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Splicing of a non-coding antisense transcript controls LEF1 gene expression.

Beltran M, Aparicio-Prat E, Mazzolini R, Millanes-Romero A, Massó P, Jenner RG, Díaz VM, Peiró S, de Herreros AG - Nucleic Acids Res. (2015)

Bottom Line: Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3.Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter.Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms.

View Article: PubMed Central - PubMed

Affiliation: Programa de Recerca en Càncer, Institut Hospital del Mar d'Investigacions Mèdiques, 08003 Barcelona, Spain UCL Cancer Institute, University College London, London, WC1E6BT, UK.

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LEF1 NAT controls the expression of LEF1 mRNA and protein. (A) Unspliced (+213/−1856) or spliced NAT were synthesized in vitro. A three-fold excess spliced NAT with respect to the unspliced form were transfected in the conditions indicated in Methods. In the control samples an irrelevant RNA (corresponding to a fragment of pcDNA3 plasmid) was transfected. After 36 h, RNA was obtained and levels of LEF1 mRNA were determined using two oligonucleotides corresponding to an amplicon present in the third exon. The results correspond to the average ± range of two experiments performed in duplicate. The asterisk indicates significant (P < 0.05). (B) RWP-1 or HT-29 M6 Snail1 cells were stably transfected with pBabe-LEF1 NAT (unspliced) or pBabe as control. RNA was collected and analyzed by RT-PCR with oligonucleotides specific for LEF1 mRNA, LEF1 NAT (total) or HPRT as control (top panel); alternatively protein extracts were prepared and analyzed by western blot with a polyclonal antibody against LEF1 (Cell Signal) or anti PyrK (Sigma) (bottom panel). (C) The migration capacity of the indicated cell populations was determined as described in Methods. The differences are significant with a P < 0.05.
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Figure 3: LEF1 NAT controls the expression of LEF1 mRNA and protein. (A) Unspliced (+213/−1856) or spliced NAT were synthesized in vitro. A three-fold excess spliced NAT with respect to the unspliced form were transfected in the conditions indicated in Methods. In the control samples an irrelevant RNA (corresponding to a fragment of pcDNA3 plasmid) was transfected. After 36 h, RNA was obtained and levels of LEF1 mRNA were determined using two oligonucleotides corresponding to an amplicon present in the third exon. The results correspond to the average ± range of two experiments performed in duplicate. The asterisk indicates significant (P < 0.05). (B) RWP-1 or HT-29 M6 Snail1 cells were stably transfected with pBabe-LEF1 NAT (unspliced) or pBabe as control. RNA was collected and analyzed by RT-PCR with oligonucleotides specific for LEF1 mRNA, LEF1 NAT (total) or HPRT as control (top panel); alternatively protein extracts were prepared and analyzed by western blot with a polyclonal antibody against LEF1 (Cell Signal) or anti PyrK (Sigma) (bottom panel). (C) The migration capacity of the indicated cell populations was determined as described in Methods. The differences are significant with a P < 0.05.

Mentions: The effect of the NAT on LEF1 mRNA expression was also determined by transfecting the spliced and unspliced forms of the transcript. We performed this analysis by two alternative experimental approaches. First, we transfected in vitro synthesized RNA corresponding to these NATs and checked LEF1 mRNA expression by strand-specific qRT-PCR. As shown in Figure 3A, the unspliced form down-regulated LEF1 mRNA whereas the spliced NAT did not have a significant effect. Co-transfection of the spliced NAT inhibited the action of the unspliced form, suggesting that the spliced NAT is acting as a dominant negative factor (Figure 3A). We also stably transfected a plasmid expressing the unspliced form of the transcript. As shown in Figure 3B (top), the unspliced NAT down-regulated LEF1 mRNA both in HT-29 M6 and RWP-1 Snail1 cells, without altering the stability of this RNA (see Supplementary Figure S3). A similar inhibition of LEF1 protein by unspliced NAT was also observed (Figure 3B bottom). Moreover, unspliced LEF1 NAT expression decreased the migration of both cell lines (Figure 3C) without affecting their growth rate (Supplementary Figure S4). Co-transfection of spliced NAT remarkably rescued the inhibition of migration caused by unpsliced NAT in RWP-1 Snail1 cells (Figure 3C). We did not detect a significant change in the phenotype of these cells. LEF1 NAT only caused a small increase in the mRNA levels of E-cadherin and other epithelial genes (Claudin4, Occludin) (Supplementary Figure S5A); E-cadherin up-regulation was also detected at protein level (Supplementary Figure S5B). No changes in the E-cadherin repressors Snail1 or Zeb1 (2,3) were observed, however ZEB2 RNA was significantly down-regulated (Supplementary Figure S5A), suggesting that LEF1 was contributing to the expression of this gene.


Splicing of a non-coding antisense transcript controls LEF1 gene expression.

Beltran M, Aparicio-Prat E, Mazzolini R, Millanes-Romero A, Massó P, Jenner RG, Díaz VM, Peiró S, de Herreros AG - Nucleic Acids Res. (2015)

LEF1 NAT controls the expression of LEF1 mRNA and protein. (A) Unspliced (+213/−1856) or spliced NAT were synthesized in vitro. A three-fold excess spliced NAT with respect to the unspliced form were transfected in the conditions indicated in Methods. In the control samples an irrelevant RNA (corresponding to a fragment of pcDNA3 plasmid) was transfected. After 36 h, RNA was obtained and levels of LEF1 mRNA were determined using two oligonucleotides corresponding to an amplicon present in the third exon. The results correspond to the average ± range of two experiments performed in duplicate. The asterisk indicates significant (P < 0.05). (B) RWP-1 or HT-29 M6 Snail1 cells were stably transfected with pBabe-LEF1 NAT (unspliced) or pBabe as control. RNA was collected and analyzed by RT-PCR with oligonucleotides specific for LEF1 mRNA, LEF1 NAT (total) or HPRT as control (top panel); alternatively protein extracts were prepared and analyzed by western blot with a polyclonal antibody against LEF1 (Cell Signal) or anti PyrK (Sigma) (bottom panel). (C) The migration capacity of the indicated cell populations was determined as described in Methods. The differences are significant with a P < 0.05.
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Figure 3: LEF1 NAT controls the expression of LEF1 mRNA and protein. (A) Unspliced (+213/−1856) or spliced NAT were synthesized in vitro. A three-fold excess spliced NAT with respect to the unspliced form were transfected in the conditions indicated in Methods. In the control samples an irrelevant RNA (corresponding to a fragment of pcDNA3 plasmid) was transfected. After 36 h, RNA was obtained and levels of LEF1 mRNA were determined using two oligonucleotides corresponding to an amplicon present in the third exon. The results correspond to the average ± range of two experiments performed in duplicate. The asterisk indicates significant (P < 0.05). (B) RWP-1 or HT-29 M6 Snail1 cells were stably transfected with pBabe-LEF1 NAT (unspliced) or pBabe as control. RNA was collected and analyzed by RT-PCR with oligonucleotides specific for LEF1 mRNA, LEF1 NAT (total) or HPRT as control (top panel); alternatively protein extracts were prepared and analyzed by western blot with a polyclonal antibody against LEF1 (Cell Signal) or anti PyrK (Sigma) (bottom panel). (C) The migration capacity of the indicated cell populations was determined as described in Methods. The differences are significant with a P < 0.05.
Mentions: The effect of the NAT on LEF1 mRNA expression was also determined by transfecting the spliced and unspliced forms of the transcript. We performed this analysis by two alternative experimental approaches. First, we transfected in vitro synthesized RNA corresponding to these NATs and checked LEF1 mRNA expression by strand-specific qRT-PCR. As shown in Figure 3A, the unspliced form down-regulated LEF1 mRNA whereas the spliced NAT did not have a significant effect. Co-transfection of the spliced NAT inhibited the action of the unspliced form, suggesting that the spliced NAT is acting as a dominant negative factor (Figure 3A). We also stably transfected a plasmid expressing the unspliced form of the transcript. As shown in Figure 3B (top), the unspliced NAT down-regulated LEF1 mRNA both in HT-29 M6 and RWP-1 Snail1 cells, without altering the stability of this RNA (see Supplementary Figure S3). A similar inhibition of LEF1 protein by unspliced NAT was also observed (Figure 3B bottom). Moreover, unspliced LEF1 NAT expression decreased the migration of both cell lines (Figure 3C) without affecting their growth rate (Supplementary Figure S4). Co-transfection of spliced NAT remarkably rescued the inhibition of migration caused by unpsliced NAT in RWP-1 Snail1 cells (Figure 3C). We did not detect a significant change in the phenotype of these cells. LEF1 NAT only caused a small increase in the mRNA levels of E-cadherin and other epithelial genes (Claudin4, Occludin) (Supplementary Figure S5A); E-cadherin up-regulation was also detected at protein level (Supplementary Figure S5B). No changes in the E-cadherin repressors Snail1 or Zeb1 (2,3) were observed, however ZEB2 RNA was significantly down-regulated (Supplementary Figure S5A), suggesting that LEF1 was contributing to the expression of this gene.

Bottom Line: Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3.Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter.Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms.

View Article: PubMed Central - PubMed

Affiliation: Programa de Recerca en Càncer, Institut Hospital del Mar d'Investigacions Mèdiques, 08003 Barcelona, Spain UCL Cancer Institute, University College London, London, WC1E6BT, UK.

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