similar to those previously obtained by dietary restriction of phosphate intake.

Acidosis↗