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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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Subcelluar distribution of CDK5RAP1 variants. (a) Representation of the two investigated CDK5RAP1 variants with mitochondrial import sequence and catalytic domain. (b) Live-cell images of HeLa cells transfected with C-terminally GFP-tagged CDK5RAP1-v1 and CDK5RAP1-v2. For the counterstain of mitochondria, cells were incubated with MitoTracker Red dye. The merge shows that CDK5RAP1-v1 colocalizes with mitochondria, while CDK5RAP1-v2 is distributed in both cytoplasm and nucleus. (c) Formaldehyde-fixed HeLa cells were stained with an antibody against the N-terminal region of CDK5RAP1-v2 (Alexa-488, green) and against CDK5 (Alexa-555, red). The localization of CDK5RAP1 is predominantly nuclear with little overlap to CDK5. (d) Again, fixed HeLa cells were stained with anti-CDK5RAP1 (green). For localization of nucleic acids, propidium iodide (red) was used. CDK5RAP1 shows an association with the mitotic spindle.
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gks240-F1: Subcelluar distribution of CDK5RAP1 variants. (a) Representation of the two investigated CDK5RAP1 variants with mitochondrial import sequence and catalytic domain. (b) Live-cell images of HeLa cells transfected with C-terminally GFP-tagged CDK5RAP1-v1 and CDK5RAP1-v2. For the counterstain of mitochondria, cells were incubated with MitoTracker Red dye. The merge shows that CDK5RAP1-v1 colocalizes with mitochondria, while CDK5RAP1-v2 is distributed in both cytoplasm and nucleus. (c) Formaldehyde-fixed HeLa cells were stained with an antibody against the N-terminal region of CDK5RAP1-v2 (Alexa-488, green) and against CDK5 (Alexa-555, red). The localization of CDK5RAP1 is predominantly nuclear with little overlap to CDK5. (d) Again, fixed HeLa cells were stained with anti-CDK5RAP1 (green). For localization of nucleic acids, propidium iodide (red) was used. CDK5RAP1 shows an association with the mitotic spindle.

Mentions: In order to unravel the unusual link between CDK5 kinase activity and RNA modification chemistry, we investigated the catalytic properties and the cellular distribution of CDK5RAP1, which exists in different splice variants (18). Variant 1 contains an N-terminal extension that is by computational methods predicted to comprise a mitochondrial import signal (score = 0.647, TargetP1.1) (19), while variant 2 lacks this extension (Figure 1a). To analyze the subcellular distribution of both splicing variants, we transfected HeLa cells with GFP fusions of these two CDK5RAP1 splice variants. The confocal analysis clearly shows a mitochondrial localization of variant 1. In order to secure the interpretation we performed a colocalization with a mitochondrial counterstain and indeed observed the colocalization of variant 1 and the MitoTracker (Figure 1b). Variant 2, which is lacking the putative import signal, we found distributed in both cytoplasm and nucleus, respectively (Figure 1b), with the stronger signal appearing in the cytoplasm. This distribution was confirmed by immunocytochemical (ICC) staining of CDK5RAP1, however with a stronger preference for nuclear localization (Figure 1c). Interestingly, in both live-cell imaging and ICC methods, CDK5RAP1 appeared to be excluded from the nucleoli. This is a somehow surprising observation since the nucleoli are the compartment where RNA processing mainly occurs. ICC staining with an antibody against CDK5 showed that there is no apparent colocalization of CDK5 with CDK5RAP1 (Figure 1c). This is in accordance with CDK5RAP1s mode of CDK5-inhibition by preventing formation of the activated CDK5 complex. In some cells, CDK5RAP1 shows an association with the mitotic spindle (Figure 1d). Such localization was previously shown also for CDK5RAP2 (20), the second CDK5R1-binding protein identified in the yeast two hybrid screen. This result suggests that CDK5RAP1 might be interacting with cell cycle regulating mitotic CDKs as well. The mitochondrial localization of CDK5RAP1 variant 1 is in full agreement with the suggested MiaB-like function in ms2i6A biosynthesis in mitochondrial tRNA.Figure 1.


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)

Subcelluar distribution of CDK5RAP1 variants. (a) Representation of the two investigated CDK5RAP1 variants with mitochondrial import sequence and catalytic domain. (b) Live-cell images of HeLa cells transfected with C-terminally GFP-tagged CDK5RAP1-v1 and CDK5RAP1-v2. For the counterstain of mitochondria, cells were incubated with MitoTracker Red dye. The merge shows that CDK5RAP1-v1 colocalizes with mitochondria, while CDK5RAP1-v2 is distributed in both cytoplasm and nucleus. (c) Formaldehyde-fixed HeLa cells were stained with an antibody against the N-terminal region of CDK5RAP1-v2 (Alexa-488, green) and against CDK5 (Alexa-555, red). The localization of CDK5RAP1 is predominantly nuclear with little overlap to CDK5. (d) Again, fixed HeLa cells were stained with anti-CDK5RAP1 (green). For localization of nucleic acids, propidium iodide (red) was used. CDK5RAP1 shows an association with the mitotic spindle.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gks240-F1: Subcelluar distribution of CDK5RAP1 variants. (a) Representation of the two investigated CDK5RAP1 variants with mitochondrial import sequence and catalytic domain. (b) Live-cell images of HeLa cells transfected with C-terminally GFP-tagged CDK5RAP1-v1 and CDK5RAP1-v2. For the counterstain of mitochondria, cells were incubated with MitoTracker Red dye. The merge shows that CDK5RAP1-v1 colocalizes with mitochondria, while CDK5RAP1-v2 is distributed in both cytoplasm and nucleus. (c) Formaldehyde-fixed HeLa cells were stained with an antibody against the N-terminal region of CDK5RAP1-v2 (Alexa-488, green) and against CDK5 (Alexa-555, red). The localization of CDK5RAP1 is predominantly nuclear with little overlap to CDK5. (d) Again, fixed HeLa cells were stained with anti-CDK5RAP1 (green). For localization of nucleic acids, propidium iodide (red) was used. CDK5RAP1 shows an association with the mitotic spindle.
Mentions: In order to unravel the unusual link between CDK5 kinase activity and RNA modification chemistry, we investigated the catalytic properties and the cellular distribution of CDK5RAP1, which exists in different splice variants (18). Variant 1 contains an N-terminal extension that is by computational methods predicted to comprise a mitochondrial import signal (score = 0.647, TargetP1.1) (19), while variant 2 lacks this extension (Figure 1a). To analyze the subcellular distribution of both splicing variants, we transfected HeLa cells with GFP fusions of these two CDK5RAP1 splice variants. The confocal analysis clearly shows a mitochondrial localization of variant 1. In order to secure the interpretation we performed a colocalization with a mitochondrial counterstain and indeed observed the colocalization of variant 1 and the MitoTracker (Figure 1b). Variant 2, which is lacking the putative import signal, we found distributed in both cytoplasm and nucleus, respectively (Figure 1b), with the stronger signal appearing in the cytoplasm. This distribution was confirmed by immunocytochemical (ICC) staining of CDK5RAP1, however with a stronger preference for nuclear localization (Figure 1c). Interestingly, in both live-cell imaging and ICC methods, CDK5RAP1 appeared to be excluded from the nucleoli. This is a somehow surprising observation since the nucleoli are the compartment where RNA processing mainly occurs. ICC staining with an antibody against CDK5 showed that there is no apparent colocalization of CDK5 with CDK5RAP1 (Figure 1c). This is in accordance with CDK5RAP1s mode of CDK5-inhibition by preventing formation of the activated CDK5 complex. In some cells, CDK5RAP1 shows an association with the mitotic spindle (Figure 1d). Such localization was previously shown also for CDK5RAP2 (20), the second CDK5R1-binding protein identified in the yeast two hybrid screen. This result suggests that CDK5RAP1 might be interacting with cell cycle regulating mitotic CDKs as well. The mitochondrial localization of CDK5RAP1 variant 1 is in full agreement with the suggested MiaB-like function in ms2i6A biosynthesis in mitochondrial tRNA.Figure 1.

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