Journal: Nucleic Acids Research

Article Title: The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA

doi: 10.1093/nar/gks240

Figure Lengend Snippet: 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.

Article Snippet: GFP-reporter plasmids for the subcellular localization of the human CDK5RAP1 variants were obtained from Origene (RG216600 and RG208724).

Techniques: Sequencing, Transfection, Incubation, Staining

Journal: Nucleic Acids Research

Article Title: The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA

doi: 10.1093/nar/gks240

Figure Lengend Snippet: CDK5RAP1 is the human homolog of the bacterial protein MiaB. ( a ) Complementation assay in E. coli. Quantification of m 6 A, i 6 A and ms 2 i 6 A in tRNA of wild-type E. coli , a MiaB-deficient strain and a MiaB-deficient strain complemented with CDK5RAP1. m 6 A was used as internal standard and levels did not change during the experiment. i 6 A level increased in ΔMiaB and compared to WT, because of lack of conversion to ms 2 i 6 A. ΔMiaB + CDK5RAP1 shows a decreased i 6 A level in comparison to ΔMiaB, generated by the complementation. By the expression of CDK5RAP1, the ms 2 i 6 A level increases 15-fold in comparison to ΔMiaB. ( b ) siRNA knockdown in HeLa cells. Cells were transfected twice with esiRNA against CDK5RAP1. Non-coding esiRNA against EGFP served as a control. After 72 h, total RNA was extracted and the modification content was determined by LC-MS. m 6 A and i 6 A remain constant, while ms 2 i 6 A decreased ∼78%.

Article Snippet: GFP-reporter plasmids for the subcellular localization of the human CDK5RAP1 variants were obtained from Origene (RG216600 and RG208724).

Techniques: Comparison, Generated, Expressing, Knockdown, Transfection, esiRNA, Control, Modification, Liquid Chromatography with Mass Spectroscopy