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RNA polymerase III drives alternative splicing of the potassium channel-interacting protein contributing to brain complexity and neurodegeneration.

Massone S, Vassallo I, Castelnuovo M, Fiorino G, Gatta E, Robello M, Borghi R, Tabaton M, Russo C, Dieci G, Cancedda R, Pagano A - J. Cell Biol. (2011)

Bottom Line: We found that IL1-α-dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III-transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel-interacting protein (KCNIP4).Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels.This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer's disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Biology, and Genetics, National Institute for Cancer Research, 16132 Genoa, Italy.

ABSTRACT
Alternative splicing generates protein isoforms that are conditionally or differentially expressed in specific tissues. The discovery of factors that control alternative splicing might clarify the molecular basis of biological and pathological processes. We found that IL1-α-dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III-transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel-interacting protein (KCNIP4). The alternative KCNIP4 isoform cannot interact with the γ-secretase complex, resulting in modification of γ-secretase activity, amyloid precursor protein processing, and increased secretion of β-amyloid enriched in the more toxic Aβ x-42 species. Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels. This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer's disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain.

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38A-induced perturbation of Aβ secretion. (A) A schematic view of p38A and pMock plasmid constructs used to generate p38A-SHSY5Y and pMock-SHSY5Y permanently transfected cell lines. (B) Real-time RT-PCR quantitative determination of 38A expression and KCNIP4 splice variant ratio in p38A-SHSY5Y and pMock-SHSY5Y permanently transfected cell lines. (C) Increased β-amyloid secretion and perturbation of the Aβ x-42/Aβ x-40 ratio in 38A-overexpressing SHSY5Y cells. X axis, transfected plasmids; y axis, quantitative determination of Aβ (picograms/milliliter) secreted in the medium 48 h after medium replacement as determined by sandwich ELISA. The resulting Aβ x-42/Aβ x-40 ratio is reported. Striped bars, samples treated with ML-60218 PolIII-specific inhibitor; shaded bars, untreated cell samples. (D) A demonstration that PolIII transcription is specifically inhibited in ML-60218-treated cells, whereas it does not affect PolII activity. 5S rRNA, 7SK RNA, 38A, and c-Myc transcript abundancies were detected by real-time RT-PCR in cells treated with ML-60218 and in untreated controls used as described in A. (E) Quantitative Western blotting analysis of APP and PS2 synthesis in pMock- and/or p38A-transfected cells in ML-60218–treated and –untreated conditions. All of the determinations were normalized to α-tubulin detections. (F and G) Suppression of the biological phenotype driven by the coexpression of 38A and KCNIP4 Var IV–specific silencing plasmid in SHSY5Y cells. Three independent determinations of Var IV decrease and the concomitant increase of Var I expression are shown together with the determinations of Aβ secretion in siVar IV silenced (+) with respect to cells transfected with a scrambled siRNA (−). The error bars were not relevant enough to be visible in the graphs; additional repetitions of the same experiment gave similar results (not depicted).
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fig5: 38A-induced perturbation of Aβ secretion. (A) A schematic view of p38A and pMock plasmid constructs used to generate p38A-SHSY5Y and pMock-SHSY5Y permanently transfected cell lines. (B) Real-time RT-PCR quantitative determination of 38A expression and KCNIP4 splice variant ratio in p38A-SHSY5Y and pMock-SHSY5Y permanently transfected cell lines. (C) Increased β-amyloid secretion and perturbation of the Aβ x-42/Aβ x-40 ratio in 38A-overexpressing SHSY5Y cells. X axis, transfected plasmids; y axis, quantitative determination of Aβ (picograms/milliliter) secreted in the medium 48 h after medium replacement as determined by sandwich ELISA. The resulting Aβ x-42/Aβ x-40 ratio is reported. Striped bars, samples treated with ML-60218 PolIII-specific inhibitor; shaded bars, untreated cell samples. (D) A demonstration that PolIII transcription is specifically inhibited in ML-60218-treated cells, whereas it does not affect PolII activity. 5S rRNA, 7SK RNA, 38A, and c-Myc transcript abundancies were detected by real-time RT-PCR in cells treated with ML-60218 and in untreated controls used as described in A. (E) Quantitative Western blotting analysis of APP and PS2 synthesis in pMock- and/or p38A-transfected cells in ML-60218–treated and –untreated conditions. All of the determinations were normalized to α-tubulin detections. (F and G) Suppression of the biological phenotype driven by the coexpression of 38A and KCNIP4 Var IV–specific silencing plasmid in SHSY5Y cells. Three independent determinations of Var IV decrease and the concomitant increase of Var I expression are shown together with the determinations of Aβ secretion in siVar IV silenced (+) with respect to cells transfected with a scrambled siRNA (−). The error bars were not relevant enough to be visible in the graphs; additional repetitions of the same experiment gave similar results (not depicted).

Mentions: In light of the findings in the previous section and considering that the proteolytic processing of APP plays a central role in AD etiology through the formation of neurotoxic Aβ peptides (Johnson et al., 1990; Selkoe, 1990; Hardy and Selkoe, 2002; St George-Hyslop and Petit, 2005; Haass and Selkoe, 2007), we decided to investigate whether the selective overexpression of 38A ncRNA would affect the formation of Aβ x-42 and/or Aβ x-40 species by measuring their relative amount in the medium of 38A-overexpressing SHSY5Y cells. As Aβ processing might be altered by the transfection procedure, we first generated p38A and pMock permanently transfected cell lines (hereafter referred to as p38A-SHSY5Y and pMock-SHSY5Y, respectively). p38A-SHSY5Y cells, harboring extra copies of the 38A transcription unit with its natural PolIII type 3 promoter, exhibit a moderate overexpression of 38A ncRNA and, as a consequence, a 3.1-fold increased KCNIP4 Var IV/Var I ratio (Fig. 5, A and B). Culture media conditioned for 48 h with p38A-SHSY5Y and/or pMock-SHSY5Y cells were tested for their Aβ peptide composition, showing that 38A significantly enhances the levels of Aβ x-42 (up to 1.91-fold with respect to the pMock-transfected control), whereas its effect on the secretion of the more soluble Aβ x-40 peptide is more modest (up to 1.4-fold with respect to the pMock-transfected control). These results indicate that the transcription of 38A and the concomitant synthesis of KCNIP4 Var IV change both quantitatively and qualitatively the Aβ secretion, leading to an increased amount of the total Aβ and, notably, to a concomitant variation of the Aβ x-42/Aβ x-40 ratio that could account for the altered ratio observed in AD (Fig. 5 C). To further demonstrate the PolIII dependency (and the association with 38A) of the perturbation of Aβ processing, a parallel experiment was performed in the presence of 20 µM ML-60218; in this condition, the increment of both Aβ x-42 and Aβ x-40 was prevented, confirming the role of 38A PolIII–dependent expression in the impairment of Aβ peptide production (Fig. 5 C). In this experiment, the specific inhibition of PolIII was confirmed, evidencing a decreased synthesis of PolIII-transcribed RNAs (5S ribosomal RNA [rRNA], 7SK RNA, and 38A RNA) and a concomitant unaffected expression of PolII-dependent genes (cMyc and glyceraldehyde 3 phosphate dehydrogenase [GAPDH]; Fig. 5 D). Next, to discriminate whether the altered secretion of Aβ in p38A-SHSY5Y cells has to be ascribed to a different level of APP processing or rather to the modulation of APP and/or PS2 synthesis, we measured their amount in the same experimental settings. Results showed that the levels of both APP and PS2 are not affected by the overexpression of 38A, either in the absence or in the presence of ML-60218, demonstrating that the perturbation of Aβ promoted by 38A is specifically determined by the increase of APP processing (Fig. 5 E). This point was also confirmed by measuring Aβ secretion in 38A-transfected APP-overexpressing HEK293 cells that stably overexpress APP. Indeed, although the amount of Aβ secreted is considerably higher in these cells, the effects of 38A overexpression on APP processing recapitulate those obtained in SHSY5Y cells (Fig. S4 F).


RNA polymerase III drives alternative splicing of the potassium channel-interacting protein contributing to brain complexity and neurodegeneration.

Massone S, Vassallo I, Castelnuovo M, Fiorino G, Gatta E, Robello M, Borghi R, Tabaton M, Russo C, Dieci G, Cancedda R, Pagano A - J. Cell Biol. (2011)

38A-induced perturbation of Aβ secretion. (A) A schematic view of p38A and pMock plasmid constructs used to generate p38A-SHSY5Y and pMock-SHSY5Y permanently transfected cell lines. (B) Real-time RT-PCR quantitative determination of 38A expression and KCNIP4 splice variant ratio in p38A-SHSY5Y and pMock-SHSY5Y permanently transfected cell lines. (C) Increased β-amyloid secretion and perturbation of the Aβ x-42/Aβ x-40 ratio in 38A-overexpressing SHSY5Y cells. X axis, transfected plasmids; y axis, quantitative determination of Aβ (picograms/milliliter) secreted in the medium 48 h after medium replacement as determined by sandwich ELISA. The resulting Aβ x-42/Aβ x-40 ratio is reported. Striped bars, samples treated with ML-60218 PolIII-specific inhibitor; shaded bars, untreated cell samples. (D) A demonstration that PolIII transcription is specifically inhibited in ML-60218-treated cells, whereas it does not affect PolII activity. 5S rRNA, 7SK RNA, 38A, and c-Myc transcript abundancies were detected by real-time RT-PCR in cells treated with ML-60218 and in untreated controls used as described in A. (E) Quantitative Western blotting analysis of APP and PS2 synthesis in pMock- and/or p38A-transfected cells in ML-60218–treated and –untreated conditions. All of the determinations were normalized to α-tubulin detections. (F and G) Suppression of the biological phenotype driven by the coexpression of 38A and KCNIP4 Var IV–specific silencing plasmid in SHSY5Y cells. Three independent determinations of Var IV decrease and the concomitant increase of Var I expression are shown together with the determinations of Aβ secretion in siVar IV silenced (+) with respect to cells transfected with a scrambled siRNA (−). The error bars were not relevant enough to be visible in the graphs; additional repetitions of the same experiment gave similar results (not depicted).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105541&req=5

fig5: 38A-induced perturbation of Aβ secretion. (A) A schematic view of p38A and pMock plasmid constructs used to generate p38A-SHSY5Y and pMock-SHSY5Y permanently transfected cell lines. (B) Real-time RT-PCR quantitative determination of 38A expression and KCNIP4 splice variant ratio in p38A-SHSY5Y and pMock-SHSY5Y permanently transfected cell lines. (C) Increased β-amyloid secretion and perturbation of the Aβ x-42/Aβ x-40 ratio in 38A-overexpressing SHSY5Y cells. X axis, transfected plasmids; y axis, quantitative determination of Aβ (picograms/milliliter) secreted in the medium 48 h after medium replacement as determined by sandwich ELISA. The resulting Aβ x-42/Aβ x-40 ratio is reported. Striped bars, samples treated with ML-60218 PolIII-specific inhibitor; shaded bars, untreated cell samples. (D) A demonstration that PolIII transcription is specifically inhibited in ML-60218-treated cells, whereas it does not affect PolII activity. 5S rRNA, 7SK RNA, 38A, and c-Myc transcript abundancies were detected by real-time RT-PCR in cells treated with ML-60218 and in untreated controls used as described in A. (E) Quantitative Western blotting analysis of APP and PS2 synthesis in pMock- and/or p38A-transfected cells in ML-60218–treated and –untreated conditions. All of the determinations were normalized to α-tubulin detections. (F and G) Suppression of the biological phenotype driven by the coexpression of 38A and KCNIP4 Var IV–specific silencing plasmid in SHSY5Y cells. Three independent determinations of Var IV decrease and the concomitant increase of Var I expression are shown together with the determinations of Aβ secretion in siVar IV silenced (+) with respect to cells transfected with a scrambled siRNA (−). The error bars were not relevant enough to be visible in the graphs; additional repetitions of the same experiment gave similar results (not depicted).
Mentions: In light of the findings in the previous section and considering that the proteolytic processing of APP plays a central role in AD etiology through the formation of neurotoxic Aβ peptides (Johnson et al., 1990; Selkoe, 1990; Hardy and Selkoe, 2002; St George-Hyslop and Petit, 2005; Haass and Selkoe, 2007), we decided to investigate whether the selective overexpression of 38A ncRNA would affect the formation of Aβ x-42 and/or Aβ x-40 species by measuring their relative amount in the medium of 38A-overexpressing SHSY5Y cells. As Aβ processing might be altered by the transfection procedure, we first generated p38A and pMock permanently transfected cell lines (hereafter referred to as p38A-SHSY5Y and pMock-SHSY5Y, respectively). p38A-SHSY5Y cells, harboring extra copies of the 38A transcription unit with its natural PolIII type 3 promoter, exhibit a moderate overexpression of 38A ncRNA and, as a consequence, a 3.1-fold increased KCNIP4 Var IV/Var I ratio (Fig. 5, A and B). Culture media conditioned for 48 h with p38A-SHSY5Y and/or pMock-SHSY5Y cells were tested for their Aβ peptide composition, showing that 38A significantly enhances the levels of Aβ x-42 (up to 1.91-fold with respect to the pMock-transfected control), whereas its effect on the secretion of the more soluble Aβ x-40 peptide is more modest (up to 1.4-fold with respect to the pMock-transfected control). These results indicate that the transcription of 38A and the concomitant synthesis of KCNIP4 Var IV change both quantitatively and qualitatively the Aβ secretion, leading to an increased amount of the total Aβ and, notably, to a concomitant variation of the Aβ x-42/Aβ x-40 ratio that could account for the altered ratio observed in AD (Fig. 5 C). To further demonstrate the PolIII dependency (and the association with 38A) of the perturbation of Aβ processing, a parallel experiment was performed in the presence of 20 µM ML-60218; in this condition, the increment of both Aβ x-42 and Aβ x-40 was prevented, confirming the role of 38A PolIII–dependent expression in the impairment of Aβ peptide production (Fig. 5 C). In this experiment, the specific inhibition of PolIII was confirmed, evidencing a decreased synthesis of PolIII-transcribed RNAs (5S ribosomal RNA [rRNA], 7SK RNA, and 38A RNA) and a concomitant unaffected expression of PolII-dependent genes (cMyc and glyceraldehyde 3 phosphate dehydrogenase [GAPDH]; Fig. 5 D). Next, to discriminate whether the altered secretion of Aβ in p38A-SHSY5Y cells has to be ascribed to a different level of APP processing or rather to the modulation of APP and/or PS2 synthesis, we measured their amount in the same experimental settings. Results showed that the levels of both APP and PS2 are not affected by the overexpression of 38A, either in the absence or in the presence of ML-60218, demonstrating that the perturbation of Aβ promoted by 38A is specifically determined by the increase of APP processing (Fig. 5 E). This point was also confirmed by measuring Aβ secretion in 38A-transfected APP-overexpressing HEK293 cells that stably overexpress APP. Indeed, although the amount of Aβ secreted is considerably higher in these cells, the effects of 38A overexpression on APP processing recapitulate those obtained in SHSY5Y cells (Fig. S4 F).

Bottom Line: We found that IL1-α-dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III-transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel-interacting protein (KCNIP4).Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels.This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer's disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Biology, and Genetics, National Institute for Cancer Research, 16132 Genoa, Italy.

ABSTRACT
Alternative splicing generates protein isoforms that are conditionally or differentially expressed in specific tissues. The discovery of factors that control alternative splicing might clarify the molecular basis of biological and pathological processes. We found that IL1-α-dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III-transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel-interacting protein (KCNIP4). The alternative KCNIP4 isoform cannot interact with the γ-secretase complex, resulting in modification of γ-secretase activity, amyloid precursor protein processing, and increased secretion of β-amyloid enriched in the more toxic Aβ x-42 species. Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels. This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer's disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain.

Show MeSH
Related in: MedlinePlus