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Regulation of expression of two LY-6 family genes by intron retention and transcription induced chimerism.

Calvanese V, Mallya M, Campbell RD, Aguado B - BMC Mol. Biol. (2008)

Bottom Line: In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript.In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms.Of interest is the fact that the 5' genes (CSNKbeta or G6F) undergo differential splicing only in the context of the chimera (CSNKbeta-LY6G5B or G6F-LY6G6C) and not on their own.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de BiologĂ­a Molecular Severo Ochoa, CSIC, Madrid, Spain. vincalv@cnio.es

ABSTRACT

Background: Regulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues.

Results: The MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD). This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC). This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKbeta-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively.

Conclusion: In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKbeta or G6F) undergo differential splicing only in the context of the chimera (CSNKbeta-LY6G5B or G6F-LY6G6C) and not on their own.

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Differential real time RT-PCR assay for the two LY6G5B splice isoforms in a panel of human tissues and cell lines. (A). Data are expressed in relation to the percentage of the intron-retained form expressed in blood. Numbers below the graph represent the percent of the correctly spliced isoform of LY6G5B relative to the total expression of the gene in each sample. PCR reactions were run in triplicates. (B) Schematic representation of the primer design for the differential assay. Forward primer (PR_3) is shared while reverse primers (PR_6 and 7) share only 4 nucleotides at the end of the first exon. X indicates the premature stop codon.
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Figure 1: Differential real time RT-PCR assay for the two LY6G5B splice isoforms in a panel of human tissues and cell lines. (A). Data are expressed in relation to the percentage of the intron-retained form expressed in blood. Numbers below the graph represent the percent of the correctly spliced isoform of LY6G5B relative to the total expression of the gene in each sample. PCR reactions were run in triplicates. (B) Schematic representation of the primer design for the differential assay. Forward primer (PR_3) is shared while reverse primers (PR_6 and 7) share only 4 nucleotides at the end of the first exon. X indicates the premature stop codon.

Mentions: The LY6G6D and LY6G5B genes express a small first intron (98 and 148 nucleotides, respectively) in the open reading frame which tends to be retained in the majority of cell lines and tissues, both in human and mouse RNAs [12]. To better understand the regulation of expression of these genes we performed a detailed analysis of the two transcripts of the LY6G5B gene and their relative levels in some cell lines and tissues by real time RT-PCR. The results shown in Figure 1 confirm that the intron-retaining form is the most abundant in all the samples analysed. The highest expression of this mis-spliced form was detected in lung, spleen, and in whole blood, and the K562 cell line. K562 cells also had the highest expression of the correctly spliced form.


Regulation of expression of two LY-6 family genes by intron retention and transcription induced chimerism.

Calvanese V, Mallya M, Campbell RD, Aguado B - BMC Mol. Biol. (2008)

Differential real time RT-PCR assay for the two LY6G5B splice isoforms in a panel of human tissues and cell lines. (A). Data are expressed in relation to the percentage of the intron-retained form expressed in blood. Numbers below the graph represent the percent of the correctly spliced isoform of LY6G5B relative to the total expression of the gene in each sample. PCR reactions were run in triplicates. (B) Schematic representation of the primer design for the differential assay. Forward primer (PR_3) is shared while reverse primers (PR_6 and 7) share only 4 nucleotides at the end of the first exon. X indicates the premature stop codon.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562388&req=5

Figure 1: Differential real time RT-PCR assay for the two LY6G5B splice isoforms in a panel of human tissues and cell lines. (A). Data are expressed in relation to the percentage of the intron-retained form expressed in blood. Numbers below the graph represent the percent of the correctly spliced isoform of LY6G5B relative to the total expression of the gene in each sample. PCR reactions were run in triplicates. (B) Schematic representation of the primer design for the differential assay. Forward primer (PR_3) is shared while reverse primers (PR_6 and 7) share only 4 nucleotides at the end of the first exon. X indicates the premature stop codon.
Mentions: The LY6G6D and LY6G5B genes express a small first intron (98 and 148 nucleotides, respectively) in the open reading frame which tends to be retained in the majority of cell lines and tissues, both in human and mouse RNAs [12]. To better understand the regulation of expression of these genes we performed a detailed analysis of the two transcripts of the LY6G5B gene and their relative levels in some cell lines and tissues by real time RT-PCR. The results shown in Figure 1 confirm that the intron-retaining form is the most abundant in all the samples analysed. The highest expression of this mis-spliced form was detected in lung, spleen, and in whole blood, and the K562 cell line. K562 cells also had the highest expression of the correctly spliced form.

Bottom Line: In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript.In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms.Of interest is the fact that the 5' genes (CSNKbeta or G6F) undergo differential splicing only in the context of the chimera (CSNKbeta-LY6G5B or G6F-LY6G6C) and not on their own.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de BiologĂ­a Molecular Severo Ochoa, CSIC, Madrid, Spain. vincalv@cnio.es

ABSTRACT

Background: Regulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues.

Results: The MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD). This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC). This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKbeta-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively.

Conclusion: In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKbeta or G6F) undergo differential splicing only in the context of the chimera (CSNKbeta-LY6G5B or G6F-LY6G6C) and not on their own.

Show MeSH
Related in: MedlinePlus