Supplementary Figure 2: CRAC analysis of bound rRNA.

(a) In vitro CRAC of recombinant His-tagged Mex67-Mtr2 reconstituted onto the purified Yvh1-FTP particle. Yvh1-FTP was incubated without or with His-tagged Mex67-Mtr2. Rsa4-FTP served as control for unspecific binding by also incubating it with His-tagged Mex67-Mtr2. After binding and washing, 10% of the flag eluates (the rest was used for in vitro UV crosslinking) were analyzed by SDS-PAGE and Coomassie staining. Asterisk (*) marks the position of the respective Coomassie stained bait protein and arrowhead shows the in vitro bound Mex67 band. (b) Autoradiogram of covalent His-Mex67-His-Mtr2::RNA complex after UV-crosslinking (1h exposure). Digested RNA crosslinked to His-tagged Mex67-Mtr2 bait was end-labeled with γ³²P-ATP. Red box shows the migration of the protein:RNA complex on the gel, which was later excised and used for RNA extraction, cDNA amplification and sequencing. (c) Same as (b), but the contrast was increased via FIJI image manipulation tool (Schindelin, J. et al., Nat Methods 9, 676-682, 2012). A background Mex67 signal can be also seen in the Rsa4-FTP lane. (d) Comparison between Mex67 in vivo CRAC data (Tuck, A.C. et al., Cell. 154, 996-1009, 2013) (lower panel) and the in vitro Mex67 CRAC data (upper panel). Total number of hits (from the in vitro CRAC data) was plotted along with the published in vivo CRAC data against the relative position along the rDNA sequence. The Y-axis is not normalized, as the two different data sets were not analyzed at the same time. The two specific sites, where Mex67-Mtr2 was significantly crosslinked to the Yvh1 particle, are labeled with '5.8S hit' and 'P0 hit'.