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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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Effects of 38A expression on A-type potassium current. (A–F) Outward potassium current in pMock-transfected control cells (A, C, and E), in 38A-overexpressing cells (B), in pETmKChIP4a (D), and in pMaLmKIS-transfected cells (F). Step depolarizations were delivered with 20-mV increments. The holding potential was −80 mV. The transient overexpression of the recombinant KCNIP4 Var IV is reported in D as resulting from real-time RT-PCR analysis. The overexpression of the recombinant N-terminal (KIS) domain is reported in F, as resulting from real-time RT-PCR analysis. Error bars are SD.
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fig3: Effects of 38A expression on A-type potassium current. (A–F) Outward potassium current in pMock-transfected control cells (A, C, and E), in 38A-overexpressing cells (B), in pETmKChIP4a (D), and in pMaLmKIS-transfected cells (F). Step depolarizations were delivered with 20-mV increments. The holding potential was −80 mV. The transient overexpression of the recombinant KCNIP4 Var IV is reported in D as resulting from real-time RT-PCR analysis. The overexpression of the recombinant N-terminal (KIS) domain is reported in F, as resulting from real-time RT-PCR analysis. Error bars are SD.

Mentions: KCNIP4 Var IV is known to influence the cell excitatory properties by abolishing the fast inactivation of A-type potassium channels on which rapid neuron firing depends (Baranauskas, 2004). Such alteration has been associated with neurodegenerative processes (Angulo et al., 2004; Waters et al., 2006). To test whether 38A expression can perturb the excitatory properties of synapses in neurons, we first searched for the proper cell culture system for the determination of the A-type current. SKNBE-NDM29 cells (a SKNBE clone with characteristics of neurons; Castelnuovo et al., 2010) turned out to be a system in which this current is well detected. We transfected these cells with plasmids harboring 38A under the control of its natural promoter (pEGFP-N1–38A) and/or without insert (pEGFP-N1). 48 h after transfection, we measured the inactivation time in a large panel of cells for each sample. Fig. 3 shows the outward potassium current elicited in control (Fig. 3 A) and in 38A cells (Fig. 3 B) by depolarization steps from a −80-mV holding potential. The inactivation time course of this current could be described by a single exponential curve. In 75% of 38A cells, the time constant of the inactivating potassium current was increased. The time constant of the current elicited by depolarizing steps to 60 mV was found to be close to 1,130 ms in the 38A cells as opposed to the time constant (580 ms) of the control cell current. Considering the transfection efficiency (70–80%), virtually all of the 38A-expressing cells are affected. When the same cells were transfected with constructs expressing the alternatively spliced KCNIP4 Var IV and/or the N-terminal KIS domain specifically coded by Var IV, both conditions produced the inactivation of potassium current, recapitulating the same effect elicited by 38A overexpression (Fig. 3, C–F). Altogether, these data demonstrate that 38A can abolish the fast inactivation of the A-type voltage-dependent potassium channels, thus affecting the transient component of their A-type current. Considering that this phenomenon significantly affects the excitatory potentiality of the cell, we concluded that the activation of 38A expression could be associated with an altered function/state of synapses, as it may occur in neurological disorders.


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)

Effects of 38A expression on A-type potassium current. (A–F) Outward potassium current in pMock-transfected control cells (A, C, and E), in 38A-overexpressing cells (B), in pETmKChIP4a (D), and in pMaLmKIS-transfected cells (F). Step depolarizations were delivered with 20-mV increments. The holding potential was −80 mV. The transient overexpression of the recombinant KCNIP4 Var IV is reported in D as resulting from real-time RT-PCR analysis. The overexpression of the recombinant N-terminal (KIS) domain is reported in F, as resulting from real-time RT-PCR analysis. Error bars are SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Effects of 38A expression on A-type potassium current. (A–F) Outward potassium current in pMock-transfected control cells (A, C, and E), in 38A-overexpressing cells (B), in pETmKChIP4a (D), and in pMaLmKIS-transfected cells (F). Step depolarizations were delivered with 20-mV increments. The holding potential was −80 mV. The transient overexpression of the recombinant KCNIP4 Var IV is reported in D as resulting from real-time RT-PCR analysis. The overexpression of the recombinant N-terminal (KIS) domain is reported in F, as resulting from real-time RT-PCR analysis. Error bars are SD.
Mentions: KCNIP4 Var IV is known to influence the cell excitatory properties by abolishing the fast inactivation of A-type potassium channels on which rapid neuron firing depends (Baranauskas, 2004). Such alteration has been associated with neurodegenerative processes (Angulo et al., 2004; Waters et al., 2006). To test whether 38A expression can perturb the excitatory properties of synapses in neurons, we first searched for the proper cell culture system for the determination of the A-type current. SKNBE-NDM29 cells (a SKNBE clone with characteristics of neurons; Castelnuovo et al., 2010) turned out to be a system in which this current is well detected. We transfected these cells with plasmids harboring 38A under the control of its natural promoter (pEGFP-N1–38A) and/or without insert (pEGFP-N1). 48 h after transfection, we measured the inactivation time in a large panel of cells for each sample. Fig. 3 shows the outward potassium current elicited in control (Fig. 3 A) and in 38A cells (Fig. 3 B) by depolarization steps from a −80-mV holding potential. The inactivation time course of this current could be described by a single exponential curve. In 75% of 38A cells, the time constant of the inactivating potassium current was increased. The time constant of the current elicited by depolarizing steps to 60 mV was found to be close to 1,130 ms in the 38A cells as opposed to the time constant (580 ms) of the control cell current. Considering the transfection efficiency (70–80%), virtually all of the 38A-expressing cells are affected. When the same cells were transfected with constructs expressing the alternatively spliced KCNIP4 Var IV and/or the N-terminal KIS domain specifically coded by Var IV, both conditions produced the inactivation of potassium current, recapitulating the same effect elicited by 38A overexpression (Fig. 3, C–F). Altogether, these data demonstrate that 38A can abolish the fast inactivation of the A-type voltage-dependent potassium channels, thus affecting the transient component of their A-type current. Considering that this phenomenon significantly affects the excitatory potentiality of the cell, we concluded that the activation of 38A expression could be associated with an altered function/state of synapses, as it may occur in neurological disorders.

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