supplementary information to the manuscript …genesdev.cshlp.org › content › suppl › 2014 ›...

SUPPLEMENTARY INFORMATION TO THE MANUSCRIPT ENTITLED:!

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A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains!

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Michael Gamalinda1,6, Uli Ohmayer2,6, Jelena Jakovljevic1, Beril Kumcuoglu1, Joshua

Woolford1,3, Bertrade Mbom1,4, Lawrence Lin1,5, and John L. Woolford, Jr.1,7

1 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA

2 Lehrstuhl für Biochemie III, Universität Regensburg, Regensburg, Germany

3 Present address: Department of Epidemiology, Tulane University, New Orleans, LA,

USA

4 Present address: Department of Molecular and Cellular Physiology, Stanford

University, Stanford, CA, USA

5 Present address: Department of Bioengineering and Therapeutic Sciences, University

of California, San Francisco, San Francisco, CA, USA

6 These authors contributed equally to this manuscript

7 Corresponding author

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+ +

A0 A1 A2

A3 B1L

B1s

C2 C2

C1 C1

5 ETS 3 ETS ITS1 ITS2

35S

27SA2 20S

27SA3

27SBS 27SBL

7SS 25.5S 7SL 25.5S

18S 5.8SS 25S 5.8SL 25S

E E

85-90% 10-15%

6SS 25S 6SL 25S D 20S

6SS 25S 6SL 25S D 20S

EA

RLY

M

IDD

LE

LATE

Gamalinda_SuppFig1

Gamalinda_SuppFig2 A. B.

C.

GAL-RPL16 NOP7-TAP

GAL-RPL33 NOP7-TAP

GAL-RPL4 RPF2-TAP

GAL-RPL6 RPF2-TAP

GAL-RPL7 RPF2-TAP

GAL-RPL8 RPF2-TAP

GAL-RPL20 RPF2-TAP

GAL-RPL32 RPF2-TAP

GAL-RPL13 RPF2-TAP

GAL-RPL17 NOP7-TAP

GAL-RPL19 NOP7-TAP

GAL-RPL25 NOP7-TAP

GAL-RPL26 NOP7-TAP

GAL-RPL27 NOP7-TAP

GAL-RPL31 NOP7-TAP

GAL-RPL35 NOP7-TAP

GAL-RPL37 NOP7-TAP

GAL-RPL2 NOP7-TAP

GAL-RPL11 NOP7-TAP

GAL-RPL21 NOP7-TAP

GAL-RPL43 NOP7-TAP

Gal Glu Gal Glu Gal Glu Gal Glu Gal Glu Gal Glu Gal Glu Gal Glu Gal Glu

Gal Glu Gal Glu Gal Glu Gal Glu Gal Glu Gal Glu Gal Glu Gal Glu

Gal Glu Gal Glu Gal Glu Gal Glu

Gamalinda_SuppFig3

Gal Glu

GAL-RPL28 NOP7-TAP

Nop4!

Nop7!

Ebp2!

Cic1!

Has1!

Tif6!

Nog1!

Nip7!

Rlp24!

Nsa2!

Nog2!

L3!

L4!

L11!

L17!

L25!

GAL-L20!RPF2-TAP!

GAL-L27!NOP7-TAP!

GAL-L32!RPF2-TAP!

GAL-L25!NOP7-TAP!

Gal! Glu! Gal! Glu! Gal! Glu! Gal! Glu!

L5!

GAL-L2!NOP7-TAP!Gal! Glu!

L28-MYC!

L14-HA!

L33-HA!

L21-MYC!

L20-MYC!

L6-MYC!

GAL-L18!RPF2-TAP!Gal! Glu!

A.

B.

Gamalinda_SuppFig4 GAL-L28!NOP7-TAP!Gal! Glu!

GAL-RPL18!RPF2-TAP!

GAL-RPL17!NOP7-TAP!

GAL-RPL11!NOP7-TAP!

Gamalinda_SuppFig5

A.

B.

Gamalinda_SuppFig6

C.

5’-5.8S

3’-25S 5’-5.8S 3’-25S

A. B.

Gamalinda_SuppFig7

L26$ L17$

L35$

L4$L16$

L25$

L2$

L3$

L11$

L1$

L5$

L12$

L28$

L10$P0$

L23$

L9$

L7$

5S$

23S rRNA

CP CP

P-stalk

L1-stalk

Subunit Interface

L1-stalk

P-stalk

Solvent Interface

Gamalinda_SuppFig8

A.

B.

180°

L5 L11

(L32) L35

(L9) (L9)

L5 L11

L28

L4 (L21) (L20)

L16

L3 L17 L26

L9

L12 (L25)

L7

(L27)

(L35)

(L33)

L25 (L17)

(L33)

(L27) L10

(L25)

L12

L9

L2 L23

(L19)

Supplementary Table 1. Summary of contacts of individual RPLs with rRNA sequences within 3.5Å. Phenotypic class Maturation step blocked RPL

Interaction with rRNA domains (% of nucleotides within 3.5Å)

5.8S I II III IV V VI 5S

Early

Cleavage of 27SA2 pre-rRNA at the A3 site

L3

0 4 0 0 9 21 66 0

Processing of 27SA3 pre-rRNA

L4 3 22 71 0 0 4 0 0 L6 0 0 66 0 0 0 34 0 L7 0 0 85 0 0 0 0 15 L8 18 29 0 6 0 47 0 0 L13 3 46 46 0 0 5 0 0 L16 0 0 43 0 0 9 48 0 L18 0 5 82 0 0 12 0 0 L20 0 0 55 0 0 0 14 31 L32 0 0 100 0 0 0 0 0 L33 0 0 64 0 0 0 36 0

Middle Cleavage of 27SB pre-rRNA at the C2 site

L9 0 0 6 0 0 19 74 0 L17 13 16 16 11 13 7 24 0 L19 0 0 14 51 27 0 8 0 L23 0 0 0 0 39 26 35 0 L25 41 16 0 35 0 8 0 0 L26 43 57 0 0 0 0 0 0 L27 0 0 0 74 0 26 0 0 L31 0 0 0 32 9 0 59 0 L34 0 2 0 91 0 7 0 0 L35 50 50 0 0 0 0 0 0 L37 36 25 25 11 4 0 0 0

Late

Processing of 7S pre-rRNA

L2 0 0 15 8 47 29 0 0 L43 0 0 45 24 28 3 0 0

Processing of 6S pre-rRNA and/or cytoplasmic release of export factors

L5 47 0 0 53 0 0 0 0 L10 0 0 46 0 0 43 0 11 L11 0 0 0 0 0 52 0 48 L21 0 0 34 0 0 57 0 9 L28 0 19 66 0 0 15 0 0 L40 0 0 20 0 0 40 40 0

Supplementary,Table,2.,Effect,of,depleting,individual,RPLs,to,pre=ribosomal,levels,of,assembly,factors

GAL$RPL3(RPF2$TAP

GAL$L18(RPF2$TAP

GAL$RPL9(NOP7$TAP

GAL$RPL23(NOP7$TAP

GAL$RPL17(NOP7$TAP

GAL$RPL28(NOG1$TAP

GAL$RPL11(NOP7$TAP

GAL$RPL10(NOG1$TAP

Nop6 Noc1 Jip5 Nop13 Jip5Noc13 Nop12 Nop12 Nop13Rrp5 Nop4

Nop6Rrp5

Arx1 Arx1 Noc2 Arx1 Afg2 Bud20 Rpf2 Cic1Brx1 Cic1 Noc3 Dbp10 Arx1 Ipi1 Rrs1 Erb1Cic1 Dbp10 Nog2 Noc2 Dbp10 Nsa1 Has1Dbp10 Drs1 Nop53 Noc3 Dbp9 Ssf1 Nop15Drs1 Noc3 Nsa2 Nog1 Mrt4 Nop16Ebp2 Nog2 Nug1 Nog2 Noc1 Nop53Erb1 Nop15 Spb1 Nop53 Noc2 Nop7Has1 Nop53 Nsa2 Noc3 Nsa1Mak16 Nop7 Nug1 Nog2 Rlp7Mrt4 Nsa1 Rlp24 Nop12 Rrp1Nip7 Nsa2 Rsa4 Nop4Noc2 Nug1 Spb1 Nop53Noc3 Rlp7 Spb4 Nsa2Nog1 Rrp1 Tif6 Nug1Nog2 Rsa4 Rrp15Nop15 Spb4 Rrp17Nop16 Rrp5Nop2 Rsa4Nop53 Spb1Nop7 Spb4Nsa1 Ssf1Nsa2Nug1Rea1Rlp24Rlp7Rpf1Rrp1Rrp14Rsa4Spb1Spb4Ssf1Tif6Ytm1

Assembly,factors

Early$acting Middle$acting Late$acting

Enriched

Diminished

Supplementary,Table,3.,Effect,of,depleting,individual,RPLs,to,pre=ribosomal,levels,of,other,RPLs

GAL$RPL3(RPF2$TAP

GAL$L18(RPF2$TAP

GAL$RPL9(NOP7$TAP

GAL$RPL23(NOP7$TAP

GAL$RPL17(NOP7$TAP

GAL$RPL28(NOG1$TAP

GAL$RPL11(NOP7$TAP

GAL$RPL10(NOG1$TAP

L10 L10 P0 P2 P2 L12 P2 P1L24 L24 P2 P1 P0 P0 P0 P0P0 P0 L12 P2 P2P2 P2L13 L13 L19 L32 L17 L28 L5 L10L16 L16 L23 L33 L19 L11L18 L18 L25 L17 L23L20 L20 L26 L19 L31L3 L32 L27 L23 L10L32 L33 L31 L25 L2L33 L6 L34 L27 L28L4 L17 L35 L31 L24L6 L19 L37 L35 L38L7 L23 L9 L9 L39L8 L26 L2 L10L17 L31 L28 L2L19 L35 L40 L21L23 L37 L36 L28L25 L9 L39 L43L26 L2 L1L27 L21 L30L31 L28 L39L34 L14L35 L39L37L9L2L21L28L1L14L15L30L36L39

Colors,correspond,to,the,known,phenotypic,class,of,the,diminished,RPL;,those,in,black,are,untested.The,enrichment,of,RPLs,that,associate,with,pre=60S,complexes,in,the,cytoplasm,might,be,due,to,the,their,ability,to,alternatively,assemble,in,the,nucleus,(Francisco=Velilla,et,al.,2013).,Another,explanation,relates,to,the,nature,of,the,experiment.Background levels of contaminating ribosomal proteins might be present when we purify pre-ribosomes under wild-type conditions. This,value,(iTRAQ,ratio),is,magnified,when,normalized,to,the,to,the,median,value,of,all,RPL,ratios,in,mutant,pre=ribosomes

RPLs

Early$acting Middle$acting Late$acting

Enriched

Diminished

Supplementary Table 4. Strain List Strain No. Genotype

Source

BY4741 MATa his3∆1 leu2∆0 ura3∆0 met15∆0

Euroscarf

JWY6147 MATa ura3-52 trp1-1 lys2-801 his3-∆200 leu2-∆1

E. Jones

JWY7758 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl3a:: KANMX4 pGAL-RPL3A LEU2

(Poll et al. 2009)

JWY8402 MATa ura3-52 trp1-1 lys2-801 his3-∆200 leu2-∆1 rpl4b::KANMX6 rpl4a::GAL-HA3-RPL4A TRP1

(Poll et al. 2009)


This study

JWY8423 MATa ura3-52 trp1-1 lys2-801 his3-∆200 leu2-∆1rpl7b::KANMX6 rpl7a::GAL-HA3-RPL7A TRP1

(Jakovljevic et al. 2012)

JWY7791 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl8a::HIS3MX6 rpl8b::KANMX4 pGAL-RPL8B LEU2

(Poll et al. 2009)

JWY7759 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl13a::HIS3MX6 rpl13b::KANMX4 pGAL-RPL13A LEU2

(Poll et al. 2009)


(Poll et al. 2009)


(Poll et al. 2009)


(Poll et al. 2009)

JWY7796 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl32::KANMX4 pGAL-RPL32 LEU2

(Poll et al. 2009)


(Poll et al. 2009)

JWY7792 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl9a::HIS3MX6 (Poll et al. 2009)

rpl9b::KANMX4 pGAL-RPL9A LEU2


(Gamalinda et al. 2013)


(Poll et al. 2009)


(Poll et al. 2009)


(Poll et al. 2009)


(Babiano et al. 2012)


(Poll et al. 2009)


This study






(Poll et al. 2009)


(Poll et al. 2009)


(Poll et al. 2009)


(Poll et al. 2009)

JWY7787 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl28::KANMX4

pGAL-RPL28 LEU2

(Poll et al. 2009)


(Poll et al. 2009)

JWY8491 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl3a:: KANMX4 pGAL-RPL3A LEU2 rpf2::RPF2-TAP URA3

This study

JWY8491 MATa ura3-52 trp1-1 lys2-801 his3-∆200 leu2-∆1 rpl4b::KANMX6 rpl4a::GAL-HA3-RPL4A TRP1 rpf2::RPF2-TAP URA3

This study


This study

JWY8492 MATa ura3-52 trp1-1 lys2-801 his3-∆200 leu2-∆1rpl7b::KANMX6 rpl7a::GAL-HA3-RPL7A TRP1 rpf2::RPF2-TAP URA3


JWY8591 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl8a::HIS3MX6 rpl8b::KANMX4 pGAL-RPL8B LEU2 rpf2::RPF2-TAP URA3


JWY8532 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl13a::HIS3MX6 rpl13b::KANMX4 pGAL-RPL13A LEU2 rpf2::RPF2-TAP URA3

This study

JWY8533 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl16a::HIS3MX6 rpl16b::KANMX4 pGAL-RPL16B LEU2 nop7::NOP7-TAP URA3

This study


This study

JWY9904 MATa ura3-52 trp1-1 lys2-801 his3-∆200 leu2-∆1 rpl18b::KANMX6 rpl18a::GAL-HA3-RPL18A TRP1 rpf2::RPF2-TAP URA3 rpl14::RPL14-3HA HIS3

This study

JWY9908 MATa ura3-52 trp1-1 lys2-801 his3-∆200 leu2-∆1 rpl18b::KANMX6 rpl18a::GAL-HA3-RPL18A TRP1 rpf2::RPF2-TAP URA3 rpl33::RPL33-3HA HIS3

This study


rpl18b::KANMX6 rpl18a::GAL-HA3-RPL18A TRP1 rpf2::RPF2-TAP URA3 rpl28::RPL28-13MYC HIS3

This study

JWY10650 MATa ura3-52 trp1-1 lys2-801 his3-∆200 leu2-∆1 rpl18b::KANMX6 rpl18a::GAL-HA3-RPL18A TRP1 rpf2::RPF2-TAP URA3 rpl6::RPL6-13MYC HIS3

This study


This study


This study

JWY8330 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl20a::HIS3MX6 rpl20b::KANMX4 pGAL-RPL20B LEU2 rpf2::RPF2-TAP URA3

This study

JWY8494 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl32::KANMX4 pGAL-RPL32 LEU2 rpf2::RPF2-TAP URA3

This study

JWY7959 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl33a::HIS3MX6 rpl33b::KANMX4 pGAL-RPL33A LEU2 nop7::NOP7-TAP URA3

This study


This study

JWY9622 MATa ura3-52 trp1-1 lys2-801 his3-∆200 leu2-∆1 rpl17b::KANMX6 rpl17a::GAL-HA3-RPL17A TRP1 nop7::NOP7-TAP URA3



This study


This study

JWY8412 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl25::KANMX4 pGAL-RPL25 LEU2 nop7::NOP7-TAP URA3

This study


rpl26b::KANMX6 rpl26a::GAL-HA3-RPL26A TRP1 nop7::NOP7-TAP URA3

(Babiano et al. 2012)


This study


This study

JWY9624 MATa ura3-52 trp1-1 lys2-801 his3-∆200 leu2-∆1 rpl35b::KANMX6 rpl35a::GAL-HA3-RPL35A TRP1 nop7::NOP7-TAP URA3


JWY8450 MATa his3∆1 leu2∆0 ura3∆0 met15∆0 rpl37a::HIS3MX6 rpl37b::KANMX4 pGAL-RPL37A nop7::NOP7-TAP URA3



This study


(Zhang et al. 2007)


This study

JWY8112 MATa ura3-52 trp1-1 lys2-801 his3-∆200 leu2-∆1 rpl11b::KANMX6 rpl11a::GAL-HA3-RPL11 TRP1 nop7::NOP7-TAP URA3

(Zhang et al. 2007)


This study


This study


This study

SUPPLEMENTARY REFERENCES: Babiano, R., Gamalinda, M., Woolford, J.L., Jr., and de la Cruz, J. 2012.

Saccharomyces cerevisiae ribosomal protein L26 is not essential for ribosome assembly and function. Mol Cell Biol. 32(16):3228-3241.

Gamalinda, M., Jakovljevic, J., Babiano, R., Talkish, J., de la Cruz, J., and Woolford,

J.L., Jr. 2013. Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing. Nucleic Acids Res 41(3): 1965-1983.

Jakovljevic, J., Ohmayer, U., Gamalinda, M., Talkish, J., Alexander, L., Linnemann, J.,

Milkereit, P., and Woolford, J.L., Jr. 2012. Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits. RNA 18(10): 1805-1822.

Poll, G., Braun, T., Jakovljevic, J., Neueder, A., Jakob, S., Woolford, J.L., Jr.,

Tschochner, H., and Milkereit, P. 2009. rRNA maturation in yeast cells depleted of large ribosomal subunit proteins. PLoS One 4(12): e8249.

Zhang, J., Harnpicharnchai, P., Jakovljevic, J., Tang, L., Guo, Y., Oeffinger, M., Rout,

M.P., Hiley, S.L., Hughes, T., and Woolford, J.L., Jr. 2007. Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes. Genes Dev 21(20): 2580-2592.

LEGENDS TO SUPPLEMENTARY FIGURES!

Supplementary Figure 1. Yeast pre-rRNA processing pathway from the nucleolus

to the cytoplasm. Endo- and exonucleolytic processing sites are indicated. Early,

middle, and late steps in processing of 27S pre-rRNAs to form mature large ribosomal

subunit rRNAs are marked.

Supplementary Figure 2. Steady state levels of pre-rRNAs in RPL depletion

mutants. (A) Detection of pre-rRNAs by primer extension and northern blotting verifies

the previously defined roles of representative RPLs in early, middle, or late steps in pre-

rRNA processing. (B) Assigning the role of previously uncharacterized RPLs (L6 and

L31) in 27S pre-rRNA processing, as well as re-classifying L13, previously reported to

function in late assembly steps. (C) Analyses of whole-cell and affinity-purified (pre-

)rRNAs by northern blotting confirm previously reported roles of RPLs in pre-rRNA

processing. Equal signal intensities Input and IP fractions correspond to 1% (35S, 27S

pre-rRNAs and 25S and 18S rRNAs), 10% (7S pre-rRNA, 5.8S, 5S rRNAs and

glutamyl-tRNA), or 25% (anti-TAP) co-purification efficiency. !

Supplementary Figure 3. SDS-PAGE and silver staining to assay relative levels of

proteins in pre-60S particles after depleting each RPL. Proteins associated with the

TAP-tagged bait assembly factors were purified from cells where the corresponding

RPL genes are either expressed (Gal) or repressed (Glu). Affinity purified fractions were

analyzed by SDS-PAGE followed by silver staining.

Supplementary Figure 4. Western blotting to assay relative levels of proteins in

pre-60S particles after depleting each RPL. Appropriate available antibodies against

assembly factors and RPLs were used to detect individual proteins by western blotting.!

Supplementary Figure 5. Cartoon illustration of the effects of depleting

representative early-, middle-, or late-acting RPLs. Spheres represent RPLs in the

60S ribosomal subunit, viewed from the subunit interface. Early-, middle-, and late-

acting proteins are colored green, magenta, and orange, respectively. Dashed circles

indicate depleted protein, faint colored spheres indicate other RPLs not stable

associated with pre-ribosomes upon depletion of representative RPLs, and bright solid

spheres indicate RPLs that remain stably associated after depletion of representative

RPLs.!

Supplementary Figure 6. The helices where each 25S/5.8S rRNA domain begins

and ends are oriented close to each other in mature 60S ribosomal subunits. (A)

Secondary structure of yeast large ribosomal subunit rRNA species. 5.8S rRNA,

domains I, II, III, IV, V, and VI are colored black, red, orange, yellow, green, blue, and

purple, respectively. Helices of the start and end of each 25S/5.8S rRNA domain are

boxed. (B) Spatial orientation of boxed root helices in the tertiary structure of mature

60S subunits. (C) Location of L3 relative to the 5’-end of 5.8S rRNA and 3’-end of 25S

rRNA.!

Supplementary Figure 7. Domain II as a platform for assembly of other rRNA

domains. Domain II of (a) yeast 25S rRNA and (b) E. coli 23S rRNA, showing

projections directed toward the subunit interface. 5.S rRNA (grey) is shown for

reference.!

Supplementary Figure 8. Similarities and difference between bacterial and yeast

large ribosomal subunit assembly. (A) Summary of collective results from studying

the interdependence of RPL assembly in yeast cells. The modified in vivo bacterial large

subunit assembly map proposed by Williamson and colleagues(Chen and Williamson

2013) was simplified to depict only bacterial RPLs with yeast homologs. Yeast

nomenclature is followed, and colored boxes indicate their role in early (green), middle

(magenta), and late (orange) steps of pre-rRNA processing. Horizontal lines subdivide

the in vivo assembly groups previously described. These six assembly groups are

designated as early (green), middle (magenta), and late (orange) steps of 50S

assembly. Dashed arrows indicate assembly relationships observed in bacteria but not

found in eukaryotes. Grey arrows indicate predicted dependencies based on their role in

early yeast pre-60S assembly events. Bacterial homologs of RPLs without connecting

arrows depend on primary binding RPLs without eukaryotic homologs. (B) Early

(green), middle (magenta), and late (orange) assembly groups of bacterial RPLs are

mapped onto the structure of E. coli 50S subunit (PDB accession number 2AW4).

Bacterial ribosomal proteins are annotated based on their yeast homologs. Bacterial

ribosomal proteins enclosed in parentheses do not have yeast homologs.

!

supplementary information to the manuscript …genesdev.cshlp.org › content › suppl › 2014 ›...

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