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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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In vitro transcription analysis of 38A. (A) Transcription products of 38A or empty vector (pNEB) were either radiolabeled during synthesis and directly visualized (lanes 1 and 2) or subjected to primer extension analysis (lanes 3–6; lane 3, no DNA; lane 5, no reverse transcription). In lane 6, the main primer extension product is indicated by a red arrowhead. Lanes 7–10 are sequencing reactions primed with the same oligonucleotide. The sequence of the 38A transcription unit is reported (PSE, TATA, and terminator in red, transcription start site in blue, and transcribed region underlined). (B) In vitro transcription and primer extension analysis with the human 7SK gene (lanes 1 and 10), promoterless 7SK (lanes 3 and 9), and the 7SK transcribed region fused with 38A promoter (lanes 2 and 8) as templates. The sequence of the hybrid transcription unit is reported (PSE, TATA, and terminator sequences in red and the 38A-derived upstream sequence in italic). The red arrowhead indicates the position of the most abundant primer extension product. (C) ChIP analysis in SHSY5Y-cultured cells using anti-PolIII, anti-SNAPc, or anti-Brf2 antibodies. Association with the GAPDH control gene and either the U6 gene (lanes 1–4) or the 38A locus (lanes 5–8) was assessed by PCR on both input (lanes 4 and 8) and immunoprecipitated DNA samples interrogated with both target-specific and control primer pairs. Target enrichment in the immunoprecipitated DNA is reported below the lanes. (A and B) Black lines indicate that intervening lanes have been spliced out. Molecular mass is indicated in base pairs..
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fig1: In vitro transcription analysis of 38A. (A) Transcription products of 38A or empty vector (pNEB) were either radiolabeled during synthesis and directly visualized (lanes 1 and 2) or subjected to primer extension analysis (lanes 3–6; lane 3, no DNA; lane 5, no reverse transcription). In lane 6, the main primer extension product is indicated by a red arrowhead. Lanes 7–10 are sequencing reactions primed with the same oligonucleotide. The sequence of the 38A transcription unit is reported (PSE, TATA, and terminator in red, transcription start site in blue, and transcribed region underlined). (B) In vitro transcription and primer extension analysis with the human 7SK gene (lanes 1 and 10), promoterless 7SK (lanes 3 and 9), and the 7SK transcribed region fused with 38A promoter (lanes 2 and 8) as templates. The sequence of the hybrid transcription unit is reported (PSE, TATA, and terminator sequences in red and the 38A-derived upstream sequence in italic). The red arrowhead indicates the position of the most abundant primer extension product. (C) ChIP analysis in SHSY5Y-cultured cells using anti-PolIII, anti-SNAPc, or anti-Brf2 antibodies. Association with the GAPDH control gene and either the U6 gene (lanes 1–4) or the 38A locus (lanes 5–8) was assessed by PCR on both input (lanes 4 and 8) and immunoprecipitated DNA samples interrogated with both target-specific and control primer pairs. Target enrichment in the immunoprecipitated DNA is reported below the lanes. (A and B) Black lines indicate that intervening lanes have been spliced out. Molecular mass is indicated in base pairs..

Mentions: The putative 38A ncRNA gene was identified in a previous computational screen for novel human PolIII target genes (Pagano et al., 2007). First, we verified 38A activity as a PolIII template in vitro using a HeLa cell nuclear extract. As shown in Fig. 1 A, in vitro transcription of a plasmid-borne 38A construct containing the upstream proximal sequence element (PSE) and the TATA box, followed by the putative 352-bp transcribed region, produced RNAs of different sizes (lane 2); primer extension analysis (lanes 3–10) revealed that transcription initiated ∼30 bp downstream of the TATA box. As further shown in Fig. 1 B, the 38A promoter region was also found to be active in directing faithful 7SK gene transcription in fusion constructs. The association of the 38A transcription unit with PolIII-specific transcription proteins was tested in vivo by chromatin immunoprecipitation (ChIP) analysis. Fig. 1 C shows the results of a ChIP analysis conducted on SHSY5Y-cultured cells using anti-Brf2 (a TFIIIB component), anti-SNAP43 (a SNAP-C component), and anti-RPC39 (a PolIII subunit) antibodies. 38A was found to be specifically enriched in all the immunoprecipitations.


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)

In vitro transcription analysis of 38A. (A) Transcription products of 38A or empty vector (pNEB) were either radiolabeled during synthesis and directly visualized (lanes 1 and 2) or subjected to primer extension analysis (lanes 3–6; lane 3, no DNA; lane 5, no reverse transcription). In lane 6, the main primer extension product is indicated by a red arrowhead. Lanes 7–10 are sequencing reactions primed with the same oligonucleotide. The sequence of the 38A transcription unit is reported (PSE, TATA, and terminator in red, transcription start site in blue, and transcribed region underlined). (B) In vitro transcription and primer extension analysis with the human 7SK gene (lanes 1 and 10), promoterless 7SK (lanes 3 and 9), and the 7SK transcribed region fused with 38A promoter (lanes 2 and 8) as templates. The sequence of the hybrid transcription unit is reported (PSE, TATA, and terminator sequences in red and the 38A-derived upstream sequence in italic). The red arrowhead indicates the position of the most abundant primer extension product. (C) ChIP analysis in SHSY5Y-cultured cells using anti-PolIII, anti-SNAPc, or anti-Brf2 antibodies. Association with the GAPDH control gene and either the U6 gene (lanes 1–4) or the 38A locus (lanes 5–8) was assessed by PCR on both input (lanes 4 and 8) and immunoprecipitated DNA samples interrogated with both target-specific and control primer pairs. Target enrichment in the immunoprecipitated DNA is reported below the lanes. (A and B) Black lines indicate that intervening lanes have been spliced out. Molecular mass is indicated in base pairs..
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3105541&req=5

fig1: In vitro transcription analysis of 38A. (A) Transcription products of 38A or empty vector (pNEB) were either radiolabeled during synthesis and directly visualized (lanes 1 and 2) or subjected to primer extension analysis (lanes 3–6; lane 3, no DNA; lane 5, no reverse transcription). In lane 6, the main primer extension product is indicated by a red arrowhead. Lanes 7–10 are sequencing reactions primed with the same oligonucleotide. The sequence of the 38A transcription unit is reported (PSE, TATA, and terminator in red, transcription start site in blue, and transcribed region underlined). (B) In vitro transcription and primer extension analysis with the human 7SK gene (lanes 1 and 10), promoterless 7SK (lanes 3 and 9), and the 7SK transcribed region fused with 38A promoter (lanes 2 and 8) as templates. The sequence of the hybrid transcription unit is reported (PSE, TATA, and terminator sequences in red and the 38A-derived upstream sequence in italic). The red arrowhead indicates the position of the most abundant primer extension product. (C) ChIP analysis in SHSY5Y-cultured cells using anti-PolIII, anti-SNAPc, or anti-Brf2 antibodies. Association with the GAPDH control gene and either the U6 gene (lanes 1–4) or the 38A locus (lanes 5–8) was assessed by PCR on both input (lanes 4 and 8) and immunoprecipitated DNA samples interrogated with both target-specific and control primer pairs. Target enrichment in the immunoprecipitated DNA is reported below the lanes. (A and B) Black lines indicate that intervening lanes have been spliced out. Molecular mass is indicated in base pairs..
Mentions: The putative 38A ncRNA gene was identified in a previous computational screen for novel human PolIII target genes (Pagano et al., 2007). First, we verified 38A activity as a PolIII template in vitro using a HeLa cell nuclear extract. As shown in Fig. 1 A, in vitro transcription of a plasmid-borne 38A construct containing the upstream proximal sequence element (PSE) and the TATA box, followed by the putative 352-bp transcribed region, produced RNAs of different sizes (lane 2); primer extension analysis (lanes 3–10) revealed that transcription initiated ∼30 bp downstream of the TATA box. As further shown in Fig. 1 B, the 38A promoter region was also found to be active in directing faithful 7SK gene transcription in fusion constructs. The association of the 38A transcription unit with PolIII-specific transcription proteins was tested in vivo by chromatin immunoprecipitation (ChIP) analysis. Fig. 1 C shows the results of a ChIP analysis conducted on SHSY5Y-cultured cells using anti-Brf2 (a TFIIIB component), anti-SNAP43 (a SNAP-C component), and anti-RPC39 (a PolIII subunit) antibodies. 38A was found to be specifically enriched in all the immunoprecipitations.

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