ABSTRACT
Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, we characterized the binding of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. We also identified a new 60-kDa tissue-specific protein that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins.
Subject(s)
Nerve Tissue Proteins/metabolism , RNA-Binding Proteins/metabolism , Ribonucleoproteins/metabolism , Trans-Activators , Amino Acid Sequence , Base Sequence , Cell Line , Chromatography, Affinity , Cloning, Molecular , Heterogeneous-Nuclear Ribonucleoprotein Group F-H , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Models, Genetic , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/isolation & purification , Nuclear Proteins/metabolism , Organ Specificity , Phylogeny , Polypyrimidine Tract-Binding Protein , Protein Binding , RNA Splicing/genetics , RNA, Heterogeneous Nuclear/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/isolation & purification , Regulatory Sequences, Nucleic Acid , Ribonucleoproteins/chemistry , Ribonucleoproteins/genetics , Ribonucleoproteins/isolation & purification , Sequence Homology, Amino AcidABSTRACT
The K homology-type splicing regulatory protein, KSRP, activates splicing through intronic splicing enhancer sequences. It is highly expressed in neural cells and is required for the neural-specific splicing of the c-src N1 exon. In this study, we mapped the gene (gene symbols KHSRP and Khsrp) to human chromosome 19 by using radiation hybrid panels and to mouse chromosome 17 by studying an interspecific backcross panel. Human KHSRP is a positional candidate gene for familial febrile convulsion and Cayman type cerebellar ataxia. Comparative analysis of the human and mouse genomes indicates that the KHSRP gene is located in regions of conserved synteny between the two species.
Subject(s)
Chromosomes, Human, Pair 19/genetics , RNA-Binding Proteins/genetics , Trans-Activators , Alternative Splicing , Animals , Chromosome Mapping , Conserved Sequence , Databases, Factual , Genetic Markers , Humans , Mice , Models, Genetic , Polymerase Chain Reaction , Polymorphism, Single-Stranded ConformationalSubject(s)
Chromosome Mapping , DNA-Binding Proteins/genetics , Animals , CHO Cells , Cricetinae , Databases, Factual , Mice , Molecular Sequence Data , Nucleic Acid Hybridization , RatsABSTRACT
We have purified and cloned a new splicing factor, KSRP. KSRP is a component of a multiprotein complex that binds specifically to an intronic splicing enhancer element downstream of the neuron-specific c-src N1 exon. This 75-kD protein induces the assembly of five other proteins, including the heterogeneous nuclear ribonucleoprotein F, onto the splicing enhancer. The sequence of the KSRP cDNA indicates that the protein contains four K homology RNA-binding domains and an unusual carboxy-terminal domain. KSRP is similar to two proteins, FUSE-binding protein and P-element somatic inhibitor. KSRP is expressed in both neural and non-neural cell lines, although it is present at higher levels in neural cells. Antibodies specific for KSRP inhibit the splicing of the N1 exon in vitro. Moreover, this inhibition of N1 splicing can be rescued by the addition of purified KSRP. KSRP is likely to regulate splicing from a number of intronic splicing enhancer sequences.
