个人简介

Ph.D., Duke University Postdoctoral Appointment, Brandeis University

研究领域

Bioorganic & Chemical Biology

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.

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Gamalinda M, Woolford JL Jr. (2015) Paradigms of ribosome synthesis: Lessons learned from ribosomal proteins. Translation (in press). de la Cruz J, Karbstein K, Woolford JL Jr. (2015) Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo. Annual Reviews of Biochemistry (in press). Gamalinda M, Woolford JL Jr. (2014) Deletion of L4 domains reveals insights into the importance of ribosomal protein extensions in eukaryotic ribosome assembly. RNA. 2014 Sep 22. Talkish J, May G, Lin Y, Woolford JL Jr, McManus CJ. (2014) Mod-seq: high-throughput sequencing for chemical probing of RNA structure. RNA. 20(5):713-20. Talkish J, Campbell IW, Sahasranaman A, Jakovljevic J, Woolford JL Jr. (2014) Ribosome assembly factors Pwp1 and Nop12 are important for folding of 5.8S rRNA during ribosome biogenesis in Saccharomyces cerevisiae. Mol Cell Biol. 34(10):1863-77. Gamalinda M, Ohmayer U, Jakovljevic J, Kumcuoglu B, Woolford J, Mbom B, Lin L, Woolford JL Jr. (2014) A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains. Genes Dev. 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., 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 ribosomal subunits. RNA 10:1805-1822. Talkish, J., Zhang, J., Jakovljevic, J., Horsey, E.W., and Woolford, J.L. Jr. (2012) Hierarchical recruitment into nascent ribosomes of assembly factors required for 27SB pre-rRNA processing in Saccharomyces cerevisiae. Nucleic Acids Research 40: 8646-8661. 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. Molecular and Cellular Biology 32:3228-3241. Shimoji,K.*, Jakovljevic, J.*, Tsuchihashi, K., Umeki, Y., Wan, K., Kawasaki, S., Talkish, J., Woolford, J.L., Jr., and Mizuta, K. (2012) Ebp2 and Brx1 function cooperatively in 60S ribosomal subunit assembly in Saccharomyces cerevisiae. Nucleic Acids Research 40:4574-4588. *equal contributors. Bartoli K, Jakovljevic J, Woolford Jr JL, Saunders W. (2011) Kinesin molecular motor Eg5 functions during the elongation phase of polypeptide synthesis. Molecular Biology of the Cell 22(18): 34240-30. Epub 2011 Jul 27. 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 30(19):4020-32. Sahasranaman A and Woolford Jr JL. (2012) Ribsome Assembly in Encyclopedia of Biological Chemistry (in press).