Quantitative analysis of wobble splicing indicates that it is not tissue specific.
Alternative splicing is an important mechanism mediating the function of genes in multicellular organisms. Recently, we discovered a new splicing-junction wobble mechanism that generates subtle alterations in mRNA by randomly selecting tandem 5' and 3' splicing-junction sites. Here we developed a sensitive approach to identify such splicing-junction wobble isoforms using polymerase chain reaction amplification with fluorescence-labeled primers encompassing the wobble-splicing boundary and capillary electrophoresis. Using the ING4 wobble isoforms as an example, we demonstrated that capillary electrophoresis can precisely separate DNA fragments with a small difference in size (<3 nt) and can be used to quantify the expression ratio, which thus measures the distribution of each splicing-junction wobble isoform in tissues. Based on our analyses of several genes, the relative ratio of each wobble-splicing isoform tends to be constant among various tissues. The occasional observed tissue heterogeneity of wobble-splicing transcripts can be generated only by genomic single-nucleotide polymorphisms around the splicing junction.
Tsai
Kuo-Wang
KW
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China; Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China.
Lin
Wen-Chang
WC
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China; Institute of Bioinformatics, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China. Electronic address: wenlin@ibms.sinica.edu.tw.
D000328:Adult; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D019075:Electrophoresis, Capillary; D005786:Gene Expression Regulation; D006131:Growth Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D022821:RNA Splice Sites; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D018797:Cell Cycle Proteins; D006131:Growth Inhibitors; D018398:Homeodomain Proteins; C483230:ING4 protein, human; D020033:Protein Isoforms; D022821:RNA Splice Sites; D025521:Tumor Suppressor Proteins