Tsai KW, Lin WC.. Quantitative analysis of wobble splicing indicates that it is not tissue specific. Genomics 88: 855-864

Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Genomics (Impact Factor: 2.28). 12/2006; 88(6):855-64. DOI: 10.1016/j.ygeno.2006.07.004
Source: PubMed


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.

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    • "Analysis of deep RNA-sequencing data by Bradley et al. (2012) confirmed that NAGNAG is highly regulated [1]. NAGNAG alternative splicing specifically targets inclusion or exclusion of three nucleotides at 3 í® í° splice sites (Figure 1), thus effecting a change in one or two amino acids encoded in the final protein [2] [3] [4] [5] [6] [7] [8] [9]. Such amino acid substitutions have been shown to affect protein function and interfere with signaling [10], affect cellular localization [11], and impact on DNA and protein binding [12] [13] [14] in both plants and mammals. "
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    ABSTRACT: NAGNAG alternative splicing plays an essential role in biological processes and represents a highly adaptable system for posttranslational regulation of gene function. NAGNAG alternative splicing impacts a myriad of biological processes. Previous studies of NAGNAG largely focused on messenger RNA. To the best of our knowledge, this is the first study testing the hypothesis that NAGNAG alternative splicing is also operative in large intergenic noncoding RNA (lincRNA). The RNA-seq data sets from recent deep sequencing studies were queried to test our hypothesis. NAGNAG alternative splicing of human lincRNA was identified while querying two independent RNA-seq data sets. Within these datasets, 31 NAGNAG alternative splicing sites were identified in lincRNA. Notably, most exons of lincRNA containing NAGNAG acceptors were longer than those from protein-coding genes. Furthermore, presence of CAG coding appeared to participate in the splice site selection. Finally, expression of the isoforms of NAGNAG lincRNA exhibited tissue specificity. Together, this study improves our understanding of the NAGNAG alternative splicing in lincRNA.
    BioMed Research International 06/2014; 2014(12):736798. DOI:10.1155/2014/736798 · 3.17 Impact Factor
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    • "Particularly, the ratio between the two short-distance splice isoforms may differ in various tissues and cell types [34], [36], depending on developmental stages [37], [38] or in response to external factors [39]. To date, a variety of methodological implementations such as polyacrylamide [40], [41] and agarose [27], [42] gel electrophoresis, capillary electrophoresis [43] as well as RNA sequencing analysis [8] have been employed in order to specify the expression ratio. However, these approaches cannot provide accurate results or they are limiting due to the sophisticated equipment requirements. "
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    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 05/2014; 9(5):e96557. DOI:10.1371/journal.pone.0096557 · 3.23 Impact Factor
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    • "However, the detailed mechanisms for alternative splicing at tandem motifs are currently unclear. Previous studies indicated that such tandem motifs at splicing junctions are common in human genes, but only a small fraction of them can generate wobble in splicing selection [7,9-11]. Our data confirmed that a high frequency of 5' wobble splicing events is located at four nucleotides from the dominant donor site and this is hypothesized to be associated with the binding affinity of U1 snRNA [9,25]. "
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    ABSTRACT: Alternative splicing is an important mechanism mediating the diversified functions of genes in multicellular organisms, and such event occurs in around 40-60% of human genes. Recently, a new splice-junction wobbling mechanism was proposed that subtle modifications exist in mRNA maturation by alternatively choosing at 5'- GTNGT and 3'- NAGNAG, which created single amino acid insertion and deletion isoforms. By browsing the Alternative Splicing Database information, we observed that most 3' alternative splice site choices occur within six nucleotides of the dominant splice site and the incidence significantly decreases further away from the dominant acceptor site. Although a lower frequency of alternative splicing occurs within the intronic region (alternative splicing at the proximal AG) than in the exonic region (alternative splicing at the distal AG), alternative AG sites located within the intronic region show stronger potential as the acceptor. These observations revealed that the choice of 3' splice sites during 3' splicing junction wobbling could depend on the distance between the duplicated AG and the branch point site (BPS). Further mutagenesis experiments demonstrated that the distance of AG-to-AG and BPS-to-AG can greatly influence 3' splice site selection. Knocking down a known alternative splicing regulator, hSlu7, failed to affect wobble splicing choices. Our results implied that nucleotide distance between proximal and distal AG sites has an important regulatory function. In this study, we showed that occurrence of 3' wobble splicing occurs in a distance-dependent manner and that most of this wobble splicing is probably caused by steric hindrance from a factor bound at the neighboring tandem motif sequence.
    BMC Molecular Biology 05/2010; 11(1):34. DOI:10.1186/1471-2199-11-34 · 2.19 Impact Factor
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