The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5 splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chromatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide sequence analysis of purified p58 protein identified it as hnRNP H. Immunoprecipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel mobility shift analysis of the enhancer complex in the presence of hnRNP H-specific antibodies, confirmed that hnRNP H is a protein component of the splicing enhancer complex. Immunoprecipitation of splicing intermediates from in vitro splicing reactions with anti-hnRNP H antibody indicated that hnRNP H remains bound to the src pre-mRNA after the assembly of spliceosome. Partial immunodepletion of hnRNP H from the nuclear extract partially inactivated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP H, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H can interact with the related protein hnRNP F, suggesting that hnRNPs H and F may exist as a heterodimer in a single enhancer complex. These two proteins presumably cooperate with each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream.Alternative RNA splicing is a process that allows the production of multiple mRNAs from a single gene through the selection of different combinations of splice sites within a precursor mRNA (pre-mRNA). This process is an important mechanism in the developmental and cell-type-specific control of gene expression. Although the control of alternative splicing is poorly understood, specific regulatory proteins have been identified in some systems. These splicing regulatory proteins are thought to bind to sequence elements in a pre-mRNA and positively or negatively affect spliceosome assembly at nearby splice sites (1,9,66,67).cis-acting RNA sequences can serve to either enhance or repress the use of certain splice sites. Splicing enhancers are classified by their location in either exons or introns. Exonic splicing enhancers interact with trans-acting factors including an important class of splicing regulators called serine-arginine (SR) proteins (28, 66). SR proteins each possess one or two RNA recognition motif-type RNA binding domains and a Cterminal domain containing numerous SR dipeptides (20, 39). The exonic splicing enhancer in the Drosophila doublesex premRNA is bound by two specific regulatory proteins, Transformer (Tra) and Transformer-2 (Tra-2) (38). Tra and Tra-2 bind to multiple elements of the enhancer and recruit ...

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 PTBmediated 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.A common mechanism of the regulation of gene expression in metazoans is the alternative splicing of pre-mRNA (6,11,22,23,34). Through alternative splicing, multiple mRNAs are generated from the same primary RNA transcript. Changes in splice site choice are regulated by proteins that bind to the pre-mRNA and affect spliceosome assembly. One well-studied family of splicing regulatory factors is the SR proteins that function in both constitutive and alternative splicing and can act at various stages of spliceosome assembly (3,10,24,48). These proteins are perhaps best known for binding exonic splicing enhancer (ESE) elements and thus stimulating spliceosome assembly at the adjacent splice sites. Some exons bind multiple SR proteins, each of which can activate splicing. Other exons are dependent on a single SR protein. Two of the best-characterized SR protein family members that bind ESEs and activate splicing are SF2/ASF and SC35.Another large group of proteins that bind to nascent premRNAs are the heterogeneous nuclear ribonucleoproteins (hnRNPs) (19,30). At least in vitro, spliceosome assembly occurs after hnRNP binding and some hnRNPs are implicated in splicing regulation. Two of these, hnRNP A1 and polypyrimidine tract binding protein (PTB or hnRNP I) bind to exoni...

Summary Distinct CD4+ T‐cell epitopes within the same protein can be optimally processed and loaded into major histocompatibility complex (MHC) class II molecules in disparate endosomal compartments. The CD1 protein isoforms traffic to these same endosomal compartments as directed by unique cytoplasmic tail sequences, therefore we reasoned that antigen/CD1 chimeras containing the different CD1 cytoplasmic tail sequences could optimally target antigens to the MHC class II antigen presentation pathway. Evaluation of trafficking patterns revealed that all four human CD1‐derived targeting sequences delivered antigen to the MHC class II antigen presentation pathway, to early/recycling, early/sorting and late endosomes/lysosomes. There was a preferential requirement for different CD1 targeting sequences for the optimal presentation of an MHC class II epitope in the following hierarchy: CD1b > CD1d = CD1c > > > CD1a or untargeted antigen. Therefore, the substitution of the CD1 ectodomain with heterologous proteins results in their traffic to distinct intracellular locations that intersect with MHC class II and this differential distribution leads to specific functional outcomes with respect to MHC class II antigen presentation. These findings may have implications in designing DNA vaccines, providing a greater variety of tools to generate T‐cell responses against microbial pathogens or tumours.

Almost every eukaryotic pre‐mRNA generated by RNA polymerase II transcription requires the removal of introns to create mRNA. The correct splicing of constitutive exons is thus critical for normal protein expression and function. Moreover, the removal of many introns by the spliceosome is controlled in a tissue‐specific or developmentally specific manner. In order to study RNA splicing at a biochemical level, it is necessary to employ an in vitro, or cell‐free, system. Cell‐free splicing systems require two main components: (1) an extract made from mammalian cell nuclei and (2) the introns and exons of the eukaryotic gene of interest. This minigene construct allows the synthesis of sufficient quantities of pre‐mRNA substrates in vitro, which are then incubated in the nuclear extract and analyzed for splicing. Nuclear extracts, first developed for studying transcription in vitro, are modified for splicing. This unit describes how to set up an in vitro splicing reaction using a mammalian nuclear extract derived from either cell line or tissue, and how to analyze the splicing reaction products.