Frequent occurrence of protein isoforms with or without a single amino acid residue by subtle alternative splicing: the case of Gln in DRPLA affects subcellular localization of the products

AbstractProtein isoforms with or without a single amino acid residue make a subtle difference. It has been documented on a few genes that alternative splicing generated such isoforms; however, the fact has attracted little attention. We became aware of a subtle sequence difference in DRPLA, a polyglutamine disease gene for dentatorubral pallidoluysian atrophy. Some reported cDNA sequences lacked 3 nucleotides (nt) (CAG), which were positioned apart from the expandable and polymorphic CAG repeats and also coded for glutamine. We experimentally confirmed that the difference was indeed generated by alternative splicing utilizing two acceptors separated by 3 nt. InDRPLA, the expression ratio of two mRNA isoforms was almost constant among tissues, with the CAG-included form being major. The glutamine-included protein isoform was more predominantly localized in the nucleus. Database searching revealed that alternative splice acceptors, as well as donors, are frequently situated very close to each other. We experimentally confirmed two mRNA isoforms of 3 nt difference in more than 200 cases by RT-PCR and found interesting features associated with this phenomena. Inclusion of 3 nt tends to result in single amino acid inclusion despite the phase of translational frame. The expression ratio sometimes varied extensively among tissues.

[1]  C. Ross,et al.  DRPLA gene (atrophin-1) sequence and mRNA expression in human brain. , 1996, Brain research. Molecular brain research.

[2]  I. Kanazawa,et al.  Dentatorubral-pallidoluysian atrophy or Naito-Oyanagi disease , 1998, Neurogenetics.

[3]  S. Tsuji,et al.  Molecular cloning of a full-length cDNA for dentatorubral-pallidoluysian atrophy and regional expressions of the expanded alleles in the CNS. , 1995, American journal of human genetics.

[4]  C. Shen,et al.  One‐codon alternative splicing of the CpG MTase Dnmt1 transcript in mouse somatic cells , 2000, FEBS letters.

[5]  T. Hashikawa,et al.  Protein processing and releases of neuregulin‐1 are regulated in an activity‐dependent manner , 2004, Journal of neurochemistry.

[6]  Y. Okamura-Oho,et al.  Extended polyglutamine selectively interacts with caspase-8 and -10 in nuclear aggregates , 2001, Cell Death and Differentiation.

[7]  C A Ross,et al.  Intranuclear Neuronal Inclusions: A Common Pathogenic Mechanism for Glutamine-Repeat Neurodegenerative Diseases? , 1997, Neuron.

[8]  Christine Van Broeckhoven,et al.  Pathogenesis of polyglutamine disorders: aggregation revisited. , 2003, Human molecular genetics.

[9]  H. Zoghbi,et al.  Trinucleotide repeats: mechanisms and pathophysiology. , 2000, Annual review of genomics and human genetics.

[10]  S. Tsuji,et al.  Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA protein with an expanded polyglutamine stretch , 1998, Nature Genetics.

[11]  K. Tadokoro,et al.  Structure and expression of the gene responsible for the triplet repeat disorder, dentatorubral and pallidoluysian atrophy (DRPLA) , 1994, Nature Genetics.

[12]  S. L. Berger,et al.  GAG triplets as splice acceptors of last resort. An unusual form of alternative splicing in prothymosin alpha pre-mRNA. , 1993, Journal of molecular biology.

[13]  Y. Okamura-Oho,et al.  Expression of extended polyglutamine sequentially activates initiator and effector caspases. , 1999, Biochemical and biophysical research communications.

[14]  R. Shamir,et al.  How prevalent is functional alternative splicing in the human genome? , 2004, Trends in genetics : TIG.

[15]  A. Sano,et al.  Dentatorubral and pallidoluysian atrophy expansion of an unstable CAG trinucleotide on chromosome 12p , 1994, Nature Genetics.

[16]  A. J. Lopez,et al.  Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation. , 1998, Annual review of genetics.

[17]  G. Condorelli,et al.  Two alternatively spliced forms of the human insulin-like growth factor I receptor have distinct biological activities and internalization kinetics. , 1994, The Journal of biological chemistry.

[18]  John Q Trojanowski,et al.  Neurodegenerative diseases: a decade of discoveries paves the way for therapeutic breakthroughs , 2004, Nature Medicine.

[19]  K. Vogan,et al.  An alternative splicing event in the Pax-3 paired domain identifies the linker region as a key determinant of paired domain DNA-binding activity , 1996, Molecular and cellular biology.

[20]  Y. Okamura-Oho,et al.  Dentatorubral Pallidoluysian Atrophy (DRPLA) Protein Is Cleaved by Caspase-3 during Apoptosis* , 1997, The Journal of Biological Chemistry.

[21]  M. Hayden,et al.  Nuclear Localization of a Non-caspase Truncation Product of Atrophin-1, with an Expanded Polyglutamine Repeat, Increases Cellular Toxicity* , 2003, The Journal of Biological Chemistry.

[22]  Y. Okamura-Oho,et al.  Dentatorubral-pallidoluysian atrophy protein interacts through a proline-rich region near polyglutamine with the SH3 domain of an insulin receptor tyrosine kinase substrate. , 1999, Human molecular genetics.

[23]  N. Miki,et al.  Regulation of pituitary growth hormone-releasing factor (GRF) receptor gene expression by GRF. , 1996, Biochemical and Biophysical Research Communications - BBRC.

[24]  E. Hell,et al.  Human Obese Gene Expression: Alternative Splicing of mRNA and Relation to Adipose Tissue Localization , 1997, Obesity surgery.

[25]  Y. Okamura-Oho,et al.  Genomic structure and alternative splicing of the insulin receptor tyrosine kinase substrate of 53-kDa protein , 2003, Journal of Human Genetics.

[26]  J. Valcárcel,et al.  Splicing Regulation at the Second Catalytic Step by Sex-lethal Involves 3′ Splice Site Recognition by SPF45 , 2002, Cell.

[27]  W. Johnson,et al.  Diffusion mechanisms in metallic supercooled liquids and glasses , 1999, Nature.

[28]  Christopher J. Lee,et al.  A genomic view of alternative splicing , 2002, Nature Genetics.

[29]  Y. Okamura-Oho,et al.  Protein binding of a DRPLA family through arginine-glutamic acid dipeptide repeats is enhanced by extended polyglutamine. , 2000, Human molecular genetics.

[30]  Michael R. Green,et al.  Functional recognition of the 3′ splice site AG by the splicing factor U2AF35 , 1999, Nature.

[31]  K. Tadokoro,et al.  PCR detection of 9 polymorphisms in the WT1 gene. , 1993, Human molecular genetics.

[32]  Thangavel Alphonse Thanaraj,et al.  ASD: the Alternative Splicing Database , 2004, Nucleic Acids Res..

[33]  International Human Genome Sequencing Consortium.  Finishing the euchromatic sequence of the human genome , 2004 .

[34]  R. Reed,et al.  An Upstream AG Determines Whether a Downstream AG Is Selected during Catalytic Step II of Splicing , 2001, Molecular and Cellular Biology.

[35]  A. Hackam,et al.  Cleavage of Atrophin-1 at Caspase Site Aspartic Acid 109 Modulates Cytotoxicity* , 1999, The Journal of Biological Chemistry.

[36]  C. Ross,et al.  Cloning and expression of the rat atrophin-I (DRPLA disease gene) homologue , 1995, Neurobiology of Disease.

[37]  D. Black.  Mechanisms of alternative pre-messenger RNA splicing. , 2003, Annual review of biochemistry.

[38]  Y. Okamura-Oho,et al.  Dentatorubral-pallidoluysian atrophy protein is phosphorylated by c-Jun NH2-terminal kinase. , 2003, Human molecular genetics.

[39]  Y. Okamura-Oho,et al.  Intracellular aggregate formation of dentatorubral-pallidoluysian atrophy (DRPLA) protein with the extended polyglutamine. , 1998, Biochemical and biophysical research communications.

[40]  Rolf Backofen,et al.  Widespread occurrence of alternative splicing at NAGNAG acceptors contributes to proteome plasticity , 2004, Nature Genetics.

[41]  Tim Hubbard.  Finishing the euchromatic sequence of the human genome , 2004 .