Limits...
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.

Show MeSH

Related in: MedlinePlus

Lack of KCNIP4–PS2 protein–protein interaction in 38A-overexpressing cells. (A) PS2 is detected by anti-PS2 IgGs in samples immunoprecipitated (IP) with anti-KCNIP4 in pMock-transfected cells, whereas PS2-KCNIP4 coimmunoprecipitation is prevented in 38A-overexpressing cells (P = 0.023, as the averaged result of five experiments). In the same samples, the lack of immunoprecipitation of KCNIP4 with a different component of the γ-secretase complex (PS1) is shown. (B) The 38A-dependent impairment of PS2/KCNIP4 protein–protein interaction is prevented by the treatment of cells with the PolIII inhibitor ML-60218. (C) Input sample demonstrating that the amount of PS2 and PS1 in the protein sample is not affected by 38A overexpression. (D) Immunofluorescence detection of KCNIP4 Var I and Var IV by splice variant–specific antibodies in pMock-transfected cells (M) and in 38A-overexpressing cells (38A). Bars, 100 µm. (E) KCNIP4 Var I and PS2 signals in 38A-overexpressing cells. Bars, 100 µm. (F) The KCNIP4 Var IV–specific antiserum does not evidence a colocalization of KCNIP4 and PS2. Bars, 100 µm. (G) Western blot quantitative analysis of the PS1 and KChIP3 amount in Mock (M) and 38A-overexpressing (38A) cells. (H) Immunofluorescence detection of PS1 and KChIP3 in Mock (M) and 38A-overexpressing (38A) cells. α-PS1, α–anti-PS1; α-KChIP3, α–anti-KChIP3. (I) Immunofluorescence detection of PS2 and KChIP3 in Mock (M) and 38A-overexpressing (38A) cells. Bars, 100 µm. The quantification of fluorescence signals shown in D–F, H, and I is reported in the corresponding histograms D′–F′, H′, and I′. The error bars (referred to the mean of 10 microscope fields) were not relevant enough to be visible in the graphs. α-PS2, α–anti-PS2.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Lack of KCNIP4–PS2 protein–protein interaction in 38A-overexpressing cells. (A) PS2 is detected by anti-PS2 IgGs in samples immunoprecipitated (IP) with anti-KCNIP4 in pMock-transfected cells, whereas PS2-KCNIP4 coimmunoprecipitation is prevented in 38A-overexpressing cells (P = 0.023, as the averaged result of five experiments). In the same samples, the lack of immunoprecipitation of KCNIP4 with a different component of the γ-secretase complex (PS1) is shown. (B) The 38A-dependent impairment of PS2/KCNIP4 protein–protein interaction is prevented by the treatment of cells with the PolIII inhibitor ML-60218. (C) Input sample demonstrating that the amount of PS2 and PS1 in the protein sample is not affected by 38A overexpression. (D) Immunofluorescence detection of KCNIP4 Var I and Var IV by splice variant–specific antibodies in pMock-transfected cells (M) and in 38A-overexpressing cells (38A). Bars, 100 µm. (E) KCNIP4 Var I and PS2 signals in 38A-overexpressing cells. Bars, 100 µm. (F) The KCNIP4 Var IV–specific antiserum does not evidence a colocalization of KCNIP4 and PS2. Bars, 100 µm. (G) Western blot quantitative analysis of the PS1 and KChIP3 amount in Mock (M) and 38A-overexpressing (38A) cells. (H) Immunofluorescence detection of PS1 and KChIP3 in Mock (M) and 38A-overexpressing (38A) cells. α-PS1, α–anti-PS1; α-KChIP3, α–anti-KChIP3. (I) Immunofluorescence detection of PS2 and KChIP3 in Mock (M) and 38A-overexpressing (38A) cells. Bars, 100 µm. The quantification of fluorescence signals shown in D–F, H, and I is reported in the corresponding histograms D′–F′, H′, and I′. The error bars (referred to the mean of 10 microscope fields) were not relevant enough to be visible in the graphs. α-PS2, α–anti-PS2.

Mentions: Because it has been previously reported that KCNIP4 interacts with PS2 (NCBI Protein accession no. P49810; Herreman et al., 2000; Morohashi et al., 2002; Bentahir et al., 2006), we tested the two alternative KCNIP4 protein forms for their possible interaction with PS2. 48 h after transfection of SHSY5Y cells with p38A or pMock constructs, we immunoprecipitated KCNIP4 by the KChIP4 L-14 IgGs and analyzed the immunoprecipitation products by SDS-PAGE coupled with Western blotting challenged by the anti-PS2 antibody (PS2 H-76). In pMock-transfected cells, PS2 coimmunoprecipitates with KCNIP4, confirming the previously reported physical interaction (Morohashi et al., 2002). Interestingly, the PS2 signal was significantly decreased in p38A-transfected cells, where KCNIP4 Var IV is predominantly synthesized and recognized by the same antiserum; as the control, another component of the γ-secretase complex (PS1) is not coimmunoprecipitated with KCNIP4 (Fig. 4 A).


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)

Lack of KCNIP4–PS2 protein–protein interaction in 38A-overexpressing cells. (A) PS2 is detected by anti-PS2 IgGs in samples immunoprecipitated (IP) with anti-KCNIP4 in pMock-transfected cells, whereas PS2-KCNIP4 coimmunoprecipitation is prevented in 38A-overexpressing cells (P = 0.023, as the averaged result of five experiments). In the same samples, the lack of immunoprecipitation of KCNIP4 with a different component of the γ-secretase complex (PS1) is shown. (B) The 38A-dependent impairment of PS2/KCNIP4 protein–protein interaction is prevented by the treatment of cells with the PolIII inhibitor ML-60218. (C) Input sample demonstrating that the amount of PS2 and PS1 in the protein sample is not affected by 38A overexpression. (D) Immunofluorescence detection of KCNIP4 Var I and Var IV by splice variant–specific antibodies in pMock-transfected cells (M) and in 38A-overexpressing cells (38A). Bars, 100 µm. (E) KCNIP4 Var I and PS2 signals in 38A-overexpressing cells. Bars, 100 µm. (F) The KCNIP4 Var IV–specific antiserum does not evidence a colocalization of KCNIP4 and PS2. Bars, 100 µm. (G) Western blot quantitative analysis of the PS1 and KChIP3 amount in Mock (M) and 38A-overexpressing (38A) cells. (H) Immunofluorescence detection of PS1 and KChIP3 in Mock (M) and 38A-overexpressing (38A) cells. α-PS1, α–anti-PS1; α-KChIP3, α–anti-KChIP3. (I) Immunofluorescence detection of PS2 and KChIP3 in Mock (M) and 38A-overexpressing (38A) cells. Bars, 100 µm. The quantification of fluorescence signals shown in D–F, H, and I is reported in the corresponding histograms D′–F′, H′, and I′. The error bars (referred to the mean of 10 microscope fields) were not relevant enough to be visible in the graphs. α-PS2, α–anti-PS2.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Lack of KCNIP4–PS2 protein–protein interaction in 38A-overexpressing cells. (A) PS2 is detected by anti-PS2 IgGs in samples immunoprecipitated (IP) with anti-KCNIP4 in pMock-transfected cells, whereas PS2-KCNIP4 coimmunoprecipitation is prevented in 38A-overexpressing cells (P = 0.023, as the averaged result of five experiments). In the same samples, the lack of immunoprecipitation of KCNIP4 with a different component of the γ-secretase complex (PS1) is shown. (B) The 38A-dependent impairment of PS2/KCNIP4 protein–protein interaction is prevented by the treatment of cells with the PolIII inhibitor ML-60218. (C) Input sample demonstrating that the amount of PS2 and PS1 in the protein sample is not affected by 38A overexpression. (D) Immunofluorescence detection of KCNIP4 Var I and Var IV by splice variant–specific antibodies in pMock-transfected cells (M) and in 38A-overexpressing cells (38A). Bars, 100 µm. (E) KCNIP4 Var I and PS2 signals in 38A-overexpressing cells. Bars, 100 µm. (F) The KCNIP4 Var IV–specific antiserum does not evidence a colocalization of KCNIP4 and PS2. Bars, 100 µm. (G) Western blot quantitative analysis of the PS1 and KChIP3 amount in Mock (M) and 38A-overexpressing (38A) cells. (H) Immunofluorescence detection of PS1 and KChIP3 in Mock (M) and 38A-overexpressing (38A) cells. α-PS1, α–anti-PS1; α-KChIP3, α–anti-KChIP3. (I) Immunofluorescence detection of PS2 and KChIP3 in Mock (M) and 38A-overexpressing (38A) cells. Bars, 100 µm. The quantification of fluorescence signals shown in D–F, H, and I is reported in the corresponding histograms D′–F′, H′, and I′. The error bars (referred to the mean of 10 microscope fields) were not relevant enough to be visible in the graphs. α-PS2, α–anti-PS2.
Mentions: Because it has been previously reported that KCNIP4 interacts with PS2 (NCBI Protein accession no. P49810; Herreman et al., 2000; Morohashi et al., 2002; Bentahir et al., 2006), we tested the two alternative KCNIP4 protein forms for their possible interaction with PS2. 48 h after transfection of SHSY5Y cells with p38A or pMock constructs, we immunoprecipitated KCNIP4 by the KChIP4 L-14 IgGs and analyzed the immunoprecipitation products by SDS-PAGE coupled with Western blotting challenged by the anti-PS2 antibody (PS2 H-76). In pMock-transfected cells, PS2 coimmunoprecipitates with KCNIP4, confirming the previously reported physical interaction (Morohashi et al., 2002). Interestingly, the PS2 signal was significantly decreased in p38A-transfected cells, where KCNIP4 Var IV is predominantly synthesized and recognized by the same antiserum; as the control, another component of the γ-secretase complex (PS1) is not coimmunoprecipitated with KCNIP4 (Fig. 4 A).

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