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The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA.

Reiter V, Matschkal DM, Wagner M, Globisch D, Kneuttinger AC, Müller M, Carell T - Nucleic Acids Res. (2012)

Bottom Line: CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics.This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist.Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Ludwig-Maximilians University, Munich 81377, Germany.

ABSTRACT
The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer's disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i(6)A) into 2-methylthio-N6-isopentenyladenosine (ms(2)i(6)A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.

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Correlation of ms2i6A content with mitochondrial activity. (a) Content of ms2i6A in tRNA of various pork tissues, sorted by descending amount. Highest values in red, lowest in green. (b) Cytochrome C oxidase activity in the same tissues. For comparison, the same color code was used. (c) Correlation plot of COX activity versus ms2 i 6A content.
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gks240-F2: Correlation of ms2i6A content with mitochondrial activity. (a) Content of ms2i6A in tRNA of various pork tissues, sorted by descending amount. Highest values in red, lowest in green. (b) Cytochrome C oxidase activity in the same tissues. For comparison, the same color code was used. (c) Correlation plot of COX activity versus ms2 i 6A content.

Mentions: The presence of ms2i6A in mitochondrial tRNA was shown previously (21), and we have further verified its localization. In order to investigate the role of ms2i6A for mitochondrial activity, we isolated tRNA from various pork tissues, fully digested these RNA fractions and analyzed the nucleoside mixture by LC-MS (Supplementary Methods). Precise quantification of ms2i6A in these lysates was enabled by adding a synthetic, isotope-labeled ms2i6A standard to the mixture as described recently by us in detail (17). As a marker for mitochondrial activity, we quantified the activity of cytochrome C oxidase (COX). The results summarized in Figure 2 clearly show a strong correlation with mitochondrial activity. Tissues with a high-energy demand like muscle and brain tissue feature also the highest concentrations of ms2i6A. We further enriched mitochondria from porcine heart and quantified ms2i6A in both mitochondrial and cytosolic tRNA. A strong enrichment was achieved in preparations of mitochondria (80 modifications per 1000 tRNA) as compared to cytosolic tRNA (6 per 1000). Residual ms2i6A in these preparations likely originates from ruptured mitochondria. This data is taken from Ref. (14).Figure 2.


The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA.

Reiter V, Matschkal DM, Wagner M, Globisch D, Kneuttinger AC, Müller M, Carell T - Nucleic Acids Res. (2012)

Correlation of ms2i6A content with mitochondrial activity. (a) Content of ms2i6A in tRNA of various pork tissues, sorted by descending amount. Highest values in red, lowest in green. (b) Cytochrome C oxidase activity in the same tissues. For comparison, the same color code was used. (c) Correlation plot of COX activity versus ms2 i 6A content.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401430&req=5

gks240-F2: Correlation of ms2i6A content with mitochondrial activity. (a) Content of ms2i6A in tRNA of various pork tissues, sorted by descending amount. Highest values in red, lowest in green. (b) Cytochrome C oxidase activity in the same tissues. For comparison, the same color code was used. (c) Correlation plot of COX activity versus ms2 i 6A content.
Mentions: The presence of ms2i6A in mitochondrial tRNA was shown previously (21), and we have further verified its localization. In order to investigate the role of ms2i6A for mitochondrial activity, we isolated tRNA from various pork tissues, fully digested these RNA fractions and analyzed the nucleoside mixture by LC-MS (Supplementary Methods). Precise quantification of ms2i6A in these lysates was enabled by adding a synthetic, isotope-labeled ms2i6A standard to the mixture as described recently by us in detail (17). As a marker for mitochondrial activity, we quantified the activity of cytochrome C oxidase (COX). The results summarized in Figure 2 clearly show a strong correlation with mitochondrial activity. Tissues with a high-energy demand like muscle and brain tissue feature also the highest concentrations of ms2i6A. We further enriched mitochondria from porcine heart and quantified ms2i6A in both mitochondrial and cytosolic tRNA. A strong enrichment was achieved in preparations of mitochondria (80 modifications per 1000 tRNA) as compared to cytosolic tRNA (6 per 1000). Residual ms2i6A in these preparations likely originates from ruptured mitochondria. This data is taken from Ref. (14).Figure 2.

Bottom Line: CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics.This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist.Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Ludwig-Maximilians University, Munich 81377, Germany.

ABSTRACT
The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer's disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i(6)A) into 2-methylthio-N6-isopentenyladenosine (ms(2)i(6)A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.

Show MeSH
Related in: MedlinePlus