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SUPPLEMENTAL INFORMATION

A Conserved Motif within Rap1 Plays Diversified Roles in Telomere Protection and Regulation in Different Organisms

Yong Chen1,2,10, Rekha Rai3,9,10, Zi-Ren Zhou4, Junko Kanoh5, Cyril Ribeyre6, Yuting Yang1,2, Hong Zheng3, Pascal Damay6, Feng Wang1,2, Hisayo Tsujii 5, Yasushi Hiraoka7, David Shore6, Hong-Yu Hu4, Sandy Chang3,8,9, Ming Lei1,2

1Howard Hughes Medical Institute, 2Department of Biological Chemistry, University of Michigan Medical School, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA 3Department of Genetics, The M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA 4State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 5Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan 6Department of Molecular Biology and NCCR Program 'Frontiers in Genetics', University of Geneva, Sciences III, Geneva, Switzerland 7Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan 8Department of Hematopathology, The M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA 9Present address; Dept. of Laboratory Medicine, Yale University School of Medicine, 330 Cedar St., New Haven, CT 06520

*These authors contribute equally to this work.

Correspondence and requests for materials should be addressed to M.L. ([email protected]) and S.C. ([email protected]).

Nature Structural & Molecular Biology: doi:10.1038/nsmb.1974

Supplementary Figure 1. Structural studies of the TRF2RBM and RAP1RCT complex. (a) TRF2RBM and RAP1RCT form a stable complex in solution. Left panel: gel filtration chromatography profile (Hiload Superdex 75) of the TRF2RBM-RAP1RCT complex. Right panel: SDS-PAGE of the TRF2RBM-RAP1RCT complex corresponding to the peak fraction in the gel filtration profile. (b) Four different orthogonal views of the TRF2RBM-RAP1RCT complex. TRF2RBM is in yellow ribbon and mesh representations and RAP1RCT in green surface representation.


Supplementary Figure 2. Mutational studies of the TRF2RBM and RAP1RCT interaction. (a) ITC data of the wild-type and mutant TRF2RBM-RAP1RCT interactions. (b) Effects of the TRF2 and RAP1 mutations on the TRF2-RAP1 interaction in a yeast two-hybrid assay. Interaction of LexA-TRF2 with GAD-RAP1 was measured as β-galactosidase activity. Data are average of three independent β-galactosidase measurements normalized to the wild-type TRF2-RAP1 interaction, arbitrarily set to 100. (c) Co-IP data show that the TRF2 L288R mutant still recruits Apollo to telomeres. 293T cells were transiently transfected with the indicated constructs, and IPs were performed with the anti-Myc antibody. IPs were analyzed by immunoblotting for interaction with Apollo (upper panel)


and for protein expression (bottom panel) with the indicated antibodies. Lanes marked ‘‘In’’ represent 5% of in the input lysate used for the IPs. (d) and (e) Mutations on the TRF2-RAP1 interface have different effects on the telomeric localization of TRF2 and RAP1. (d) Localization of transiently expressed HA-tagged wild-type RAP1 and the I318R and F336R mutants of RAP1 when co-transfected with wild-type Myc-TRF2 in HeLa cells. (e) Localization of transiently expressed Myc-tagged wild-type TRF2 and the TRF2 L288R mutant in HeLa cells. (f) Western blots showed that all the HA-RAP1 proteins were overexpressed at comparable levels in HeLa cells with different combinations of TRF2 and RAP1 mutations.


Supplementary Figure 3. Telomere localization of murine Rap1 requires Trf2. (a) Protein levels of endogenous Trf1 and Rap1 and the phosphorylation level of Chk2 in vector control, Trf2-shRNA-treated MEFs, or Trf2-shRNA-treated MEFs expressing Trf2 or Trf2 L286R. (b) and (c) Telomere localization of retrovirally expressed wild-type and the L286R mutant of Trf2 (b) and endogenous Rap1 (c) in Trf2-shRNA-treated MEFs. Wild-type Trf2 or Trf2 L286R expression was detected by anti-Trf2 antibody. Endogenous Rap1 was detected by anti-Rap1 antibody. IF-FISH was performed using a TAM-OO-(CCCTAA)4 PNA telomere probe.


Supplementary Figure 4. Expression of a chimeric protein Rap1-Trf2 L286R rescued the telomere fusion phenotype observed in Trf2-shRNA-treated TRf2 L286R expressing cells. (a) Chimeric Rap1-Trf2 L286R construct used in the in vivo assay. Domains of Trf2 and Rap1 are colored as in Fig. 1a. (b) MEFs expressing the Rap1-Trf2 L286R cDNA were treated with Trf2 shRNA, metaphases prepared and telomere fusions were visualized by telomere PNA-FISH (red) and DAPI (blue). (c) Quantification of the percent of cells with five or more γ-H2AX-positive TIFs from representative images shown in b and Fig. 3c. Error bars, s.d.; n ≥ 300 nuclei analyzed per sample. * P < 0.005 calculated using a two-tailed Student's t-test. (d) Quantification of the percent of mataphases with end-to-end telomere fusions from representative images shown in b and Fig. 3c. Error bars, s.d.; n > 1,600 telomeres analyzed per sample. * P < 0.005 calculated using a two-tailed Student's t-test.


Supplementary Figure 5. Structural study of the fission yeast Taz1RBM-SpRap1RCT complex. (a) The 1H-15N HSQC spectrum of the Taz1RBM-SpRap1RCT complex. (b) Ribbon diagram of the 20 lowest-energy NMR structures superimposed using backbone heavy atoms. Sir3RBM is colored in orange and SpRap1RCT in cyan. (c) Gel filtration chromatographic analyses of the Taz1RBM-SpRap1RCT fusion protein and the Rap1RCT-Taz1RBM complex (co-expressed and co-purified from E. coli) showed that the fusion


protein migrated at roughly the same position as the complex, indicating that the stable cores of the fusion protein and the complex have similar conformations. (d) The final calculated 20 NMR structures show that the linker region (in red) is flexible and long enough so that the interacting cores of both Taz1RBM and SpRap1RCT are well folded with unique conformations. The RMSD of the cores of Taz1RBM and SpRap1RCT are 0.51 Å for backbone atoms and 1.46 Å for heavy atoms. (e) The Taz1RBM helix binds in a hydrophobic groove formed by helices α1 and α2 of SpRap1RCT. The Taz1RBM binding site of SpRap1RCT is shown in surface representation and colored according to its electrostatic surface potential (positive potential, blue; negative potential, red).


Supplementary Figure 6. Mutational analyses of the Taz1-SpRap1 interaction. (a) and (b) Western blot showed that all the mutant Taz1 (a) and SpRap1 (b) proteins were expressed at near wild-type levels in yeast cells, suggesting that residues at the Taz1-


SpRap1 interface are not required for protein stability. Anti-GFP was used to detect GFP tagged Taz1 proteins and anti-SpRap1 was used to detect SpRap1. (c) Colocalization analysis of wild-type or mutant GFP-tagged SpRap1 and mCherry-tagged Taz1 in yeasts cells in the presence or absence of Poz1. (d) Quantification of the percentile of cells that show SpRap1 foci in wild-type, taz1Δ, poz1Δ, or taz1Δpoz1Δ cells from representative images shown in c. (e) Quantification of the percentile of cells with SpRap1-GFP colocalized with Taz1-2mCherry from representative images shown in c.


Supplementary Figure 7. Structural study of the budding yeast Sir3RBM-ScRap1RCT complex. (a) The Sir3RBM-ScRap1RCT complex with a 1:2 ratio in the asymmetric unit. The two ScRap1RCT molecules are colored in light cyan (ScRap1RCT-A) and light blue


(ScRap1RCT-B), respectively. The simulated annealing omit map shows that residues 461-480 of Sir3RBM are ordered. The refined model of the Sir3RBM peptide is superimposed on the map. Contours are draw at the 1.0 σ level. The peptide is shown as a stick representation with carbon colored in salmon, nitrogen blue, and oxygen red. (b) In vitro ITC measurements of the interaction between Sir3RBM with two deletion mutants of ScRap1RCT (left panel: Sir3RBMΔC; right panel: Sir3RBMΔN) (nd: not detectable by ITC). (c) The complementary surfaces of Sir3RBM and ScRap1RCT around Gly760 of ScRap1RCT. The surface of Sir3RBM is colored in light orange. ScRap1RCT in both ribbon and mesh representations is colored in light cyan. ScRap1RCT Gly760 is in sphere model with carbon in green, nitrogen in blue, and oxygen in red.


Supplementary Table 1. S. pombe strains used in this study.

Strain Genotype

JK317 h- leu1-32 ura4-D18

JK325 h- leu1-32 ura4-D18 rad3::ura4+ JK702 h- leu1-32 ura4-D18 taz1::ura4+

TM1513 h- leu1-32 ura4-D18 lig4::ura4+ (from F. Ishikawa) JK3111 h- leu1-32 ura4-D18 rad3::kanr taz1::ura4+

HT343 h- leu1-32 ura4-D18 rap1::ura4+ JP400 h- leu1-32 ura4-D18 taz1-I379R

JP401 h- leu1-32 ura4-D18 taz1-L380R JP402 h- leu1-32 ura4-D18 taz1-L383R

JP403 h- leu1-32 ura4-D18 taz1-V387R JP407 h- leu1-32 ura4-D18 rap1-V651R

JP408 h- leu1-32 ura4-D18 rap1-I655R HT430 h- leu1-32 ura4-D18 taz1-GFP:kanr

HT432 h- leu1-32 ura4-D18 taz1-I379R-GFP:kanr HT434 h- leu1-32 ura4-D18 taz1-L380R-GFP:kanr

HT436 h- leu1-32 ura4-D18 taz1-L383R-GFP:kanr HT438 h- leu1-32 ura4-D18 taz1-V387R-GFP:kanr

JP500 h- leu1-32 ura4-D18 lig4::ura4+ taz1:: hygr JP503 h- leu1-32 ura4-D18 lig4::ura4+ rap1::hygr

JP506 h- leu1-32 ura4-D18 rad3::kanr rap1::ura4+ JP514 h- leu1-32 ura4-D18 exo1::hygr

JP516 h- leu1-32 ura4-D18 exo1::hygr taz1::ura4+ JP519 h- leu1-32 ura4-D18 exo1::hygr rap1::ura4+

JP708 h- leu1-32 ura4-D18 taz1::ura4+ rap1-GFP:kanr JP734 h- leu1-32 ura4-D18 taz1-2mCherry:hygr rap1-GFP:kanr

JP736 h- leu1-32 ura4-D18 taz1-I379R-2mCherry:hygr rap1-GFP:kanr JP738 h- leu1-32 ura4-D18 taz1-L380R-2mCherry:hygr rap1-GFP:kanr

JP740 h- leu1-32 ura4-D18 taz1-L383R-2mCherry:hygr rap1-GFP:kanr JP742 h- leu1-32 ura4-D18 taz1-V387R-2mCherry:hygr rap1-GFP:kanr


JP744 h- leu1-32 ura4-D18 taz1-2mCherry:hygr rap1-V651R-GFP:kanr JP746 h- leu1-32 ura4-D18 taz1-2mCherry:hygr rap1-I655R-GFP:kanr

JP753 h- leu1-32 ura4-D18 poz1::ura4+ taz1-2mCherry:hygr rap1-GFP:kanr JP755 h- leu1-32 ura4-D18 poz1:: hygr taz1::ura4+ rap1-GFP:kanr ___________________________________________________________________


supplemental information a conserved motif within rap1 ... · supplemental information a conserved...

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