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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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Nested RT-PCR to characterise chimeric products between CSNK2B and LY6G5B. (A) Schematic representation of the cloned and sequenced products on the genomic structure. Arrows represent primers used whose number corresponds with the number in Table 2. On the right are reported the length of the product as represented in the figure. Dashed boxes indicate inframe protein sequence. Each set of primers was tested in the indicated cell lines: primers from exon 2 (B), exon 5 (C) or exon 6 (D) of CSNK2B with exon 3 of LY6G5B. In E we report the RT-PCR for the CSNK2B ORF, from exon 2 to 7, is shown. X indicates premature stop codon.
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Figure 6: Nested RT-PCR to characterise chimeric products between CSNK2B and LY6G5B. (A) Schematic representation of the cloned and sequenced products on the genomic structure. Arrows represent primers used whose number corresponds with the number in Table 2. On the right are reported the length of the product as represented in the figure. Dashed boxes indicate inframe protein sequence. Each set of primers was tested in the indicated cell lines: primers from exon 2 (B), exon 5 (C) or exon 6 (D) of CSNK2B with exon 3 of LY6G5B. In E we report the RT-PCR for the CSNK2B ORF, from exon 2 to 7, is shown. X indicates premature stop codon.

Mentions: To prove the presence of the chimeric transcript for LY6G5B we performed RT-PCR using primers from the second, fifth and sixth exons of the CSNK2B gene and the third exon of LY6G5B (Figure 6A). We found a defined pattern of bands (Figure 6B) in Raji, K562 and U937 cells whose sequences represent many combinations of exons from the two genes (Figure 6A). Three main bands of 1090, 936 and 900 bp were found when the nested RT-PCR was performed for the whole chimeric transcript. The first (1090 bp) corresponds to exons 2 to 6 of CSNK2B spliced to exons 2 and 3 of LY6G5B though the resulting chimeric transcript is not in frame with the LY6G5B ORF. The other two bands of 936 bp and 900 bp correspond to exons 2 to 5 of CSNK2B spliced to the last 36 nucleotides of exon 1 and exons 2 and 3 of LY6G5B (936 bp), or directly to exons 2 and 3 of LY6G5B (900 bp) which maintain the LY6G5B ORF. Other less abundant transcripts were also detected (see Figure 6A) which were confirmed when primers from exons 5 or 6 of CSNK2B were used in the PCR reactions (Figures 6C and 6D). Amplification under the same conditions of the CSNK2B gene using primers from exons 2 and 7 resulted in the appearance of a single band of 645 bp (Figure 6E) corresponding to only one RNA form, the one described in the literature [29].


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)

Nested RT-PCR to characterise chimeric products between CSNK2B and LY6G5B. (A) Schematic representation of the cloned and sequenced products on the genomic structure. Arrows represent primers used whose number corresponds with the number in Table 2. On the right are reported the length of the product as represented in the figure. Dashed boxes indicate inframe protein sequence. Each set of primers was tested in the indicated cell lines: primers from exon 2 (B), exon 5 (C) or exon 6 (D) of CSNK2B with exon 3 of LY6G5B. In E we report the RT-PCR for the CSNK2B ORF, from exon 2 to 7, is shown. X indicates premature stop codon.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Nested RT-PCR to characterise chimeric products between CSNK2B and LY6G5B. (A) Schematic representation of the cloned and sequenced products on the genomic structure. Arrows represent primers used whose number corresponds with the number in Table 2. On the right are reported the length of the product as represented in the figure. Dashed boxes indicate inframe protein sequence. Each set of primers was tested in the indicated cell lines: primers from exon 2 (B), exon 5 (C) or exon 6 (D) of CSNK2B with exon 3 of LY6G5B. In E we report the RT-PCR for the CSNK2B ORF, from exon 2 to 7, is shown. X indicates premature stop codon.
Mentions: To prove the presence of the chimeric transcript for LY6G5B we performed RT-PCR using primers from the second, fifth and sixth exons of the CSNK2B gene and the third exon of LY6G5B (Figure 6A). We found a defined pattern of bands (Figure 6B) in Raji, K562 and U937 cells whose sequences represent many combinations of exons from the two genes (Figure 6A). Three main bands of 1090, 936 and 900 bp were found when the nested RT-PCR was performed for the whole chimeric transcript. The first (1090 bp) corresponds to exons 2 to 6 of CSNK2B spliced to exons 2 and 3 of LY6G5B though the resulting chimeric transcript is not in frame with the LY6G5B ORF. The other two bands of 936 bp and 900 bp correspond to exons 2 to 5 of CSNK2B spliced to the last 36 nucleotides of exon 1 and exons 2 and 3 of LY6G5B (936 bp), or directly to exons 2 and 3 of LY6G5B (900 bp) which maintain the LY6G5B ORF. Other less abundant transcripts were also detected (see Figure 6A) which were confirmed when primers from exons 5 or 6 of CSNK2B were used in the PCR reactions (Figures 6C and 6D). Amplification under the same conditions of the CSNK2B gene using primers from exons 2 and 7 resulted in the appearance of a single band of 645 bp (Figure 6E) corresponding to only one RNA form, the one described in the literature [29].

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