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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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Increased APP processing in 38A-overexpressing cells. (A) Quantitative Western blotting analysis of APP C-terminal fragments in pMock- and/or p38A-transfected cells (P = 0.023, as the averaged result of five experiments). Error bars represent SD. (B) Quantitative determination of APP and PS2 synthesis in pMock- and/or p38A-transfected cells by Western blotting analysis. All of the determinations were normalized to α-tubulin expression. Error bars represent SD. (C) Determination of APP processing by pGAL4-activated luciferase assay: c−, pLuc + pRL were transfected; pMock, pGal4-APP + pGAL4-Luc + pMock + pRL were transfected; and p38A, pGal4-APP + pGAL4-Luc + p38A + pRL were transfected.
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fig6: Increased APP processing in 38A-overexpressing cells. (A) Quantitative Western blotting analysis of APP C-terminal fragments in pMock- and/or p38A-transfected cells (P = 0.023, as the averaged result of five experiments). Error bars represent SD. (B) Quantitative determination of APP and PS2 synthesis in pMock- and/or p38A-transfected cells by Western blotting analysis. All of the determinations were normalized to α-tubulin expression. Error bars represent SD. (C) Determination of APP processing by pGAL4-activated luciferase assay: c−, pLuc + pRL were transfected; pMock, pGal4-APP + pGAL4-Luc + pMock + pRL were transfected; and p38A, pGal4-APP + pGAL4-Luc + p38A + pRL were transfected.

Mentions: To validate the hypothesis that 38A affects Aβ secretion by altering APP processing, we tested the possible change of the C-terminal fragments profile generated by APP cleavage in 38A-overexpressing cells. To better detect these small C-terminal fragments in SDS-PAGE, we transiently transfected the 38A-expressing plasmid in HEK293-APP cells that harbor extra copies of the APP transcription unit. As shown in Fig. 6 A, 48 h after transfection, p38A–HEK293-APP cells are characterized by a significant increase of both α and β APP C-terminal fragments detected at 10 and 12 kD, respectively, and generated by an increased processing of APP (2.82-fold).


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)

Increased APP processing in 38A-overexpressing cells. (A) Quantitative Western blotting analysis of APP C-terminal fragments in pMock- and/or p38A-transfected cells (P = 0.023, as the averaged result of five experiments). Error bars represent SD. (B) Quantitative determination of APP and PS2 synthesis in pMock- and/or p38A-transfected cells by Western blotting analysis. All of the determinations were normalized to α-tubulin expression. Error bars represent SD. (C) Determination of APP processing by pGAL4-activated luciferase assay: c−, pLuc + pRL were transfected; pMock, pGal4-APP + pGAL4-Luc + pMock + pRL were transfected; and p38A, pGal4-APP + pGAL4-Luc + p38A + pRL were transfected.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: Increased APP processing in 38A-overexpressing cells. (A) Quantitative Western blotting analysis of APP C-terminal fragments in pMock- and/or p38A-transfected cells (P = 0.023, as the averaged result of five experiments). Error bars represent SD. (B) Quantitative determination of APP and PS2 synthesis in pMock- and/or p38A-transfected cells by Western blotting analysis. All of the determinations were normalized to α-tubulin expression. Error bars represent SD. (C) Determination of APP processing by pGAL4-activated luciferase assay: c−, pLuc + pRL were transfected; pMock, pGal4-APP + pGAL4-Luc + pMock + pRL were transfected; and p38A, pGal4-APP + pGAL4-Luc + p38A + pRL were transfected.
Mentions: To validate the hypothesis that 38A affects Aβ secretion by altering APP processing, we tested the possible change of the C-terminal fragments profile generated by APP cleavage in 38A-overexpressing cells. To better detect these small C-terminal fragments in SDS-PAGE, we transiently transfected the 38A-expressing plasmid in HEK293-APP cells that harbor extra copies of the APP transcription unit. As shown in Fig. 6 A, 48 h after transfection, p38A–HEK293-APP cells are characterized by a significant increase of both α and β APP C-terminal fragments detected at 10 and 12 kD, respectively, and generated by an increased processing of APP (2.82-fold).

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