2020 Theses Doctoral

Single-particle cryo-electron microscopy studies of ribosomes with fragmented 28S rRNA

Gutierrez-Vargas, Cristina

In the past five years, single-particle cryo-electron microscopy (cryo-EM) has revolutionized structural biology. Recent advances in detector technology and powerful computational methods now allow images of unprecedented detail to be recorded and structures to be determined at near-atomic resolution. For my PhD studies, I took advantage of this technique to study the structure of uniquely fragmented ribosomes.

Ribosomes, are large macromolecular complexes that translate genetic information carried by messenger RNAs (mRNAs) into polypeptide chains. They are the protein production factories of all living cells and are thus involved in virtually all aspects of cellular development and maintenance. By virtue of their core role in the cell, ribosomes share a highly evolutionarily conserved core that carries out the fundamental processes of protein synthesis [1]. However, outside of this core, ribosome composition varies considerably. The main differences among eukaryotic ribosomes are due to rRNA expansion segments (ESs) and variations of ribosomal proteins (r-proteins) [1, 2]. Further, rRNA fragmentation occurring in regions of high variability has been reported in several organisms from bacteria to protozoa, insects, helminths, fish, and surprisingly mammals [3-15]. Recently, the naked mole-rat (Heterocephalus glaber) was discovered to have unusual cleavage sites in its 28S rRNA resulting in the deletion of the major part of the D6 variable region (ES15L) and leaving the two rRNA fragments disconnected [14]. The cleaved 28S rRNA has been associated with the naked mole rat’s increased translational fidelity [14]. The only other known mammals having fragmented rRNA are the tuco-tuco rodent (of the genus Ctenomys) and the degu (in the related genus Octodontomys) [13]. Here we present the high-resolution structures of the naked mole-rat, tuco-tuco, and guinea pig (Cavia porcellus) ribosomes. Guinea pig, which has canonical (non-fragmented) 28S rRNA is used as a rodent model for comparisons to the naked mole-rat and tuco-tuco ribosomes.

During my PhD studies, I also looked at another uniquely fragmented ribosome, that of the protozoan parasite, Trypanosoma cruzi (T. cruzi), the causative agent of Chagas disease. The T. cruzi large subunit (LSU) rRNA is assembled from 8 pieces-5S, 5.8S, and six pieces forming jointly the 28S rRNA. Together with my colleagues from Joachim Frank’s and Liang Tong’s research groups, we solved the structure of the T. Cruzi LSU and identified distinctive trypanosome interactions, which allowed us to propose a tentative model for assembly of the large ribosomal subunit (60S) [16, 17].