The Karbstein Lab

The Karbstein Lab - Research

Research Overview

Ribosomes are large macromolecular machines that catalyze protein synthesis in all cells. Their function depends on communication between distant sites on the ribosomes, and thus requires correct assembly from the 80+ constituent components. In our lab, we study how assembly occurs within cells, how it is regulated, and what the mechanism are for quality control of assembly. Importantly, when cells cannot assembly the number of ribosomes needed for normal growth, or if the ribosomes that are assembled are compromised, diseases such as Diamond Blackfan anemia, 5q- syndrome and congenital asplenia occur. These are also associated with a ~30-fold higher cancer incidence.

Ribosome assembly in eukaryotic cells requires a large macromolecular machinery, comprising over 200 protein and RNA factors, which transiently associate with assembling ribosomes to facilitate the transcription of pre-rRNA, the modification and cleavage of rRNA precursors, the folding of rRNA as well as the binding of r-proteins. They also provide an interface to allow for regulation and quality control. While most of these assembly factors (AFs) are essential (~ 20% of essential genes in yeast are involved in ribosome assembly), and conserved from yeast to humans, their function remains often unknown.

We use a combination of approaches – including biochemistry, protein engineering, cryo-EM and crystallography, yeast genetics and mechanistic enzymology - to study eukaryotic ribosome assembly at the molecular level. In order to uncover regulatory hubs and quality control mechanisms, we are deciphering the function of assembly factors, and the order of events during assembly.

Dissecting the cytoplasmic maturation steps

Much of our efforts have concentrated on late cytoplasmic steps in the assembly of the small ribosomal subunit. By determining the structure of an assembly intermediate purified from yeast (Figure 1), we have shown that AFs bound to late pre-40S subunits are positioned to prevent premature translation initiation, by blocking the binding of translation initiation factors, the recruitment of mRNA and initiator tRNA, and subunit joining (Strunk et al., Science 2011).

Figure 1
Figure 1: Cryo-EM structure of pre-40S subunits from S. cerevisiae.

Excitingly, we have shown that this translationally repressed assembly intermediate is matured and released into the translating pool via a translation-like cycle (Strunk et al., Cell 2012). In this cycle the translation initiation factor eIF5B promotes joining of pre-40S subunits with 60S subunits. Maturation occurs in these 80S-like particles. However, because they contain neither mRNA nor tRNA, they do not produce protein. Release of newly assembled 40S subunits into the translating pool then requires the termination factors Rli1 and Dom34 (Figure 2). We are currently investigating the steps of this pathway in more detail.

Figure 2 

Figure 2: Testing maturing 40S ribosomes in a translation-like cycle. 40S precursors (dark orange) join 60S subunits (yellow) in a reaction catalyzed by eIF5B. The nuclease Nob1 promotes 40S maturation (to light orange 40S subunits), before Rli1 separates 40S and 60S subunits.


Ribosome Assembly as a Target for Novel Cancer Drugs

Conversion of the stable translationally inactive assembly intermediate in Figure 1 to mature ribosomes is initiated by the dissociation of the assembly factor Ltv1 (Strunk et al., Cell 2012). We have recently shown that this dissociation is mediated by the casein kinase 1d homolog Hrr25 (Ghalei et al., in revision). Importantly, Hrr25 is not essential when Ltv1 is deleted from yeast, demonstrating that the essential function of Hrr25 lies in ribosome assembly, and not any of the other cellular processes including DNA repair, cell cycle progression etc. Excitingly, the role of Hrr25/CK1δ is conserved in human cells. In collaboration with the Cleveland and Roush labs we have shown that a small molecules currently in development against triple negative breast cancer works through the CK1δ/Ltv1 circuit. This validates the ribosome assembly pathway as a novel cancer drug target. We are currently developing additional novel ways to target ribosome assembly in cancer cells.

Early nucleolar maturation steps and DEAD-box proteins

We have used the DEAD-box protein Rok1 and its co-factor Rrp5 as a model system to study how DEAD-box proteins are regulated by their cofactors. DEAD-box proteins are involved in all aspects of RNA biology and have highly conserved structures, which recognize the phosphate backbone of their substrate RNAs. This leads to the question of how these proteins recognize specific RNA substrates. Our work demonstrates that co-factors can increase the specificity of DEAD-box proteins by changing the way DEAD-box proteins interact with their RNA substrates (Young et al., Proceedings of the National Academy 2013).  

Using a combination of structural approaches and biochemical experiments we have addressed the role of Rok1 in ribosome assembly. Our results demonstrate that Rok1 uses an ATP-dependent switch to release Rrp5 from pre-40S subunits and thereby coordinates Rrp5’s roles in 40S and 60S subunit assembly (Khoshnevis et al., in revision).  We are now interested in a further structural characterization of earlier assembly intermediates using cryo-EM. This work is carried out in collaboration with the Stroupe lab at Florida State University.

Announcements

Postdoc Position Available

 Max wins Best Poster at the Scripps Research Fest!

 Graduate Student, Haina Huang, joins the lab! Welcome Haina!

Congratulations to Homa on her position as Assistant Professor in the Department of Biochemistry at Emory University (https://www.biochem.emory.edu/) on October 1, 2017.

Katrin is named an HHMI Faculty Scholar! hhmi

Miriam Koch joins the lab!  Welcome Miriam!

Nickan Khoshnevis is born!  Congratulations Homa and Sohail!

Joanne Doherty joins the lab as Staff Scientist!  Welcome Joanne!

Graduate Student, Melissa Parker, joins the lab!  Welcome Melissa!

Jason Collins joins the lab!  Welcome Jason!

Sohail's poster wins best poster prize at Research Fest!

Ribosome Assembly Meeting in Brussels, August 19-23, 2015!  http://ribosome.scripps.edu

Graduate Student, Lizz Miguet, joins the lab!  Welcome Lizz!

Scripps awarded an NSF REU grant for undergraduate research.

Katrin wins tenure!

Homa's poster wins prize at the Scripps Research Fest!

Homa's poster wins prize at the Ribosome Meeting!

Graduate Student, Max Ferretti, joins the lab!  Welcome Max!

Karbstein Lab

TSRI FL


Donation Link for Karbstein Lab