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HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition.

Bonomi S, di Matteo A, Buratti E, Cabianca DS, Baralle FE, Ghigna C, Biamonti G - Nucleic Acids Res. (2013)

Bottom Line: Notably, hnRNP A1, by inhibiting the production of ΔRon, activates the reversal program, namely the mesenchymal-to-epithelial transition, which instead occurs at the final metastasis sites.Also, hnRNP A1 affects Ron splicing by regulating the expression level of hnRNP A2/B1, which similarly to SRSF1 can promote ΔRon production.These results shed light on how splicing regulation contributes to the tumor progression and provide potential targets to develop anticancer therapies.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (IGM-CNR), 27100 Pavia, Italy, International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy and Division of Regenerative Medicine, Stem Cells, and Gene Therapy, Dulbecco Telethon Institute at San Raffaele Scientific Institute, 20132 Milan, Italy.

ABSTRACT
Epithelial-to-mesenchymal transition (EMT) is an embryonic program used by cancer cells to acquire invasive capabilities becoming metastatic. ΔRon, a constitutively active isoform of the Ron tyrosine kinase receptor, arises from skipping of Ron exon 11 and provided the first example of an alternative splicing variant causatively linked to the activation of tumor EMT. Splicing of exon 11 is controlled by two adjacent regulatory elements, a silencer and an enhancer of splicing located in exon 12. The alternative splicing factor and oncoprotein SRSF1 directly binds to the enhancer, induces the production of ΔRon and activates EMT leading to cell locomotion. Interestingly, we now find an important role for hnRNP A1 in controlling the activity of the Ron silencer. HnRNP A1 is able to antagonize the binding of SRSF1 and prevent exon skipping. Notably, hnRNP A1, by inhibiting the production of ΔRon, activates the reversal program, namely the mesenchymal-to-epithelial transition, which instead occurs at the final metastasis sites. Also, hnRNP A1 affects Ron splicing by regulating the expression level of hnRNP A2/B1, which similarly to SRSF1 can promote ΔRon production. These results shed light on how splicing regulation contributes to the tumor progression and provide potential targets to develop anticancer therapies.

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Related in: MedlinePlus

HnRNP A1 affects the MET program. (A) HEK-293 cells not treated (NT) or transfected with hnRNP A1 (A1) or with control siRNAs (Ctr). Total cell extracts were probed by western blotting with the following antibodies: anti hnRNP A1 (9H10), anti-hnRNP H, anti-SRSF1 (mAb96) and anti-α-tubulin; the three histograms on the right show the relative level of hnRNP A1, SRSF1 and hnRNP H transcripts quantified by qRT-PCR. (B) The same cells (not treated or transfected with siRNA oligos) were analyzed in (i) RT-PCR to determine the splicing profile of the endogenous Ron transcripts as in Figure 3A and (ii) qRT-PCR for the expression level of the indicated EMT markers. Cells morphologies were examined under a phase-contrast microscope (objective 10× with additional magnification 1.6×). (C) HeLa cells were transfected with T7-hnRNP A1 or with the empty vector (‘Vector’) with an efficiency of ∼60%; RNAs were analyzed with RT-PCR (as in Figure 3A) to determine the splicing profile of Ron exon 11 and cell extracts were analyzed in western blotting to assess the abundance of the indicated proteins and the expression of T7-hnRNP A1. The same cells were also analyzed in qRT-PCR for the expression levels of selected EMT markers. Panel on the right shows the results of the wound healing assay. Transient transfected HeLa cells were grown to confluence and wounded by dragging a 200 μl pipette tip through the monolayer (0 h); cell migration was measured at designated times (5, 10 and 24 h) after wounding. (D) MDA-MB-435S cells stably transfected with T7-SRSF1 (Cl.SF2) or with the empty vector (Cl.Vector) (29). Cl.SF2 and Cl.Vector cells were transfected with T7-hnRNP A1 or with the empty vector (‘Vector’) as indicated; total cell extracts were analyzed in western blotting to assess the level of the indicated proteins. (E) Total RNAs from the same cells were analyzed by RT-PCR (as in Figure 3A); the histogram below shows the ΔRon/Ron ratio. Expression level of the epithelial marker E-cadherin in the different cells as assessed by qRT-PCR is also shown.
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gkt579-F4: HnRNP A1 affects the MET program. (A) HEK-293 cells not treated (NT) or transfected with hnRNP A1 (A1) or with control siRNAs (Ctr). Total cell extracts were probed by western blotting with the following antibodies: anti hnRNP A1 (9H10), anti-hnRNP H, anti-SRSF1 (mAb96) and anti-α-tubulin; the three histograms on the right show the relative level of hnRNP A1, SRSF1 and hnRNP H transcripts quantified by qRT-PCR. (B) The same cells (not treated or transfected with siRNA oligos) were analyzed in (i) RT-PCR to determine the splicing profile of the endogenous Ron transcripts as in Figure 3A and (ii) qRT-PCR for the expression level of the indicated EMT markers. Cells morphologies were examined under a phase-contrast microscope (objective 10× with additional magnification 1.6×). (C) HeLa cells were transfected with T7-hnRNP A1 or with the empty vector (‘Vector’) with an efficiency of ∼60%; RNAs were analyzed with RT-PCR (as in Figure 3A) to determine the splicing profile of Ron exon 11 and cell extracts were analyzed in western blotting to assess the abundance of the indicated proteins and the expression of T7-hnRNP A1. The same cells were also analyzed in qRT-PCR for the expression levels of selected EMT markers. Panel on the right shows the results of the wound healing assay. Transient transfected HeLa cells were grown to confluence and wounded by dragging a 200 μl pipette tip through the monolayer (0 h); cell migration was measured at designated times (5, 10 and 24 h) after wounding. (D) MDA-MB-435S cells stably transfected with T7-SRSF1 (Cl.SF2) or with the empty vector (Cl.Vector) (29). Cl.SF2 and Cl.Vector cells were transfected with T7-hnRNP A1 or with the empty vector (‘Vector’) as indicated; total cell extracts were analyzed in western blotting to assess the level of the indicated proteins. (E) Total RNAs from the same cells were analyzed by RT-PCR (as in Figure 3A); the histogram below shows the ΔRon/Ron ratio. Expression level of the epithelial marker E-cadherin in the different cells as assessed by qRT-PCR is also shown.

Mentions: Our results suggest that hnRNP A1 can inhibit the production of ΔRon transcripts by antagonizing the activity of the adjacent ESE element. As a first step to verify this hypothesis, we used previously validated siRNAs (28) to downregulate hnRNP A1 expression in HEK-293 cells that display a high level of hnRNP A1 and a low ΔRon/Ron mRNAs ratio (Figure 4A). This treatment did not affect the levels of SRSF1 and hnRNP H, another factor recently shown to control splicing of Ron exon 11 (12). As expected from our model, downregulation of hnRNP A1 was accompanied by skipping of Ron exon 11 and a higher ΔRon/Ron ratio (Figure 4A). We have previously shown that the level of ΔRon is a critical determinant of the transition from an epithelial to a mesenchymal cellular phenotype (EMT) (8). Thus, we asked whether down-regulation of hnRNP A1, by inducing the production of ΔRon, could trigger EMT. As shown in Figure 4B, down-regulation of hnRNP A1, in addition to stimulate ΔRon production, had evident morphological consequences. Upon transfection with siRNA against hnRNP A1 cells with long cytoplasmic protrusions and elongated, spindle-shaped morphology were detectable, whereas cells transfected with the control oligo were rounded and grew in clusters. This effect was accompanied by strong reduction of expression levels of the epithelial marker E-cadherin and increased expression of the mesenchymal markers Slug, Snail1 and ZEB1, three repressors of E-cadherin (9,10).Figure 4.


HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition.

Bonomi S, di Matteo A, Buratti E, Cabianca DS, Baralle FE, Ghigna C, Biamonti G - Nucleic Acids Res. (2013)

HnRNP A1 affects the MET program. (A) HEK-293 cells not treated (NT) or transfected with hnRNP A1 (A1) or with control siRNAs (Ctr). Total cell extracts were probed by western blotting with the following antibodies: anti hnRNP A1 (9H10), anti-hnRNP H, anti-SRSF1 (mAb96) and anti-α-tubulin; the three histograms on the right show the relative level of hnRNP A1, SRSF1 and hnRNP H transcripts quantified by qRT-PCR. (B) The same cells (not treated or transfected with siRNA oligos) were analyzed in (i) RT-PCR to determine the splicing profile of the endogenous Ron transcripts as in Figure 3A and (ii) qRT-PCR for the expression level of the indicated EMT markers. Cells morphologies were examined under a phase-contrast microscope (objective 10× with additional magnification 1.6×). (C) HeLa cells were transfected with T7-hnRNP A1 or with the empty vector (‘Vector’) with an efficiency of ∼60%; RNAs were analyzed with RT-PCR (as in Figure 3A) to determine the splicing profile of Ron exon 11 and cell extracts were analyzed in western blotting to assess the abundance of the indicated proteins and the expression of T7-hnRNP A1. The same cells were also analyzed in qRT-PCR for the expression levels of selected EMT markers. Panel on the right shows the results of the wound healing assay. Transient transfected HeLa cells were grown to confluence and wounded by dragging a 200 μl pipette tip through the monolayer (0 h); cell migration was measured at designated times (5, 10 and 24 h) after wounding. (D) MDA-MB-435S cells stably transfected with T7-SRSF1 (Cl.SF2) or with the empty vector (Cl.Vector) (29). Cl.SF2 and Cl.Vector cells were transfected with T7-hnRNP A1 or with the empty vector (‘Vector’) as indicated; total cell extracts were analyzed in western blotting to assess the level of the indicated proteins. (E) Total RNAs from the same cells were analyzed by RT-PCR (as in Figure 3A); the histogram below shows the ΔRon/Ron ratio. Expression level of the epithelial marker E-cadherin in the different cells as assessed by qRT-PCR is also shown.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3794575&req=5

gkt579-F4: HnRNP A1 affects the MET program. (A) HEK-293 cells not treated (NT) or transfected with hnRNP A1 (A1) or with control siRNAs (Ctr). Total cell extracts were probed by western blotting with the following antibodies: anti hnRNP A1 (9H10), anti-hnRNP H, anti-SRSF1 (mAb96) and anti-α-tubulin; the three histograms on the right show the relative level of hnRNP A1, SRSF1 and hnRNP H transcripts quantified by qRT-PCR. (B) The same cells (not treated or transfected with siRNA oligos) were analyzed in (i) RT-PCR to determine the splicing profile of the endogenous Ron transcripts as in Figure 3A and (ii) qRT-PCR for the expression level of the indicated EMT markers. Cells morphologies were examined under a phase-contrast microscope (objective 10× with additional magnification 1.6×). (C) HeLa cells were transfected with T7-hnRNP A1 or with the empty vector (‘Vector’) with an efficiency of ∼60%; RNAs were analyzed with RT-PCR (as in Figure 3A) to determine the splicing profile of Ron exon 11 and cell extracts were analyzed in western blotting to assess the abundance of the indicated proteins and the expression of T7-hnRNP A1. The same cells were also analyzed in qRT-PCR for the expression levels of selected EMT markers. Panel on the right shows the results of the wound healing assay. Transient transfected HeLa cells were grown to confluence and wounded by dragging a 200 μl pipette tip through the monolayer (0 h); cell migration was measured at designated times (5, 10 and 24 h) after wounding. (D) MDA-MB-435S cells stably transfected with T7-SRSF1 (Cl.SF2) or with the empty vector (Cl.Vector) (29). Cl.SF2 and Cl.Vector cells were transfected with T7-hnRNP A1 or with the empty vector (‘Vector’) as indicated; total cell extracts were analyzed in western blotting to assess the level of the indicated proteins. (E) Total RNAs from the same cells were analyzed by RT-PCR (as in Figure 3A); the histogram below shows the ΔRon/Ron ratio. Expression level of the epithelial marker E-cadherin in the different cells as assessed by qRT-PCR is also shown.
Mentions: Our results suggest that hnRNP A1 can inhibit the production of ΔRon transcripts by antagonizing the activity of the adjacent ESE element. As a first step to verify this hypothesis, we used previously validated siRNAs (28) to downregulate hnRNP A1 expression in HEK-293 cells that display a high level of hnRNP A1 and a low ΔRon/Ron mRNAs ratio (Figure 4A). This treatment did not affect the levels of SRSF1 and hnRNP H, another factor recently shown to control splicing of Ron exon 11 (12). As expected from our model, downregulation of hnRNP A1 was accompanied by skipping of Ron exon 11 and a higher ΔRon/Ron ratio (Figure 4A). We have previously shown that the level of ΔRon is a critical determinant of the transition from an epithelial to a mesenchymal cellular phenotype (EMT) (8). Thus, we asked whether down-regulation of hnRNP A1, by inducing the production of ΔRon, could trigger EMT. As shown in Figure 4B, down-regulation of hnRNP A1, in addition to stimulate ΔRon production, had evident morphological consequences. Upon transfection with siRNA against hnRNP A1 cells with long cytoplasmic protrusions and elongated, spindle-shaped morphology were detectable, whereas cells transfected with the control oligo were rounded and grew in clusters. This effect was accompanied by strong reduction of expression levels of the epithelial marker E-cadherin and increased expression of the mesenchymal markers Slug, Snail1 and ZEB1, three repressors of E-cadherin (9,10).Figure 4.

Bottom Line: Notably, hnRNP A1, by inhibiting the production of ΔRon, activates the reversal program, namely the mesenchymal-to-epithelial transition, which instead occurs at the final metastasis sites.Also, hnRNP A1 affects Ron splicing by regulating the expression level of hnRNP A2/B1, which similarly to SRSF1 can promote ΔRon production.These results shed light on how splicing regulation contributes to the tumor progression and provide potential targets to develop anticancer therapies.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (IGM-CNR), 27100 Pavia, Italy, International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy and Division of Regenerative Medicine, Stem Cells, and Gene Therapy, Dulbecco Telethon Institute at San Raffaele Scientific Institute, 20132 Milan, Italy.

ABSTRACT
Epithelial-to-mesenchymal transition (EMT) is an embryonic program used by cancer cells to acquire invasive capabilities becoming metastatic. ΔRon, a constitutively active isoform of the Ron tyrosine kinase receptor, arises from skipping of Ron exon 11 and provided the first example of an alternative splicing variant causatively linked to the activation of tumor EMT. Splicing of exon 11 is controlled by two adjacent regulatory elements, a silencer and an enhancer of splicing located in exon 12. The alternative splicing factor and oncoprotein SRSF1 directly binds to the enhancer, induces the production of ΔRon and activates EMT leading to cell locomotion. Interestingly, we now find an important role for hnRNP A1 in controlling the activity of the Ron silencer. HnRNP A1 is able to antagonize the binding of SRSF1 and prevent exon skipping. Notably, hnRNP A1, by inhibiting the production of ΔRon, activates the reversal program, namely the mesenchymal-to-epithelial transition, which instead occurs at the final metastasis sites. Also, hnRNP A1 affects Ron splicing by regulating the expression level of hnRNP A2/B1, which similarly to SRSF1 can promote ΔRon production. These results shed light on how splicing regulation contributes to the tumor progression and provide potential targets to develop anticancer therapies.

Show MeSH
Related in: MedlinePlus