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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 Biología 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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Related in: MedlinePlus

Real Time-PCR to quantify the amount of intron-retained and intron-spliced forms of the LY6G5B transcripts in K562 (A, B) and Raji (C, D) cell lines harvested at times 0, 0.5, 1, 2 and 4 hrs post treatment with Actinomycin D. Data are expressed as the percentage of each form at time 0 with no treatment. PCR reactions were run in triplicate.
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Figure 3: Real Time-PCR to quantify the amount of intron-retained and intron-spliced forms of the LY6G5B transcripts in K562 (A, B) and Raji (C, D) cell lines harvested at times 0, 0.5, 1, 2 and 4 hrs post treatment with Actinomycin D. Data are expressed as the percentage of each form at time 0 with no treatment. PCR reactions were run in triplicate.

Mentions: We confirmed this experiment by measuring the levels of the two LY6G5B transcripts by a real time-PCR assay (Figure 3). In this case expression levels of the two splicing isoforms were normalised [25] to either GAPDH (Figure 3A and 3C) or β-Actin (Figure 3B and 3D) levels in K562 and Raji cells. As the transcripts for these two control genes also have their own kinetics of degradation we cannot measure an absolute stability of LY6G5B, but a relative stability compared to the control genes. In all cases we observed an increase in the relative expression of the LY6G5B isoforms with time, allowing us to conclude that the LY6G5B transcripts are more stable than Actin and GAPDH mRNA (Figure 3).


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)

Real Time-PCR to quantify the amount of intron-retained and intron-spliced forms of the LY6G5B transcripts in K562 (A, B) and Raji (C, D) cell lines harvested at times 0, 0.5, 1, 2 and 4 hrs post treatment with Actinomycin D. Data are expressed as the percentage of each form at time 0 with no treatment. PCR reactions were run in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Real Time-PCR to quantify the amount of intron-retained and intron-spliced forms of the LY6G5B transcripts in K562 (A, B) and Raji (C, D) cell lines harvested at times 0, 0.5, 1, 2 and 4 hrs post treatment with Actinomycin D. Data are expressed as the percentage of each form at time 0 with no treatment. PCR reactions were run in triplicate.
Mentions: We confirmed this experiment by measuring the levels of the two LY6G5B transcripts by a real time-PCR assay (Figure 3). In this case expression levels of the two splicing isoforms were normalised [25] to either GAPDH (Figure 3A and 3C) or β-Actin (Figure 3B and 3D) levels in K562 and Raji cells. As the transcripts for these two control genes also have their own kinetics of degradation we cannot measure an absolute stability of LY6G5B, but a relative stability compared to the control genes. In all cases we observed an increase in the relative expression of the LY6G5B isoforms with time, allowing us to conclude that the LY6G5B transcripts are more stable than Actin and GAPDH mRNA (Figure 3).

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 Biología 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