Figure S1 : Limited Proteolysis of the Rpf2-Rrs1 Complex

Legends to Supplementary Figures

Figure S1 : Limited proteolysis of the Rpf2-Rrs1 complex

(A) Purified Rpf2-Rrs1 complex was partially digested using trypsin in 1/1000 mass ratio and incubated at 30°C for 1, 2, 5, 10, 15,20, 30, 45 and 60 minutes. The different peptides obtained were then analyzed by coomassie staining and mass spectrometry. (B) Partially digested peptides recovered after trypsic digestion of Rpf2/Rrs1 complex and solved by X-ray crystallography are indicated underneath full-length proteins. Unstructured loop absent from the solved structure are indicated as dashed squares.

Figure S2 : Electrostatic potential of the Rpf2-rrs1 Complex

Electrostatic potential mapped on the structure of the Rpf2-Rrs1 complex in the same orientation as Figure 1, panel A,B and C.

Figure S3 : Complementary CD analysis

(A) CD spectra of yRNA 5S (dark blue) and bRNA 5S (light blue) scanned in the range of 200 to 300 nm. (B) CD spectra of yRNA 5S obtained when the protein Rpf2 and the yRNA are present in two separate cuvette compartments (dark purple) and after mixing the two compartments (light purple). (C) CD spectra of yRNA 5S obtained when the protein Rrs1 and the yRNA are present in two separate cuvette compartments (dark pink) and after mixing the two compartments (light pink).

(D) Filter-binding assay of fluorescently labelled wild-type E-loop (WT) or G77U mutant (G77U) to Rpf2/Rrs1 complex. Free RNA molecules hybridize to nylon filter (unBound) and RNA associated to different concentration of proteins (0, 25, 50, 100, 250, 1000 and 2000nM) are retained on protran filter (Bound). (E) Secondary structure of E-loop used in the filter-binding assay. Position of the fluorescent label and two additional residues used to strengthen the loop are indicated as a circled F and with a grey color respectively.

Figure S4: Comparison of Rpf2 5S RNA binding with TFIIIA and aaRS proteins

(A) Superposition of TFIIIA (PDB 1UN6) and Rpf2-Rrs1 bound to 5S shows that binding is mutually exclusive. TFIIIA is represented in yellow. (B) The threonyl-tRNA synthetase-tRNA(Thr) complex (PDB 1Q6F) was superposed on the BID domain of Rpf2. The Rpf2-Rrs1 complex is omitted for clarity. The binding of the aaRS anticodon binding domain to tRNA shows a different binding structure compared to the 5S RNA.

Figure S5: Rpf2 binding to 5S RNA is not compatible with its conformation in mature ribosomes.

The Proteins are colored as in Figure 5. The Rpf2-Rrs1 complex is superposed on the 5S RNP of mature 60S structure (Ben-Shem et al., 2011). The ribosome rRNA is represented in surface representation, the proteins have been omitted for clarity.

Figure S6: Rpf2 C-terminal Domain is required for proper binding to pre-60S ribosomes

(A) Drop test analysis of expression of Rpf2 full-length (FL), Rpf2 N-terminal domain (NTD, 1-252) and Rpf2 C-terminal domain (CTD, 252-344) mutants. Effect on growth of the expression of the different mutants was analysed either in presence (galactose) or in absence (glucose) of the endogenous copy of RPF2. (B,C and D) To test the capacity of the different mutants of RPF2-HTP, an immuno-precipitation experiment was performed using extract from cells grown in absence of the endogenous copy (Glucose). (B) Correct expression and precipitations of the different proteins was assessed by western-blot using anti-PAP antibodies. (C) RNAs co-precipitated with the different mutants were analysed on acrylamide gel stained by Ethidium Bromide, extract from cell transformed with empty vector was used as control. 5S and 5.8S species are indicated. (D) Ratio between percentage of 5S and 5.8S bound to the different Rpf2 mutants were quantified and reported as bars or as not determined (ND) when no amount was detected. Standard deviations between the different experiments are reported as error bars.