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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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ncRNA-induced alternative splicing analysis. (A) Real-time RT-PCR detection of KCNIP4 splice variant synthesis in SHSY5Y and/or in SKNBE cells transiently transfected with either 38A or 17A (a noncorrelated PolIII-transcribed ncRNA used as a negative control) expression plasmids. Altogether, these results indicate that each ncRNA specifically influences the splicing of its corresponding protein-coding gene. (B) Western blotting analysis of the 38A-dependent alternative splicing of KCNIP4. In pMock-transfected cells, the relative abundance of the 250-residue-long form of KCNIP4 (Var I, striped bars) is predominant with respect to the alternatively spliced 229-residue-long form (Var IV, shaded bars) that, in turn, is the main KCNIP4 form synthesized by p38A-transfected cells. Quantitative bars and the correspondent error bars (SD) are referred to the mean of three independent experimental determinations (A and B). The p38A-dependent change of Var IV versus Var I protein form ratio is quantitatively reported (inset in B). (C and D) Immunofluorescence detection of KCNIP4 alternative splicing. Antibodies raised against the KCNIP4 N-terminal fragment of the protein form I show a clearly detectable signal in GFP-expressing cells only in the absence of a concomitant 38A overexpression (arrows in C); on the contrary, the signal is absent or very weak in cells transfected with a construct coexpressing 38A and GFP (arrows in D). Untransfected cells, which do not express GFP and 38A, are positive for KCNIP4 (arrowheads in D).
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fig2: ncRNA-induced alternative splicing analysis. (A) Real-time RT-PCR detection of KCNIP4 splice variant synthesis in SHSY5Y and/or in SKNBE cells transiently transfected with either 38A or 17A (a noncorrelated PolIII-transcribed ncRNA used as a negative control) expression plasmids. Altogether, these results indicate that each ncRNA specifically influences the splicing of its corresponding protein-coding gene. (B) Western blotting analysis of the 38A-dependent alternative splicing of KCNIP4. In pMock-transfected cells, the relative abundance of the 250-residue-long form of KCNIP4 (Var I, striped bars) is predominant with respect to the alternatively spliced 229-residue-long form (Var IV, shaded bars) that, in turn, is the main KCNIP4 form synthesized by p38A-transfected cells. Quantitative bars and the correspondent error bars (SD) are referred to the mean of three independent experimental determinations (A and B). The p38A-dependent change of Var IV versus Var I protein form ratio is quantitatively reported (inset in B). (C and D) Immunofluorescence detection of KCNIP4 alternative splicing. Antibodies raised against the KCNIP4 N-terminal fragment of the protein form I show a clearly detectable signal in GFP-expressing cells only in the absence of a concomitant 38A overexpression (arrows in C); on the contrary, the signal is absent or very weak in cells transfected with a construct coexpressing 38A and GFP (arrows in D). Untransfected cells, which do not express GFP and 38A, are positive for KCNIP4 (arrowheads in D).

Mentions: We then tested whether the overexpression of 38A causes a gene-specific alternative splicing shift of KCNIP4. To this end, we focused on the two physiologically relevant splice variants Var I and Var IV. We transiently transfected SHSY5Y and SKNBE neuroblastoma cells (Biedler et al., 1973; Biedler and Spengler, 1976) with a construct expressing 38A transcript (driven by its natural promoter) and measured changes in the levels of Var I and Var IV mRNAs by real-time RT-PCR. As shown in Fig. 2 A, 48 h after transfection, a remarkable alteration of Var IV versus Var I ratio was detected (21.1 vs. 1) in SHSY5Y cells as a consequence of an increased generation of Var IV and a decreased synthesis of Var I. The same result was obtained with SKNBE cells, although this cell type transcribes 38A less efficiently. Overexpression of 17A, a distinct PolIII-transcribed regulatory RNA that maps in an unrelated locus, did not exert any effect on KCNIP4 pre-mRNA maturation. Altogether, these results indicate that 38A overexpression is sufficient to drive the alternative splicing shift of KCNIP4 mRNAs, promoting the synthesis of the Var IV isoform. With respect to Var I, this isoform encompasses a KIS domain in its N terminus that enables it to inhibit the fast kinetics of inactivation of A-type voltage-dependent potassium channels (Holmqvist et al., 2002).


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)

ncRNA-induced alternative splicing analysis. (A) Real-time RT-PCR detection of KCNIP4 splice variant synthesis in SHSY5Y and/or in SKNBE cells transiently transfected with either 38A or 17A (a noncorrelated PolIII-transcribed ncRNA used as a negative control) expression plasmids. Altogether, these results indicate that each ncRNA specifically influences the splicing of its corresponding protein-coding gene. (B) Western blotting analysis of the 38A-dependent alternative splicing of KCNIP4. In pMock-transfected cells, the relative abundance of the 250-residue-long form of KCNIP4 (Var I, striped bars) is predominant with respect to the alternatively spliced 229-residue-long form (Var IV, shaded bars) that, in turn, is the main KCNIP4 form synthesized by p38A-transfected cells. Quantitative bars and the correspondent error bars (SD) are referred to the mean of three independent experimental determinations (A and B). The p38A-dependent change of Var IV versus Var I protein form ratio is quantitatively reported (inset in B). (C and D) Immunofluorescence detection of KCNIP4 alternative splicing. Antibodies raised against the KCNIP4 N-terminal fragment of the protein form I show a clearly detectable signal in GFP-expressing cells only in the absence of a concomitant 38A overexpression (arrows in C); on the contrary, the signal is absent or very weak in cells transfected with a construct coexpressing 38A and GFP (arrows in D). Untransfected cells, which do not express GFP and 38A, are positive for KCNIP4 (arrowheads in D).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: ncRNA-induced alternative splicing analysis. (A) Real-time RT-PCR detection of KCNIP4 splice variant synthesis in SHSY5Y and/or in SKNBE cells transiently transfected with either 38A or 17A (a noncorrelated PolIII-transcribed ncRNA used as a negative control) expression plasmids. Altogether, these results indicate that each ncRNA specifically influences the splicing of its corresponding protein-coding gene. (B) Western blotting analysis of the 38A-dependent alternative splicing of KCNIP4. In pMock-transfected cells, the relative abundance of the 250-residue-long form of KCNIP4 (Var I, striped bars) is predominant with respect to the alternatively spliced 229-residue-long form (Var IV, shaded bars) that, in turn, is the main KCNIP4 form synthesized by p38A-transfected cells. Quantitative bars and the correspondent error bars (SD) are referred to the mean of three independent experimental determinations (A and B). The p38A-dependent change of Var IV versus Var I protein form ratio is quantitatively reported (inset in B). (C and D) Immunofluorescence detection of KCNIP4 alternative splicing. Antibodies raised against the KCNIP4 N-terminal fragment of the protein form I show a clearly detectable signal in GFP-expressing cells only in the absence of a concomitant 38A overexpression (arrows in C); on the contrary, the signal is absent or very weak in cells transfected with a construct coexpressing 38A and GFP (arrows in D). Untransfected cells, which do not express GFP and 38A, are positive for KCNIP4 (arrowheads in D).
Mentions: We then tested whether the overexpression of 38A causes a gene-specific alternative splicing shift of KCNIP4. To this end, we focused on the two physiologically relevant splice variants Var I and Var IV. We transiently transfected SHSY5Y and SKNBE neuroblastoma cells (Biedler et al., 1973; Biedler and Spengler, 1976) with a construct expressing 38A transcript (driven by its natural promoter) and measured changes in the levels of Var I and Var IV mRNAs by real-time RT-PCR. As shown in Fig. 2 A, 48 h after transfection, a remarkable alteration of Var IV versus Var I ratio was detected (21.1 vs. 1) in SHSY5Y cells as a consequence of an increased generation of Var IV and a decreased synthesis of Var I. The same result was obtained with SKNBE cells, although this cell type transcribes 38A less efficiently. Overexpression of 17A, a distinct PolIII-transcribed regulatory RNA that maps in an unrelated locus, did not exert any effect on KCNIP4 pre-mRNA maturation. Altogether, these results indicate that 38A overexpression is sufficient to drive the alternative splicing shift of KCNIP4 mRNAs, promoting the synthesis of the Var IV isoform. With respect to Var I, this isoform encompasses a KIS domain in its N terminus that enables it to inhibit the fast kinetics of inactivation of A-type voltage-dependent potassium channels (Holmqvist et al., 2002).

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