Abstract:Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 10 of the gene is an alternatively spliced cassette that is adult-specific and that codes for a microtubule binding domain. Recently, mutations that affect splicing of exon 10 have been shown to cause inherited frontotemporal dementia (FTDP). In this study, we establish the endogenous expression patterns of exon 10 in human tissue; by reconstituting naturally occurring FTDP mutants in the homologous context of exon 10, we show that the cis determinants of exon 10 splicing regulation include an exonic silencer within the exon, its 5Ј splice site, and the relative affinities of its flanking exons to it. By cotransfections in vivo, we demonstrate that several splicing regulators affect the ratio of tau isoforms by inhibiting exon 10 inclusion. Key Words: Microtubule-associated protein tauExpression pattern of regulated isoforms-Microtubule binding domain-Regulation of alternative splicingFrontotemporal dementia. J. Neurochem. 74, 490 -500 (2000).Alternative splicing is a versatile and widespread mechanism for generating multiple mRNAs from a single transcript (reviewed by Krämer, 1996;Manley and Tacke, 1996;Grabowski, 1998;López, 1998). Splicing choices are regulated by both tissue/cell type and developmental stage; the mRNAs arising from such processing produce functionally diverse protein isoforms.Splicing occurs in two transesterification reactions, with the participation of the spliceosome, a large complex of proteins and small RNAs [small nuclear riboproteins (snRNPs)] (reviewed by Sharp, 1994;Reed, 1996). The snRNPs play an important role in exon definition and splice site hierarchies (Black, 1995;Reed, 1996). In addition to the invariant 5Ј and 3Ј splice sites, another splicing signal is the branch point, normally located 30 -50 nucleotides upstream of the 3Ј splice site. The branch point is the site of formation of the first splicing intermediate (known as the lariat because of its unusual chemical configuration), and it is usually followed by a polypyrimidine (polyY) tract Reed, 1996).A major unanswered question in the splicing field is what distinguishes a cryptic splicing site from an authentic one (reviewed by Black, 1995). Such a distinction is important because mammalian splice sites are loosely defined with respect to sequence and thus redundant in the genome. The exon definition model (in which the snRNPs are postulated to recognize exon boundaries by attachment at the 3Ј splice site of an exon and scanning downstream for a 5Ј splice site within a certain distance) has provided a partial explanation for splice site authentication (Berget, 1995). However, in alternative splicing, splice sites that adhere to the consensus sequences are either used or bypassed when the primary transcript is processed .When alternatively spliced genes are expressed in inappropriate contexts, they are not spliced constitutively, but produce one of the possible mRNAs, called the defa...

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We identified the rat Sam68-like mammalian protein (rSLM-2), a member of the STAR (signal transduction and activation of RNA) protein family as a novel splicing regulatory protein. Using the yeast two-hybrid system, coimmunoprecipitations, and pull-down assays, we demonstrate that rSLM-2 interacts with various proteins involved in the regulation of alternative splicing, among them the serine/arginine-rich protein SRp30c, the splicing-associated factor YT521-B and the scaffold attachment factor B. rSLM-2 can influence the splicing pattern of the CD44v5, human transformer-2␤ and tau minigenes in cotransfection experiments. This effect can be reversed by rSLM-2-interacting proteins. Employing rSLM-2 deletion variants, gel mobility shift assays, and linker scan mutations of the CD44 minigene, we show that the rSLM-2-dependent inclusion of exon v5 of the CD44 pre-mRNA is dependent on a short purinerich sequence. Because the related protein of rSLM-2, Sam68, is believed to play a role as an adapter protein during signal transduction, we postulate that rSLM-2 is a link between signal transduction pathways and pre-mRNA processing.Prior to export to the cytosol, pre-mRNA generated from most eukaryotic genes undergoes maturation processes such as splicing, in which intronic sequences are removed and exonic sequences are rejoined, as well as polyadenylation and 5Ј-end capping. There is increasing evidence that transcription, pre-mRNA processing, and RNA transport are coupled in a highly coordinated manner (1-3). Recent results indicate a direct interaction among RNA polymerase II, transcription, capping, splicing, and polyadenylation factors (3-6). These complexes are possibly attached to chromatin, for example by the scaffold attachment factor B (SAF-B 1 ) (7). This supports the model of a large RNA processing unit (8, 9) termed RNA factory (3). Pre-mRNA splicing is characterized by a high fidelity and can be modulated in a cell type-or development-specific way to use exons alternatively. Although the exact mechanisms governing splice site selection are still not fully understood, recent results indicate that loosely defined signals on the pre-mRNA known as splicing enhancers/silencers, play a crucial role in splice site selection (10 -12). An important class of proteins that recognize splicing enhancers/silencers is the serine/arginine-rich (SR) and SR-related protein family that is involved in both constitutive and alternative splicing (13,14). In addition, it has also been shown that an increasing number of heterogenous nuclear ribonucleoproteins (hnRNPs) are involved in the regulation of alternative splicing. For example, splicing regulation of the neuron-specific exon N1 or the src pre-mRNA is under the control of the hnRNPs hnRNP I (polypyrimidine tract binding protein), hnRNP F, and hnRNP H (15, 16). SR proteins and hnRNPs can change alternative splicing patterns in a concentration-dependent manner both in vivo and in vitro (for review, see Refs. 13 and 14). Because the relative expression levels of SR proteins and...

Tau is a microtubule-associated protein (MAP) whose transcript undergoes complex regulated splicing in the mammalian nervous system. Our previous work with exon 6 established that tau shows a unique expression pattern and splicing regulation profile, and that it utilizes alternative splice sites in several human tissues. The mRNAs from these splicing events, if translated, would result in truncated tau variants that lack the microtubule-binding domain. In this study, we demonstrate that at least one of these tau variants is present as a stable protein in several tissues. The novel isoform shows a localization distinct from that of canonical tau in SH-SY5Y neuroblastoma cells which stably overexpress it. In both normal and Alzheimer's hippocampus, the novel isoform is found in dentate gyrus granular cells and CA1/CA3 pyramidal cells. However, it does not co-localize with canonical tau but, rather, partly co-localizes with MAP2.

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