John L. Woolford, Jr.
616 Mellon Institute
Department of Biological Sciences
Carnegie Mellon University
4400 Fifth Avenue
Pittsburgh, PA 15213
Ph.D., Duke University
Postdoctoral Appointment, Brandeis University
Our laboratory is studying the pathway of assembly of ribosomes in eukaryotes. We use the yeast Saccharomyces cerevisiae as an experimental organism in which to study this essential and highly conserved process. Thus we can utilize state-of-the-art genomics, proteomics, and molecular biology, as well as classical genetic and biochemical approaches.
Ribosomes consist of two ribonucleoprotein subunits. In eukaryotes, the 40S subunit contains a single 18S rRNA and 32 different ribosomal proteins, while the 60S subunit contains the 5S, 5.8S, and 25S rRNAs and 48 different ribosomal proteins. The 18S, 5.8S, and 25S rRNAs are derived from a single 35S transcript, by a series of endonucleolytic and exonucleolytic processing steps. The 5S pre-rRNA is synthesized from separate genes. The genes for 5S and 35S rRNA are linked in yeast and present in ~100 to 150 tandem repeats on chromosome XII. Transcription and processing of pre-rRNA and most steps of assembly of ribosomal proteins with rRNA occur in the nucleolus, a subdomain of the nucleus. Late steps in ribosome assembly occur upon release of the immature ribosomal subunits from the nucleolus into the nucleoplasm, including changes that allow the subunits to be exported from the nucleus to the cytoplasm. Several final steps of cytoplasmic maturation are required to produce ribosomal subunits capable of functioning in protein synthesis.
Our goals are to identify and characterize proteins that are necessary for the biogenesis of ribosomes but do not end up as constituents of the mature ribosomes. Such "non-ribosomal" molecules might be required for a number of processes in order to assemble a ribosome. These include: (1) rearrangements of rRNA structure or of protein-rRNA interactions that occur during sequential assembly of ribosomal proteins with rRNA (e.g. RNA-dependent helicases/ATPases, GTPases or protein isomerases), (2) cleavage and processing of RNA, (3) sensing and reporting the progress and fidelity of RNA processing and ribosome assembly (quality control, kinases, phosphatases, GTPases) and (4) transport of nascent ribosomes within the nucleolus and nucleoplasm and export to the cytoplasm.
We have identified a number of such factors in genetic screens or selections for mutants defective in ribosome biogenesis. Most of these proteins are essential and have homologues in humans, as well as in other metazoans. An important recent breakthrough in our lab has been the development of methods to purify ribosome assembly intermediates and identify their RNA and protein constituents. These genetic and biochemical tools now enable us to investigate in much more detail than before the order in which ribosomes assemble and the precise function of each non-ribosomal protein in the biogenesis of ribosomes.
An interesting outcome of this much expanded catalogue of ribosome assembly factors is the discovery that many of these proteins are also important for cell growth or proliferation. This should not be entirely surprising, since ribosome biogenesis was known to be tightly coordinated with cell growth and cell cycle progression. We are beginning to investigate in more detail how ribosome biogenesis and cell growth and proliferation might be coordinated through these molecules.
Tartakoff AM, Chen L, Raghavachari S, Gitiforooz D, Dhinakaran A, Ni CL, Pasadyn C, Mahabeleshwar GH, Pasadyn V, Woolford JL Jr. (2021). The nucleolus as a polarized coaxial cable in which the rDNA axis is surrounded by dynamic subunit-specific phases. Current Biology 31: 2507-2519.
Wilson, D.M., Li, Y., LaPeruta, A., Gamalinda, M., Gao, N., and Woolford, J.L.Jr. (2020). Structural insights into assembly of the ribosomal polypeptide exit tunnel. Nature Communications 2020 Oct 9;11(1):5111.
Micic, J., Li,Y., Wu, S., Wilson,D., Tutuncuoglu, B., Gao, N., and Woolford, J.L.,Jr. (2020). Coupling of 5SRNP rotation with maturation of functional centers during large ribosomal subunit assembly. Nature Communications 2020 Jul 27;11(1):3751.
Klinge,S. and Woolford, J.L.,Jr. (2019). Ribosome assembly coming into focus. Nature Reviews Molecular Cell Biology 20: 116–131
Biedka S, Micic J, Wilson D, Brown H, Diorio-Toth L, Woolford JL Jr. Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes. J Cell Biol. 2018 Jul 2;217(7):2503-2518. doi: 10.1083/jcb.201711037. Epub 2018 Apr 24. PMID: 29691304
Shan Wu, Beril Tutuncuoglu, Kaige Yan, Hailey Brown, Yixiao Zhang, Dan Tan, Michael Gamalinda, Yi Yuan, Zhifei Li, Jelena Jakovljevic, Chengying Ma, Jianlin Lei, Meng-Qiu Dong, John L. Woolford Jr & Ning Gao. Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes. Nature. In press.
Talkish J, Biedka S, Jakovljevic J, Zhang J, Tang L, Strahler JR, Andrews PC, Maddock JR, Woolford JL Jr. Disruption of ribosome assembly in yeast blocks cotranscriptional pre-rRNA processing and affects the global hierarchy of ribosome biogenesis. RNA. 2016 Jun;22(6):852-66.
Kormuth KA, Woolford JL Jr, Armitage BA. Homologous PNA Hybridization to Noncanonical DNA G-Quadruplexes. Biochemistry. 2016 Mar 29;55(12):1749-57.
Gamalinda M, Woolford JL Jr. Paradigms of ribosome synthesis: Lessons learned from ribosomal proteins. Translation, DOI: 10.4161/21690731.2014.975018
Woolford J. Assembly of ribosomes in eukaryotes. RNA. 2015 Apr;21(4):766-8.
de la Cruz J, Karbstein K, Woolford JL Jr. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo. Annu Rev Biochem. 2015;84:93-129.
Gamalinda M, Woolford JL Jr. Deletion of L4 domains reveals insights into the importance of ribosomal protein extensions in eukaryotic ribosome assembly. RNA. 2014 Sep 22.
Talkish J, Campbell IW, Sahasranaman A, Jakovljevic J, Woolford JL Jr. Ribosome assembly factors Pwp1 and Nop12 are important for folding of 5.8S rRNA during ribosome biogenesis in Saccharomyces cerevisiae. Mol Cell Biol. 2014 May;34(10):1863-77.
Gamalinda M, Ohmayer U, Jakovljevic J, Kumcuoglu B, Mbom B, Lin L, Woolford JL Jr. A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains. Genes Dev. 2014 Jan 15;28(2):198-210.
Woolford, J.L., Jr. and Baserga, S. (2013) Ribosome biogenesis in the yeast Saccharomyces cerevisiae. Genetics 195: 643-681.
Dembowski, J.A., Ramesh, M., McManus, C. J., and Woolford, J.L. Jr. (2013) Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae. RNA 19:1639-1647.
Ohmayer, U., Gamalinda, M., Sauert,M., Ossowski,J., Poll,G., Linnemann,J., Hierlmeier,T., Perez-Fernandez,J., Kumcuoglu,B., Leger-Silvestre, I., Faubladier,M., Griesenbeck,M. Woolford,J.L.Jr., Tschochner,H., and Milkereit,P. (2013) Assembly characteristics of large subunit ribosomal proteins in S. cerevisiae. PLOS One 8:e68412.
Sahasranaman, A., and Woolford, J.L. Jr. (2013) "Ribosome Assembly" in Encyclopedia of Biological Chemistry, 2nd edition (Lennarz, W.J. and Lane, M.D. eds.) Elsevier, Inc
Dembowski, J.A., Kuo,B., and Woolford,J.L. Jr. (2013) Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits. Nucleic Acids Research 41:7889-7904.
Curtis,R.E., Kim,S., Woolford,J.L.Jr., Xu,W., and Xing,E.P. (2013) Structured association analysis leads to insight into Saccharomyces cerevisiae gene regulation by finding multiple contributing eQTL hotspots associated with functional gene modules. BMC Genomics 14: 196.
Gamalinda, M., Jakovljevic, J., Talkish, J., Babiano, R., 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 Research 41:1965-1983.
Jakovljevic J, Ohmayer U, Gamalinda M, Talkish J, Alexander L, Linneman J, Milkereit P, Woolford JL Jr. 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, 2012. (in press)
Talkish J, Zhang J, Jakovljevic J, Horsey EW, Woolford JL Jr. Hierarchical recruitment into nascent ribosomes of assembly factors required for 27SB pre-rRNA processing in Saccharomyces cerevisiae. Nucleic Acids Res. 2012 Jun 26.
Babiano R, Gamalinda M, Woolford JL Jr, de la Cruz J. Saccharomyces cerevisiae ribosomal protein L26 is not essential for ribosome assembly and function. Mol Cell Biol. 2012 Jun 11.
Shimoji K, Jakovljevic J, Tsuchihashi K, Umeki Y, Wan K, Kawasaki S, Talkish J, Woolford JL Jr, Mizuta K. Ebp2 and Brx1 function cooperatively in 60S ribosomal subunit assembly in Saccharomyces cerevisiae. Nucleic Acids Res. 2012 Feb 8.
Sahasranaman A, Dembowski J, Strahler J, Andrews P, Maddock J and Woolford Jr JL. 2011. Assembly of Saccharomyces cerevisiae 60S ribosomal subunits: role of factors required for 27S pre-rRNA processing. EMBO J. 2011 Sep 16;30(19):4020-32.
Poll G, Braun T, Jakovljevic J, Neueder A, Jakob S, Woolford Jr JL, Tschochner H, Milkereit P. rRNA maturation in yeast cells depleted of large ribosomal subunit proteins. PLOS One 4: e8249, 2009.
Talkish J and Woolford Jr JL. The Rea1 tadpole loses its tail. Cell 138: 832-834, 2009.
Staley J and Woolford Jr JL. Assembly of ribosomes and spliceosomes:complex ribonucleoprotein machines. Current Opinion in Cell Biology 21:109-118, 2009.
Tang L, Sahasranaman A, Jakovljevic J, Schleifman E and Woolford Jr JL. Interactions among Ytm1, Erb1, and Nop7 Required for Assembly of the Nop7-Subcomplex in Yeast Preribosomes. Molecular Biology of the Cell, 19:2844-2856, 2008.
Zhang J, Harnpicharnchai P, Jakovljevic J, Tang L, Guo Y, Oeffinger M, Rout MP, Hiley SL, Hughes T and Woolford Jr JL. Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes. Genes and Development, 21:2580-2592, 2007.
Miles TD, Jakovljevic J, Horsey E, Harnpicharnchai P, Tang L and Woolford Jr JL. Ytm1,Nop7,and Erb1 Form a Complex Necessary for Maturation of 66S Preribosomes. Molecular and Cellular Biology, 25:10419-10432, 2005.
Jakovljevic J, Antunez de Mayolo P, Miles TD, Nguyen TM-L, Leger-Silvestre, I, Gas N and Woolford Jr. JL. The carboxyl-terminal extension of yeast ribosomal protein S14 is necessary for maturation of 43S preribosomes. Molecular Cell, 14:331-342, 2004.
Woolford JL Jr. Chaperoning Ribosome Assembly. Molecular Cell 10: 8-10, 2003.
Adams CA, Jakovljevic J, Roman J, Harnpicharnchai P, Woolford JL Jr. Saccharomyces cerevisiae Nucleolar Protein Nop7p is Necessary for Biogenesis of 60S Ribosomal Subunits. RNA, 8: 150-165, 2002.
Harnpicharnchai P, Jakovljevic J, Horsey E, Miles T, Roman J, Rout M, Meagher D, Imai B, Guo Y, Brame C, Shabanowitz J, Hunt DF, Woolford JL Jr. Composition and Functional Characterization of Yeast 66S Ribosome Assembly Intermediates. Molecular Cell, 8: 505-515, 2001.
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