Competing RNA secondary structures are required for mutually exclusive splicing of the Dscam exon 6 cluster

Alternative splicing of eukaryotic pre-mRNAs is an important mechanism for generating proteome diversity and regulating gene expression. The Drosophila melanogaster Down Syndrome Cell Adhesion Molecule (Dscam) gene is an extreme example of mutually exclusive splicing. Dscam contains 95 alternatively spliced exons that potentially encode 38,016 distinct mRNA and protein isoforms. We previously identified two sets of conserved sequence elements, the docking site and selector sequences in the Dscam exon 6 cluster, which contains 48 mutually exclusive exons. These elements were proposed to engage in competing RNA secondary structures required for mutually exclusive splicing, though this model has not yet been experimentally tested. Here we describe a new system that allowed us to demonstrate that the docking site and selector sequences are indeed required for exon 6 mutually exclusive splicing and that the strength of these RNA structures determines the frequency of exon 6 inclusion. We also show that the function of the docking site has been conserved for ~500 million years of evolution. This work demonstrates that conserved intronic sequences play a functional role in mutually exclusive splicing of the Dscam exon 6 cluster.     

Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2β in development

Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl); Nestin-Cre(tg/+)). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.     

Alternative splicing of ADAM15 regulates its interactions with cellular SH3 proteins

A Disintegrin And Metalloprotease (ADAM15) is a member of the adamalysin protein family and has been associated with cancer, possibly via its role in ectodomain shedding of cadherins. Alternative mRNA splicing generates several ADAM15 isoforms containing different combinations of putative Src homology‐3 (SH3) domain binding sites in their cytosolic tails. Here we present a comprehensive characterization of SH3 binding potential of different ADAM15 isoforms. Alternative use of ADAM15 exons was found to profoundly influence selection of SH3‐containing cellular partner proteins, including the avid interactions with nephrocystin and sorting nexin‐33 (SNX33 a.k.a. SNX30). Specifically, strong co‐precipitation of nephrocystin from cell lysates was specific to ADAM15 isoforms i4, i5, and i6. These isoforms contain one or both of the two almost identical proline‐rich regions encoded by exons 20 and 21, wherein the residues RxLPxxP were found to be indispensable for nephrocystin SH3 binding. Similarly, robust cellular association with SNX33 was observed only for ADAM15 isoforms containing the most carboxyterminal proline cluster lacking in isoforms i1 and i3. Thus, alternative mRNA splicing provides a versatile mechanism for regulation of intracellular protein interactions and thereby likely the cellular functions of ADAM15, which could explain the association with cancer of some but not all ADAM15 isoforms. J. Cell. Biochem. 108: 877–885, 2009. © 2009 Wiley‐Liss, Inc.     

Mechanisms of Drosophila Dscam mutually exclusive splicing regulation

Alternative splicing of pre-mRNA is a major mechanism to increase protein diversity in higher eukaryotes. Dscam, the Drosophila homologue of human DSCAM (Down's syndrome cell adhesion molecule), generates up to 38016 isoforms through mutually exclusive splicing in four variable exon clusters. This enormous molecular diversity is functionally important for wiring of the nervous system and phagocytosis of invading pathogens. Current models explaining this complex splicing regulation include a default repressed state of the variable exon clusters to prevent the splicing together of adjacent exons, the presence of RNA secondary structures important for the release of one specific variable exon from the repressed state and combinatorial interaction of RNA-binding proteins for choosing a specific exon.     

Multiple isoforms of beta-TrCP display differential activities in the regulation of Wnt signaling

The F-box proteins beta-TrCP1 and 2 (beta-transducin repeat containing protein) have 2 and 3 isoforms, respectively, due to alternative splicing of exons encoding the N-terminal region. We identified an extra exon in between the previously known exons 1 and 2 of beta-TrCP1 and beta-TrCP2. Interestingly, sequence analysis suggested that many more isoforms are produced than previously identified, via the alternative splicing of all possible combination of exons II to V of beta-TrCP1 and exons II to IV of beta-TrCP2. Different mouse tissues show specific expression patterns of the isoforms, and the level of expression of the isoform that has been used in most published papers was very low. Yeast two-hybrid assays show that beta-TrCP1 isoforms containing exon III, which are the most highly expressed isoforms in most tissues, do not interact with Skp1. Indirect immunofluorescence analysis of transiently expressed beta-TrCP1 isoforms suggests that the presence of exon III causes beta-TrCP1 to localize in nuclei. Consistent with the above findings, isoforms including exon III showed a reduced ability to block ectopic embryonic axes induced via injection of Wnt8 or beta-catenin in Xenopus embryos. Overall, our data suggest that isoforms of beta-TrCPs generated by alternative splicing may have different biological roles.     

Identification of a Novel Zn2+-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin

Missense mutations in park2, encoding the parkin protein, account for ∼50% of autosomal recessive juvenile Parkinson disease (ARJP) cases. Parkin belongs to the family of RBR (RING-between-RING) E3 ligases involved in the ubiquitin-mediated degradation and trafficking of proteins such as Pael-R and synphillin-1. The proposed architecture of parkin, based largely on sequence similarity studies, consists of N-terminal ubiquitin-like and C-terminal RBR domains. These domains are separated by a ∼160-residue unique parkin sequence having no recognizable domain structure. We used limited proteolysis experiments on bacterially expressed and purified parkin to identify a new domain (RING0) within the unique parkin domain sequence. RING0 comprises two distinct, conserved cysteine-rich clusters between Cys¹⁵⁰–Cys¹⁶⁹ and Cys¹⁹⁶–His²¹⁵ consisting of CX₂-₃CX₁₁CX₂C and CX_4–6CX_10–16-CX₂(H/C) motifs. The positions of the cysteine/histidine residues in this region bear similarity to parkin RING1 and RING2 domains, as well as other E3 ligase RING domains. However, in parkin a 26-residue linker region separates the motifs, which is not typical of other RING domain structures. Further, the RING0 domain includes all but one of the known ARJP mutation sites between the ubiquitin-like and RBR regions of parkin. Using electrospray ionization mass spectrometry and inductively coupled plasma-atomic emission spectrometry analysis, we determined that the RING0, RING1, IBR, and RING2 domains each bind two Zn²⁺ ions, the first observation of an E3 ligase with the ability to bind eight metal ions. Removal of the zinc from parkin causes near complete unfolding of the protein, an observation that rationalizes cysteine-based ARJP mutations found throughout parkin, including RING0 (C212Y) that form cellular inclusions and/or are defective for ubiquitination likely because of poor zinc binding and misfolding. The identification of the RING0 domain in parkin provides a new overall domain structure for the protein that will be important in assessing the roles of ARJP mutations and designing experiments aimed at understanding the disease.Autosomal recessive juvenile Parkinson disease (ARJP)² is a neurodegenerative disorder arising from the loss of dopaminergic neurons in the substantia nigra of the midbrain. ARJP is characterized by the onset of Parkinsonian symptoms such as tremors, rigidity, and bradykinesia. It is distinguished from the idiopathic form of Parkinson disease by the onset of symptoms, prior to the age of forty. The hereditary nature of ARJP implicates a number of mutations in the genes encoding the proteins parkin, PINK1, LRRK2, and DJ-1 as the cause of dopaminergic neurodegeneration (1–4). A variety of deletion, truncation, and point mutations distributed throughout the park2 gene, which encodes the protein parkin, have been reported in ARJP patients (1, 5–18).Parkin functions as a ubiquitin ligase (E3) and belongs to a family of RBR (RING-between-RING) ubiquitin ligase enzymes involved in proteosome-mediated protein degradation (19–21). The currently accepted domain architecture of parkin, deduced from multiple sequence alignment, shows that the C terminus of the protein is characterized by two ∼50-residue RING (really interesting new gene) domains separated by a 51-residue IBR (In-Between-RING) domain (22, 23). The RING domains of parkin are proposed to interact with the ubiquitin-conjugating enzymes UbcH7, UbcH8, Ubc7, and Ubc13 and control parkin-mediated ubiquitination of a variety of substrates such as Pael-R, synphilin-1, Sept5, and PICK1 among others (24–31). Other members of the RBR family include the human homolog of Drosophila Ariadne (HHARI), DORFIN, and HOIL-1, which share close domain architecture (32–35). Traditionally, RING domains coordinate two Zn²⁺ ions through a C₃HC₄ metal-binding consensus sequence. However, the RING2 domain of HHARI binds a single Zn²⁺ (36), and because this is the only RING2 structure available for an RBR protein, it suggests that there may be variability in the number of Zn²⁺ ions coordinated by different RING domains. The recent three-dimensional structure of the parkin IBR domain (23) revealed a two-site zinc-binding motif with a novel fold compared with other zinc-binding motifs (37). However, despite the potential importance of zinc binding to the RING domains (or other portions) of parkin, the ability and capacity for zinc coordination or its impact on structure has not been identified for parkin.The N terminus of parkin comprises a ubiquitin-like domain (UblD) proposed to facilitate the delivery and degradation of ubiquitinated substrates by the 26 S proteosome via interactions with the S5a subunit (38, 39). The central ∼150 residues of parkin separating the UblD from the RBR region are referred to as the unique parkin domain (UPD). This segment of parkin is essential for function, and ARJP associated mutations within this region have been shown to lead to dysfunction of parkin E3 ligase activity (40, 41). However, the absence of any sequence similarity to other proteins or the identification of a distinct domain within the UPD has made these experiments difficult to interpret. Other than the isolated UblD and IBR domains of parkin, there has been limited success with the purification and characterization of parkin, especially when lacking affinity tags in the final purified form. Bacterially expressed parkin typically shows a heterogeneous mixture of full-length and degraded protein species, making characterization of the protein difficult (42). In this work we have used purified parkin to identify a novel zinc-binding C₄C₃(C/H) domain upstream of the RBR region and within the UPD. We have used limited proteolysis and electrospray ionization mass spectrometry (ESI-MS) to show that this domain coordinates two Zn²⁺ ions in addition to six other Zn²⁺ ions in the RBR C terminus. The presence of a new parkin-specific zinc-binding domain provides insight into the structure of parkin and opens the door to establish the importance of this domain in ARJP for this new subclass of RBR E3 ligases.     

Impact of autosomal recessive juvenile Parkinson's disease mutations on the structure and interactions of the parkin ubiquitin-like domain

Autosomal recessive juvenile parkinsonism (ARJP) is an early onset familial form of Parkinson's disease. Approximately 50% of all ARJP cases are attributed to mutations in the gene park2, coding for the protein parkin. Parkin is a multidomain E3 ubiquitin ligase with six distinct domains including an N-terminal ubiquitin-like (Ubl) domain. In this work we examined the structure, stability, and interactions of the parkin Ubl domain containing most ARJP causative mutations. Using NMR spectroscopy we show that the Ubl domain proteins containing the ARJP substitutions G12R, D18N, K32T, R33Q, P37L, and K48A retained a similar three-dimensional fold as the Ubl domain, while at least one other (V15M) had altered packing. Four substitutions (A31D, R42P, A46P, and V56E) result in poor folding of the domain, while one protein (T55I) showed evidence of heterogeneity and aggregation. Further, of the substitutions that maintained their three-dimensional fold, we found that four of these (V15M, K32T, R33Q, and P37L) lead to impaired function due to decreased ability to interact with the 19S regulatory subunit S5a. Three substitutions (G12R, D18N, and Q34R) with an uncertain role in the disease did not alter the three-dimensional fold or S5a interaction. This work provides the first extensive characterization of the structural effects of causative mutations within the ubiquitin-like domain in ARJP.     

Developmentally induced, muscle-specific trans factors control the differential splicing of alternative and constitutive troponin T exons

Alternative RNA splicing is a ubiquitous process permitting single genes to encode multiple protein isoforms. Here we report experiments in which a gene construct, containing combinatorial Troponin T (TnT) exons that manifest an exceptional diversity of alternative splicing in vivo, has been transfected into muscle and nonmuscle cells. Analyses of the spliced RNAs show that the alternative TnT exons retain their capacity for differential splicing in the modified minigene context when introduced into a variety of nonmuscle and muscle cells. The patterns of alternative splicing differ depending on cell type. Only in differentiated myotubes are the alternative exons normally incorporated during splicing, reproducing their behavior in the native gene; they are excluded in nonmuscle cells and myoblasts that do not express the endogenous TnT. These results provide proof that trans factors required for correct alternative splicing are induced during myogenesis. Surprisingly, such factors are also required for the correct splicing of constitutive TnT exons.     

Parkin Expression Profile in Dopamine D3 Receptor Knock-Out Mice Brains

Patients affected by autosomic recessive juvenile parkinsonism (ARJP) exhibit parkin gene mutations with brain decrease in dopamine D2/D3 binding sites. To date, there are no data indicating whether the reduction in dopamine D3 receptors (DRD3) may be associated with the expression of specific parkin variants. In the present study we investigated parkin expression profile in DRD3 knock-out mice brains. RT-PCR analysis was performed to assess qualitative changes in parkin isoforms’ distribution pattern and in exons’ expression both in wild type controls and dopamine D3 receptor’s knock-out mice. Real-time PCR was performed to quantify single exons mRNA. Results demonstrated that exons 1, 2, 4, 6, 7, 8, were more expressed in wild type compared to dopamine D3 receptor KO mice brains while some other (3, 9, 10) were lower expressed. The expression levels of exons 5, 11 and 12 did not change in both animal groups. Our analysis was confirmed by western blot, which showed that parkin protein levels were influenced by the absence of DRD3.     

Comparative genomic analysis of the arthropod muscle myosin heavy chain genes allows ancestral gene reconstruction and reveals a new type of 'partially' processed pseudogene

Background  

Alternative splicing of mutually exclusive exons is an important mechanism for increasing protein diversity in eukaryotes. The insect Mhc (myosin heavy chain) gene produces all different muscle myosins as a result of alternative splicing in contrast to most other organisms of the Metazoa lineage, that have a family of muscle genes with each gene coding for a protein specialized for a functional niche.     

Structure and evolution of the alternatively spliced fast troponin T isoform gene.

The vertebrate fast skeletal muscle troponin T gene, TnTf, produces a complexity of isoforms through differential mRNA splicing. The mechanisms that regulate splicing and the physiological significance of TnTf isoforms are poorly understood. To investigate these questions, we have determined the complete sequence structure of the quail TnTf gene, and we have characterized the developmental expression of alternatively spliced TnTf mRNAs in quail embryonic muscles. We report the following: 1) the quail TnTf gene is significantly larger than the rat TnTf gene and has 8 non-homologous exons, including a pectoral muscle-specific set of alternatively spliced exons; 2) specific sequences are implicated in regulated exon splicing; 3) a 900-base pair sequence element, composed primarily of intron sequence flanking the pectoral muscle-specific exons, is tandemly repeated 4 times and once partially, providing direct evidence that the pectoral-specific TnT exon domain arose by intragenic duplications; 4) a chicken repeat 1 retrotransposon element resides upstream of this repeated intronic/pectoral exon sequence domain and is implicated in transposition of this element into an ancestral genome; and 5) a large set of novel isoforms, produced by regulated exon splicing, is expressed in quail muscles, providing insights into the developmental regulation, physiological function, and evolution of the vertebrate TnTf isoforms.