Article

Quantitative analysis of wobble splicing indicates that it is not tissue specific.

Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Genomics (Impact Factor: 3.01). 12/2006; 88(6):855-64. DOI: 10.1016/j.ygeno.2006.07.004
Source: PubMed

ABSTRACT 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.

0 Bookmarks
 · 
55 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: NAGNAG alternative splicing is one type of alternative splicing in mammals and plants. There are two opposite arguments regarding the mechanism of this NAGNAG event, i.e. whether splice variation is controllable by the cell or is just biological noise. In this paper, we systematically investigated NAGNAG acceptors in Arabidopsis thaliana using both cDNA/EST and RNA-Seq data. We identified 9,473 NAGNAG motifs, including 529 cDNA/EST-confirmed NAGNAG acceptors. A nomenclature tree for this type of alternative splicing was defined based on the cDNA/EST validation, location in the exon, sequence and expression level. Low expression of some NAGNAG motifs was observed in various tissues or pathogen-infected samples, indicating the existence of background splicing. Tissue-specific or treatment-specific differences in the dynamic profiles suggest that some NAGNAG acceptors are highly regulated.
    Planta 10/2013; · 3.35 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase κ transcript (RNase κ-02) which lacks four consecutive bases compared to the previously isolated RNase κ isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase κ-02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms.
    PLoS ONE 01/2014; 9(5):e96557. · 3.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Thousands of tandem alternative splice sites (TASS) give rise to mRNA insertion/deletion variants with small size differences. Recent work has concentrated on the question of biological relevance in general, and the physiological regulation of TASS in particular. We have quantitatively studied 11 representative TASS cases in comparison to one mutually exclusive exon case and two cassette exons (CEs) using a panel of human and mouse tissues, as well as cultured cell lines. Tissues show small but significant differences in TASS isoform ratios, with a variance 4- to 20-fold lower than seen for CEs. Remarkably, in cultured cells, all studied alternative splicing (AS) cases showed a cell-density-dependent shift of isoform ratios with similar time series profiles. A respective genome-wide co-regulation of TASS splicing was shown by next-generation mRNA sequencing data. Moreover, data from human and mouse organs indicate that this co-regulation of TASS occurs in vivo, with brain showing the strongest difference to other organs. Together, the results indicate a physiological AS regulation mechanism that functions almost independently from the splice site context and sequence.
    Nucleic Acids Research 07/2014; · 8.28 Impact Factor

Full-text

View
0 Downloads
Available from