Search PubMed⌕ Search

Biomedical subjects

Douglas L Black

Publications and source records attributed to Douglas L Black.

14 recordsLinked to original sources
PubMed2007

MicroRNAs regulate the expression of the alternative splicing factor nPTB during muscle development.

Alternative pre-mRNA splicing determines many changes in gene expression during development. Two regulators known to control splicing patterns during neuron and muscle differentiation are the polypyrimidine tract-binding protein (PTB) and its neuronal homolog nPTB. These proteins repress certain exons in early myoblasts, but upon differentiation of mature myotubes PTB/nPTB expression is reduced, leading to increased inclusion of their target exons. We show here that the repression of nPTB expression during myoblast differentiation results from its targeting by the muscle-restricted microRNA miR-133. During differentiation of C2C12 myoblasts, nPTB protein but not mRNA expression is strongly reduced, concurrent with the up-regulation of miR-133 and the induction of splicing for several PTB-repressed exons. Introduction of synthetic miR-133 into undifferentiated C2C12 cells leads to a decrease in endogenous nPTB expression. Both the miR-133 and the coexpressed miR-1/206 microRNAs are extremely conserved across animal species, and PTB proteins are predicted targets for these miRNAs in Drosophila, mice, and humans. There are two potential miR-133-responsive elements (MRE) within the nPTB 3' untranslated region (UTR), and a luciferase reporter carrying this 3' UTR is repressed by miR-133 in an MRE-dependent manner. Transfection of locked nucleic acid (LNA) oligonucleotides designed to block the function of miR-133 and miR-1/206 increases expression of nPTB and decreases the inclusion of PTB dependent exons. These results indicate that miR-133 directly down-regulates a key splicing factor during muscle development and establishes a role for microRNAs in the control of a developmentally dynamic splicing program.

3' Untranslated Regions↗
PubMed2006

Maps, codes, and sequence elements: can we predict the protein output from an alternatively spliced locus?

Alternative splicing choices are governed by splicing regulatory protein interactions with splicing silencer and enhancer elements present in the pre-mRNA. However, the prediction of these choices from genomic sequence is difficult, in part because the regulators can act as either enhancers or silencers. A recent study describes how for a particular neuronal splicing regulatory protein, Nova, the location of its binding sites is highly predictive of the protein's effect on an exon's splicing.

Alternative Splicing↗
PubMed2005

Molecular basis of RNA recognition by the human alternative splicing factor Fox-1.

The Fox-1 protein regulates alternative splicing of tissue-specific exons by binding to GCAUG elements. Here, we report the solution structure of the Fox-1 RNA binding domain (RBD) in complex with UGCAUGU. The last three nucleotides, UGU, are recognized in a canonical way by the four-stranded beta-sheet of the RBD. In contrast, the first four nucleotides, UGCA, are bound by two loops of the protein in an unprecedented manner. Nucleotides U1, G2, and C3 are wrapped around a single phenylalanine, while G2 and A4 form a base-pair. This novel RNA binding site is independent from the beta-sheet binding interface. Surface plasmon resonance analyses were used to quantify the energetic contributions of electrostatic and hydrogen bond interactions to complex formation and support our structural findings. These results demonstrate the unusual molecular mechanism of sequence-specific RNA recognition by Fox-1, which is exceptional in its high affinity for a defined but short sequence element.

Alternative Splicing↗
PubMed2005

Structure of PTB bound to RNA: specific binding and implications for splicing regulation.

The polypyrimidine tract binding protein (PTB) is a 58-kilodalton RNA binding protein involved in multiple aspects of messenger RNA metabolism, including the repression of alternative exons. We have determined the solution structures of the four RNA binding domains (RBDs) of PTB, each bound to a CUCUCU oligonucleotide. Each RBD binds RNA with a different binding specificity. RBD3 and RBD4 interact, resulting in an antiparallel orientation of their bound RNAs. Thus, PTB will induce RNA looping when bound to two separated pyrimidine tracts within the same RNA. This leads to structural models for how PTB functions as an alternative-splicing repressor.

Alternative Splicing↗
PubMed2005

Polypyrimidine tract binding protein blocks the 5' splice site-dependent assembly of U2AF and the prespliceosomal E complex.

Polypyrimidine tract binding protein (PTB) represses some alternatively spliced exons by direct occlusion of splice sites. In repressing the splicing of the c-src N1 exon, we find that PTB acts by a different mechanism. PTB does not interfere with U1 snRNP binding to the N1 5' splice site. Instead, PTB prevents formation of the prespliceosomal early (E) complex across the intervening intron by preventing the assembly of the splicing factor U2AF on the 3' splice site of exon 4. When the unregulated 5' splice site of the upstream exon 3 is present, U2AF binding is restored and splicing between exons 3 and 4 proceeds in spite of the N1 exon bound PTB. Thus, rather than directly blocking the N1 splice sites, PTB prevents the 5' splice site-dependent assembly of U2AF into the E complex. This mechanism likely occurs in many other alternative exons.

5' Untranslated Regions↗
PubMed2005

Homologues of the Caenorhabditis elegans Fox-1 protein are neuronal splicing regulators in mammals.

A vertebrate homologue of the Fox-1 protein from C. elegans was recently shown to bind to the element GCAUG and to act as an inhibitor of alternative splicing patterns in muscle. The element UGCAUG is a splicing enhancer element found downstream of numerous neuron-specific exons. We show here that mouse Fox-1 (mFox-1) and another homologue, Fox-2, are both specifically expressed in neurons in addition to muscle and heart. The mammalian Fox genes are very complex transcription units that generate transcripts from multiple promoters and with multiple internal exons whose inclusion is regulated. These genes produce a large family of proteins with variable N and C termini and internal deletions. We show that the overexpression of both Fox-1 and Fox-2 isoforms specifically activates splicing of neuronally regulated exons. This splicing activation requires UGCAUG enhancer elements. Conversely, RNA interference-mediated knockdown of Fox protein expression inhibits splicing of UGCAUG-dependent exons. These experiments show that this large family of proteins regulates splicing in the nervous system. They do this through a splicing enhancer function, in addition to their apparent negative effects on splicing in vertebrate muscle and in worms.

Animals↗
PubMed2005

A consensus CaMK IV-responsive RNA sequence mediates regulation of alternative exons in neurons.

Neurons make extensive use of alternative pre-mRNA splicing to regulate gene expression and diversify physiological responses. We showed previously in a pituitary cell line that the Ca(++)/calmodulin-dependent protein kinase CaMK IV specifically repressed splicing of the BK channel STREX exon. This repression is dependent on a CaMK IV-responsive RNA element (CaRRE) within the STREX 3' splice site. Here, we report that similar Ca(++) regulation of splicing, mediated by L-type calcium channels and CaM kinase IV, occurs in cultured neurons and in the brain. We identify a critical CaRRE motif (CACATNRTTAT) that is essential for conferring CaMK IV repression on an otherwise constitutive exon. Additional Ca(++)-regulated exons that carry this consensus sequence are also identified in the human genome. Thus, the Ca(++)/CaMK IV pathway in neurons controls the alternative splicing of a group of exons through this short CaRRE consensus sequence. The functions of some of these exons imply that splicing control through the CaMK IV pathway will alter neuronal activity.

Alternative Splicing↗
PubMed2005

Exon repression by polypyrimidine tract binding protein.

Polypyrimidine tract binding protein (PTB) is known to silence the splicing of many alternative exons. However, exons repressed by PTB are affected by other RNA regulatory elements and proteins. This makes it difficult to dissect the structure of the pre-mRNP complexes that silence splicing, and to understand the role of PTB in this process. We determined the minimal requirements for PTB-mediated splicing repression. We find that the minimal sequence for high affinity binding by PTB is relatively large, containing multiple polypyrimidine elements. Analytical ultracentrifugation and proteolysis mapping of RNA cross-links on the PTB protein indicate that most PTB exists as a monomer, and that a polypyrimidine element extends across multiple PTB domains. The high affinity site is bound initially by a PTB monomer and at higher concentrations by additional PTB molecules. Significantly, this site is not sufficient for splicing repression when placed in the 3' splice site of a strong test exon. Efficient repression requires a second binding site within the exon itself or downstream from it. This second site enhances formation of a multimeric PTB complex, even if it does not bind well to PTB on its own. These experiments show that PTB can be sufficient to repress splicing of an otherwise constitutive exon, without binding sites for additional regulatory proteins and without competing with U2AF binding. The minimal complex mediating splicing repression by PTB requires two binding sites bound by an oligomeric PTB complex.

Alternative Splicing↗
PubMed2003

Differentiation-induced colocalization of the KH-type splicing regulatory protein with polypyrimidine tract binding protein and the c-src pre-mRNA.

We have examined the subcellular localization of the KH-type splicing regulatory protein (KSRP). KSRP is a multidomain RNA-binding protein implicated in a variety of cellular processes, including splicing in the nucleus and mRNA localization in the cytoplasm. We find that KSRP is primarily nuclear with a localization pattern that most closely resembles that of polypyrimidine tract binding protein (PTB). Colocalization experiments of KSRP with PTB in a mouse neuroblastoma cell line determined that both proteins are present in the perinucleolar compartment (PNC), as well as in other nuclear enrichments. In contrast, HeLa cells do not show prominent KSRP staining in the PNC, even though PTB labeling identified the PNC in these cells. Because both PTB and KSRP interact with the c-src transcript to affect N1 exon splicing, we examined the localization of the c-src pre-mRNA by fluorescence in situ hybridization. The src transcript is present in specific foci within the nucleus that are presumably sites of src transcription but are not generally perinucleolar. In normally cultured neuroblastoma cells, these src RNA foci contain PTB, but little KSRP. However, upon induced neuronal differentiation of these cells, KSRP occurs in the same foci with src RNA. PTB localization remains unaffected. This differentiation-induced localization of KSRP with src RNA correlates with an increase in src exon N1 inclusion. These results indicate that PTB and KSRP do indeed interact with the c-src transcript in vivo, and that these associations change with the differentiated state of the cell.

Animals↗
PubMed2003

Protein kinase A phosphorylation modulates transport of the polypyrimidine tract-binding protein.

The heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins play important roles in mRNA processing in eukaryotes, but little is known about how they are regulated by cellular signaling pathways. The polypyrimidine-tract binding protein (PTB, or hnRNP I) is an important regulator of alternative pre-mRNA splicing, of viral RNA translation, and of mRNA localization. Here we show that the nucleo-cytoplasmic transport of PTB is regulated by the 3',5'-cAMP-dependent protein kinase (PKA). PKA directly phosphorylates PTB on conserved Ser-16, and PKA activation in PC12 cells induces Ser-16 phosphorylation. PTB carrying a Ser-16 to alanine mutation accumulates normally in the nucleus. However, export of this mutant protein from the nucleus is greatly reduced in heterokaryon shuttling assays. Conversely, hyperphosphorylation of PTB by coexpression with the catalytic subunit of PKA results in the accumulation of PTB in the cytoplasm. This accumulation is again specifically blocked by the S16A mutation. Similarly, in Xenopus oocytes, the phospho-Ser-16-PTB is restricted to the cytoplasm, whereas the non-Ser-16-phosphorylated PTB is nuclear. Thus, direct PKA phosphorylation of PTB at Ser-16 modulates the nucleo-cytoplasmic distribution of PTB. This phosphorylation likely plays a role in the cytoplasmic function of PTB.

3T3 Cells↗
PubMed2003

Mechanisms of alternative pre-messenger RNA splicing.

Alternative pre-mRNA splicing is a central mode of genetic regulation in higher eukaryotes. Variability in splicing patterns is a major source of protein diversity from the genome. In this review, I describe what is currently known of the molecular mechanisms that control changes in splice site choice. I start with the best-characterized systems from the Drosophila sex determination pathway, and then describe the regulators of other systems about whose mechanisms there is some data. How these regulators are combined into complex systems of tissue-specific splicing is discussed. In conclusion, very recent studies are presented that point to new directions for understanding alternative splicing and its mechanisms.

Alternative Splicing↗
PubMed2003

Roles for SR proteins and hnRNP A1 in the regulation of c-src exon N1.

The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5' half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.

Animals↗