Figure 2

Gis2p preferentially binds to coding sequences that bear GAN repeats. (a) Conserved sequence element in the ORF of Gis2p targets identified with MEME. The E-value reflects the probability to detect the motif by chance. (b) RNA-protein complexes formed between biotinylated RNA fragments and Gis2-TAP were purified on streptavidin beads and monitored by immunoblot analysis. Representative experiments from at least three biological replicates are shown. Biotin labeled fragments comprising the 5'-UTRs (lanes 2 and 5), ORFs (lanes 3 and 6), and 3'-UTRs (lanes 4 and 7) of Erv25 and Fcy1 were incubated with extracts of Gis2-TAP expressing cells (lane 1). Eno2-5'UTR (lane 8) is a negative control RNA derived from the 'non-target' ENO2 (lane 8) and a sample without RNA (lane 9) was used to control for RNA-independent binding to the beads. (c) RNA pull-downs with RNA fragments derived from NOP53. Nop53-GAN (lanes 3 and 7 to 9) contains a GAN-rich sequence element whereas the similarly sized fragment Nop53-ctrl does not (lanes 4 and 10). Erv25-ORF (lane 2) and Eno2-ORF (lane 5) are positive and negative control RNAs, respectively. Binding of Gis2-TAP to Nop53-GAN was competed with a ten-fold excess of non-biotinylated Nop53-GAN (lane 8) but not with excess of Nop53-ctrl (lane 9). (d) RNA pull-downs with two fragments derived from the RAS2 ORF. Biotinylated RNAs were incubated with extracts from yeast cells expressing Gis2-TAP (eGis2-TAP, lanes 1 to 3) or with Gis2-His expressed and purified from Escherichia coli (pGis2-His, lanes 4 and 5). A fragment derived from the ORF of SNF5 was used as a negative control RNA (lane 3).