John Woolford-The Center for Nucleic Acids Science and Technology - Carnegie Mellon University

John Woolford

Professor of Biological Sciences & Co-Director of CNAST

Phone: (412) 268-3412

Research

The Woolford lab is investigating the mechanism of assembly of eukaryotic ribosomes in vivo. We use the yeast Saccharomyces cerevisiae as our model organism, to facilitate  molecular genetic and proteomic approaches. Ribosomes are complex ribonucleoprotein particles containing four ribosomal RNAs and 79 different ribosomal  proteins. Assembly requires folding of the rRNA, coupled with removal of transcribed spacer sequences  from the RNA and binding of the ribosomal proteins to the RNA. More than 200 assembly factors associate with nascent ribosomes and are required for their maturation. Our research is guided in part by high resolution crystal structures recently determined for yeast ribosomes.

We are collaborating with the groups of Philipp Milkereit in Germany and Jesus de la Cruz in Spain, to systematically investigate the roles of each ribosomal protein in assembly of the large ribosomal subunit. We have found that the timing of the function of each protein correlates with its location in the ribosome. Proteins that are required for early steps in assembly bind to domains I and II of rRNA, along the solvent-exposed domain. R-proteins that function in middle steps are located along the bottom of the subunit, bound primarily to domain III of rRNA. R-proteins required for late steps are positioned near the central protuberance and along the subunit interface, near the active site of the ribosome.  Currently we are investigating roles of specific r-proteins in structuring  specific rRNA neighborhoods. 

A second project of the Woolford lab is to investigate functions of assembly factors, including RNA binding proteins that help knit together rRNA tertiary structure, and RNA helicases that drive stable assembly of neighborhoods of r-proteins, during early stages of large subunit maturation.

Tools that we use include affinity purification of ribosome assembly intermediates, mass spectrometry to assay protein constituents of pre-ribosomes, in vivo cross linking and high throughput RNA sequencing to map protein-RNA interactions, two-hybrid assays and protein cross-linking to identify protein-protein interactions, chemical probing in concert with high throughput sequencing to map rRNA structure changes during assembly, and site-directed mutagenesis to assess  the importance of molecular interactions driving ribosome biogenesis.

Publications

Curtis, R.E.; Kim,S.; Woolford, J.L., Jr.; Xu, W.; Xing, E.P. Structured association analysis leads insight into Saccharomyces cerevisiae gene regulation by finding multiple contributing eQTL hotspots associated with functional gene modules. BMC Genomics 2013, 14 (in press).

Sahasranaman, A.; Woolford, J.L., Jr. Ribosome Assembly in Encyclopedia of Biological Chemistry. 2013 (in press).

Babiano, R.; Gamlinda, M.; Woolford, J.L., Jr.; de la Cruz, J. Saccharomyces cerevisiae ribosomal protein L26 is not essential for ribosome assembly and function (2102). Molecular and Cellular Biology 2012, 32: 3228-3241.

Gamalinda, M.; Jakovljevic, J.; Babiano, R.; Talkish, J.; de la Cruz, J.; Woolford, J.L., Jr. 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 2012, 41: 1965-1983.

Jakovljevic, J.; Ohmayer, U.; Gamalinda, M.; Talkish, J.; Alexander, L.; Linneman, J.; Milkereit, P.; Woolford, J.L., Jr. Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and I of 25S rRNA in yeast 60S ribosomal subunits. RNA 2012, 18: 1805-1822.

Talkish, J.; Zhang, J.; Jakovljevic, J.; Horsey, E.; Woolford, J.L., Jr. Hierarchical recruitment into nascent ribosomes of assembly factors required for 27SB pre-rRNA processing in Saccharomyces cerevisiae. Nucleic Acids Research 2012, 40: 8646-8661.

Shimoji, K.; Jakovljevic, J.; Tsuchihashi, K.; Umeki,Y.; Wan, K.; Kawasaki, S.; Talkish, J.; Woolford, J.L., Jr.; Mizuta, K. Ebp2 and Brx1 function cooperatively in 60S ribosomal subunit assembly in Saccharomyces cerevisiae. Nucleic Acids Research 2012, 40: 4574-4588.