NORMAL GENOMIC

• Yu W et al.
• Characterization of three splice variants and genomic organization of themouse BMAL1 gene.
• Biochem Biophys Res Commun. 1999; 260: 760-7
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• The BMAL1 gene encodes a member of the basic helix-loop-helix/PER-ARNT-SIM(bHLH/PAS) family of transcription factors. It is a key regulator ofcircadian rhythms. Using sequence information from human BMAL1 (hBMAL1)cDNAs previously reported by our laboratory, we have isolated andcharacterized cDNAs encoding three splice variants of the mouse BMAL1(mBMAL1) gene. Of the three splice variants, mBMAL1b extends for 1878 bpin the coding sequence, which is 91% identical to that of hBMAL1b; itsdeduced amino acid sequence is 626 residues long and is 98% identical tothat of hBMAL1b, and sequence identities in the bHLH, PAS-A, and PAS-Bregions are 98, 100, and 100%, respectively. mBMAL1b' arises fromalternative usage of exon 2, which results in a 7-amino-acid insertion andalternative splice acceptor usage at the intron 9/exon 10 splice junction,which causes an alanine residue deletion. mBMAL1b' encodes 632 amino acidsand contains the bHLH/PAS domains. mBMAL1g' is generated by alternativesplice acceptor usage at the intron 6/exon 7 splice junction, whichresults in a 28-bp deletion adjacent to the 5' end of the PAS domain.Since the 28-bp deletion shifts the reading frame, mBMAL1g' is predictedto encode a product of only 222 amino acids that lacks the PAS domain. Thetissue distributions of the three splice variants showed some variation.The variations in the tissue distributions and predicted amino acidsequences suggest that the three splice variants may have differentfunctions. Direct sequencing of the genomic mBMAL1 clones indicated thatthe coding sequence of mBMAL1 spans 32 kb and includes 17 exons. Anunusual exon/intron donor sequence was found in intron 14, which beginswith GC at the 5' end. Comparison with the bHLH/PAS family genes revealedthat the intron/exon splice pattern of mBMAL1 most closely matches that ofthe mAhr, which suggests that BMAL1 and Ahr belong to the same subclassand may be derived from a common primordial gene.

• Lynch KW, Maniatis T
• Synergistic interactions between two distinct elements of a regulatedsplicing enhancer.
• Genes Dev. 1995; 9: 284-93
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• Regulated alternative splicing of doublesex (dsx) pre-mRNA requires asplicing enhancer designated the dsx repeat element (dsxRE) that containssix copies of a 13-nucleotide repeat sequence. Previous studies have shownthat the activity of the dsxRE requires the splicing regulatorsTransformer (Tra) and Transformer 2 (Tra2), and one or more members of theSR family of general splicing factors. In this paper we identify apurine-rich enhancer (PRE) sequence within the dsxRE, and show that thiselement functionally synergizes with the repeat sequences. In vitrobinding studies show that the PRE is required for specific binding of Tra2to the dsxRE, and that Tra and SR proteins bind cooperatively to the dsxREin the presence or absence of the PRE. Thus positive control of dsxpre-mRNA splicing requires the Tra- and Tra2-dependent assembly of amultiprotein complex on at least two distinct enhancer elements.

• Takahara K, Lyons GE, Greenspan DS
• Bone morphogenetic protein-1 and a mammalian tolloid homologue (mTld) areencoded by alternatively spliced transcripts which are differentiallyexpressed in some tissues.
• J Biol Chem. 1994; 269: 32572-8
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• Bone morphogenetic protein-1 (BMP-1) is a metalloprotease purified fromextracts capable of inducing ectopic bone formation. In humans, it has adomain structure similar to that of the Drosophila dorsal-ventralpatterning gene-product tolloid (Tld), but is considerably shorter. Herewe show that, in humans and mice, alternatively spliced transcripts encodeBMP-1 and a longer protein, designated mammalian tolloid (mTld), with adomain structure identical to that of Drosophila Tld. A thirdalternatively spliced product, in which a novel domain is inserted nearthe BMP-1 C terminus, is also reported. Low levels of transcripts for mTldwere found in all adult human tissues surveyed, while BMP-1 transcriptswere detectable in all adult tissues except brain. This differentialexpression was mirrored in embryonic mouse tissues where in situhybridization found high levels of mTld transcripts, but was unable todetect BMP-1 transcripts, in the floor plate of the neural tube of thedeveloping central nervous system. The third alternatively spliced formwas not detected in adult human tissues. In situ hybridizations foundpunctate signals for all three forms localized to trophoblast giant cellsin 17.5-day mouse placenta, with highest levels of expression, especiallyfor BMP-1, near the maternal interface.

• Thackeray JR, Ganetzky B
• Developmentally regulated alternative splicing generates a complex arrayof Drosophila para sodium channel isoforms.
• J Neurosci. 1994; 14: 2569-78
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• The para locus encodes the predominant class of sodium channels expressedin Drosophila neurons. Previous sequence analysis of para cDNAs indicatedthe occurrence of alternative splicing at several sites within the openreading frame. Here we report a detailed analysis of this alternativesplicing and its regulation during development. We have used a combinationof RNA-PCR and sequence analysis to examine a 1.7 kilobase region of thepara mRNA that encompasses the previously reported sites of alternativesplicing. Five sites of alternative splicing were identified; 48 differentsplice variants could be generated by the differential exon usageobserved. The number of splice forms and their relative frequency in vivowere characterized in RNA samples of both embryos and adults. The range ofsplice types was found to be much more diverse in adults than in embryos;of a total of 19 different combinations of alternative exons, 11 splicetypes were found in embryos and 18 in adults. Usage of some individualalternative exons changed during development; a newly identified exon,which is found in one of two forms either 24 or 30 base pairs long, waspresent in about 85% of para transcripts from embryos but only 7% of thosein adults. These data suggest that a wide variety of subtly distinct Nachannel isoforms are present in Drosophila, and that these may provide arange of voltage-gated sodium channel functions. Although multiple sodiumchannel genes have already been described in both Drosophila and mammaliansystems, this study provides a clear indication that sodium channelvariability may be much greater than previously thought.

• Vellard M, Sureau A, Soret J, Martinerie C, Perbal B
• A potential splicing factor is encoded by the opposite strand of thetrans-spliced c-myb exon.
• Proc Natl Acad Sci U S A. 1992; 89: 2511-5
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• We previously established that the expression of a thymic c-myb mRNAspecies requires the intermolecular recombination of coding sequencesexpressed from transcriptional units localized on different chromosomes,in both chicken and human. We now report that a putative splicing factor(PR264), extremely well conserved in chicken and human, is encoded by theopposite strand of the c-myb trans-spliced exon. The PR264 polypeptide,which contains a typical ribonucleoprotein 80 and an arginine/serine-richdomain, is highly homologous to the Drosophila splicing regulators tra,tra-2, and su(wa) and to the human alternative splicing factor ASF/SF2.Furthermore, we show that PR264-specific mRNAs are expressed in normalhematopoietic cells of chicken and human origin and that the relativeproportion of the PR264 transcripts is developmentally regulated inchicken.

• Tartof KD, Henikoff S
• Trans-sensing effects from Drosophila to humans.
• Cell. 1991; 65: 201-3

• Krainer AR, Mayeda A, Kozak D, Binns G
• Functional expression of cloned human splicing factor SF2: homology toRNA-binding proteins, U1 70K, and Drosophila splicing regulators.
• Cell. 1991; 66: 383-94
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• SF2 is a protein factor essential for constitutive pre-mRNA splicing inHeLa cell extracts and also activates proximal alternative 5' splice sitesin a concentration-dependent manner. This latter property suggests a rolefor SF2 in preventing exon skipping, ensuring the accuracy of splicing,and regulating alternative splicing. Human SF2 cDNAs have been isolatedand overexpressed in bacteria. Recombinant SF2 is active in splicing andstimulates proximal 5' splice sites. SF2 has a C-terminal region rich inarginine-serine dipeptides, similar to the RS domains of the U1 snRNP 70Kpolypeptide and the Drosophila alternative splicing regulatorstransformer, transformer-2, and suppressor-of-white-apricot. Liketransformer-2 and 70K, SF2 contains an RNP-type RNA recognition motif.

• Ge H, Zuo P, Manley JL
• Primary structure of the human splicing factor ASF reveals similaritieswith Drosophila regulators.
• Cell. 1991; 66: 373-82
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• We described previously the purification of a human protein, calledalternative splicing factor (ASF), that can switch utilization ofalternative 5' splice sites in an SV40 early pre-mRNA. We now report theisolation of a cDNA, designated ASF-1, that encodes this protein. ASF-1consists of 248 amino acid residues, including an 80 residue RNA-bindingdomain at its N-terminus and a 50 residue C-terminal region that is 80%serine plus arginine. ASF-1 produced in E. coli can activate splicing invitro and switch 5' splice-site utilization, establishing that therecombinant protein is sufficient to supply these activities. Analysis ofadditional cDNAs revealed that ASF pre-mRNA can itself be alternativelyspliced, surprisingly, by utilization of a shared 5' splice site and twoclosely spaced 3' splice sites. Use of the upstream site results in asecond mRNA (ASF-2) in which translation of the downstream exon occursextensively in an alternative reading frame distinct from ASF-1.

• Waring GL, Hawley RJ, Schoenfeld T
• Multiple proteins are produced from the dec-1 eggshell gene in Drosophilaby alternative RNA splicing and proteolytic cleavage events.
• Dev Biol. 1990; 142: 1-12
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• The defective chorion-1 gene (dec-1) in Drosophila encodes follicle cellproteins necessary for proper eggshell assembly. A distinctive feature ofthe gene is the production of multiple products by both alternative RNAsplicing and proteolytic processing events. DNA and protein sequencingstudies have revealed several dec-1 protein products. The predominanttranslation product, fc106, has a vitelline membrane-like N-terminaldomain followed by a glutamine, methionine-rich central region, largely inthe form of 26 amino acid repeats. During late stage 10 the N-terminalportion of fc106 is cleaved, yielding s80, a major eggshell protein.Conceptual translation of the DNA sequence as well as molecular analysesof several dec-1 mutants suggest that the less abundant alternativelyspliced RNAs encode primary translation products with different carboxyterminal ends. These results are discussed with respect to previousgenetic analyses of dec-1 mutants as well as with respect to potentialprotein-protein interactions which may underlie stabilization of thiscomplex extracellular structure.