Heterogeneous Nuclear Ribonucleoprotein (hnRNP) K Is a Component of an Intronic Splicing Enhancer Complex That Activates the Splicing of the Alternative Exon 6A from Chicken β-Tropomyosin Pre-mRNA

Abstract

Splicing of the chicken ␤-tropomyosin exon 6A is stimulated, both in vivo and in vitro, by an intronic pyrimidine-rich element (S4) located 37 nucleotides downstream of exon 6A. Several pyrimidine-rich sequences are able to substitute for the natural S4 enhancer with various stimulatory effects. We show that the different enhancer sequences recruit U1 small nuclear ribonucleoprotein (SnRNP) to the exon 6A 5 splice site, with an efficiency that correlates with the splicing activation. By using RNA affinity and two-dimensional gel electrophoresis, we characterized several proteins that bind to the different enhancer sequences. Heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP I (polypyrimidine track-binding protein, PTB) exhibit a higher level of interaction with the strong enhancer sequences (S4) than with the weakest enhancers. Functional analysis shows that hnRNP K is a component of the enhancer complex that promotes exon 6A splicing through the wild-type S4 sequence. The addition of recombinant hnRNP K to nuclear extracts preincubated with poly(rC) RNA competitor completely restores splicing efficiency to the original level. hnRNP I (PTB) was also found associated with the strong enhancer sequences. Its function in the splicing of exon 6A is discussed.Alternative splicing of pre-mRNAs generates different mRNAs from the same primary transcript and as such contributes to protein diversity (1-3). This process is thought to be important for regulation of the gene expression and has been enlightened by the discovery that human genome contains only 30,000 -40,000 genes. From these studies, it has been suggested that more than 50% of the genes are alternatively spliced (4). In many cases, alternative splicing is regulated in a tissue developmental stage or sex-specific manner and responds differently to metabolic stimuli (2, 3). Studies of different splicing events show that the bona fide regulation is orchestrated by multicomponent complexes that promote or repress the use of alternative splice sites through binding to regulatory sequences (1,5,6). The SR protein family is a well characterized class of proteins that are involved in both constitutive and alternative splicing (7,8). It has been shown that the binding of members of the SR proteins to purine enhancer sequences promotes the inclusion of the corresponding exon by facilitating the recognition of weak 3Ј splice sites (9). Another group of proteins that affect splice site selection are the hnRNPs.1 They constitute a large group of RNA-binding proteins that associate with nascent transcripts, and they can hinder or assist the splicing machinery (10, 11). Early experiments have shown that hnRNP A1 is able to antagonize the activity of ASF/SF2 by promoting a shift toward the selection of distal 5Ј splice sites (12, 13). More recently, it has been found that hnRNP A1 interacts with silencer elements to repress splicing of several pre-mRNAs (14 -18). In the case of human immunodeficiency virus type 1 tat splicing, it has been proposed that the binding o...

Heterogeneous nuclear ribonucleoprotein K is over expressed, aberrantly localised and is associated with poor prognosis in colorectal cancer

“…Heterogeneous ribonucleoprotein K involvement in splicing was demonstrated using the chicken b tropomyosin gene, whose alternative splicing of exon 6A is mediated by a downstream intronic enhancer. Heterogeneous ribonucleoprotein K was identified as a protein present in the intronic enhancer complex whose function is to activate splicing of exon 6A (Expert-Bezancon et al, 2002). Heterogeneous ribonucleoprotein K also mediates translation, in HeLa cells hnRNP K silences translation of a luciferase reporter mRNA containing the differentiation control element present in the 3 0 UTR of the 15 lipoxygenase gene (Ostareck-Lederer et al, 1994).…”

Direct Recruitment of ERK Cascade Components to Inducible Genes Is Regulated by Heterogeneous Nuclear Ribonucleoprotein (hnRNP) K

“…The K protein has been implicated to regulate many processes including transcription (17,18), mRNA splicing (19), and stability (20). K protein in addition to RNA-binding domains contains protein-interacting modules that, on one hand, bind kinases (21)(22)(23)(24) and, on the other hand, associate with transcription (18,25), RNA processing (26), and translation (27,28) factors.…”

Poly(C)-Binding Protein 1, a Novel N pro -Interacting Protein Involved in Classical Swine Fever Virus Growth

“…Mammalian cells express five evolutionarily related KH domain-containing proteins: hnRNP K and PCBP1 to PCBP4. Members of the KH domain superfamily have been shown to be involved in processing of pre-mRNA (11), shuttling of mRNA between the nucleus and the cytoplasm (12), mRNA stabilization (13)(14)(15), translational activation (16,17) and silencing (18,19), and transcriptional regulation (20)(21)(22)(23)(24). PCBP1 is a single-stranded DNA-binding protein that participates in neuronal -opioid receptor gene expression at the transcriptional level (21) and has been shown to be expressed throughout the cytoplasm and nucleus in neuronal cells, with preferential nuclear expression (12).…”