P. Böck-Taferner, H. Wank
GAPDH enhances group II intron splicing in vitro
Group II introns are autocatalytic RNAs which selfsplice
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 bI1
group II intron (domains IVVI) 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 bI1 group II intron by up
to three times. However, in vivo GAPDH is not a group II
intronsplicing 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.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 385, 07/2004
Pages: 615 - 621
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