Woolford Laboratory  

 
 
Department of Biological Sciences l Carnegie Mellon University


RESEARCH

Ribosomes are ribonucleoprotein nanomachines that translate the genetic code and catalyze the synthesis of proteins in all organisms. Because both efficient and accurate protein synthesis are central to cellular homeostasis, major defects in assembly or function of ribosomes result in embryonic lethality or a variety of diseases, including cancers and neurodegeneration.

The major goal of research in our lab is to understand the mechanism of eukaryotic ribosome assembly in vivo, using the large ribosomal subunit (LSU) of the yeast Saccharomyces cerevisiae as our model system.  We want to identify and understand mechanisms of remodeling events that drive LSU assembly forward efficiently, as well as checkpoints that might be activated to delay or prevent irreversible steps when upstream assembly events fail to occur properly.

Biogenesis of LSU’s in yeast is an extremely complex pathway involving more than 75 assembly factors and 46 ribosomal proteins.  Assembly begins in the nucleolus, a subdomain of the nucleus, then continues as nascent ribosomes transit into the nucleoplasm, and finally into the cytoplasm. Our strategy has been to focus on a discrete interval representative of a few major remodeling events necessary for completion of assembly, in order to drill deeply into mechanisms. Thus, we are concentrating on late nuclear stages of assembly, beginning with the exit of pre-ribosomes from the nucleolus to the nucleoplasm, leading up to the final steps immediately before nuclear export.  To do so, we are taking advantage of our recent near atomic resolution cryo-electron microscopy structures (3.1 Angstroms) of late nuclear pre-ribosomes. These structures revealed three successive conformers of nascent subunits, as well as the location and structure of most of the assembly factors present in pre-ribosomes at this stage of subunit biogenesis,

In order to study remodeling and quality control during ribosome assembly, we take advantage of the ability to readily manipulate the yeast genome. Guided by our high-resolution structures of pre-ribosomes, we construct mutations in assembly factors or ribosomal proteins that function in late nuclear steps and then assay the resulting defects as follows:

  • Pre-rRNA folding is interrogated by chemical probing, followed by high throughput RNA sequencing.
  • Pre-rRNA processing is assayed by northern blotting and primer extension.
  • Association of assembly factors and ribosomal proteins with pre-ribosomes is measured by affinity purification of assembly intermediates, followed by western blotting and semi-quantitative mass spectrometry.
  • Pre-rRNP remodeling events are directly visualized by near atomic resolution cryo-electron microscopy.
  • Intracellular trafficking of pre-ribosomes and assembly factors is monitored by high-resolution fluorescence microscopy.

 

 

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