GAPDH enhances group II intron splicing in vitro.
Group II introns are autocatalytic RNAs which self-splice in vitro. However, in vivo additional protein factors might be involved in the splicing process. We used an affinity chromatography method called 'StreptoTag' to identify group II intron binding proteins from Saccharomyces cerevisiae. This method uses a hybrid RNA consisting of a streptomycin-binding affinity tag and the RNA of interest, which is bound to a streptomycin column and incubated with yeast protein extract. After several washing steps the bound RNPs are eluted by addition of streptomycin. The eluted RNPs are separated and the proteins identified by mass-spectrometric analysis. Using crude extract from yeast in combination with a substructure of the bl1 group II intron (domains IV-VI) we were able to identify four glycolytic enzymes; glucose-6-phosphate isomerase (GPI), 3-phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI). From these proteins GAPDH increases in vitro splicing of the bl1 group II intron by up to three times. However, in vivo GAPDH is not a group II intron-splicing factor, since it is not localised in yeast mitochondria. Therefore, the observed activity reflects an unexpected property of GAPDH. Band shift experiments and UV cross linking demonstrated the interaction of GAPDH with the group II intron RNA. This novel activity expands the reaction repertoire of GAPDH to a new RNA species.
Böck-Taferner
Petra
P
Max F. Perutz Laboratories, University of Vienna, Department of Microbiology and Genetics, Vienna Biocenter, Dr. Bohrgasse 9/4, A-1030 Vienna, Austria.
Wank
Herbert
H
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005956:Glucose-6-Phosphate Isomerase / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D007438:Introns / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D012326:RNA Splicing / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D013307:Streptomycin / Q000737:chemistry; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000378:metabolism / Q000502:physiology
D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010735:Phosphoglycerate Kinase; D014305:Triose-Phosphate Isomerase; D005956:Glucose-6-Phosphate Isomerase; D013307:Streptomycin