Ribosomes catalyze protein synthesis in all cells. Consequently, mature and assembling ribosomes are the target for many antibiotics. Ribosome assembly defects also underlie many serious human diseases. Thus, regulation and ensuing quality control in ribosome assembly, as in other steps of translation, is of key importance. The goal of this proposal is to uncover the basis for ordered assembly steps, which likely form the basis for quality control, and to dissect the function of three regulatory proteins during the final assembly step of the 40S ribosomal subunit in yeast. The step under investigation is relatively simple and well-defined, involving two changes in the primary sequence of the RNA. Yet, it requires at least nine accessory factors, which are essential in yeast. Some of these factors have activities typically considered regulatory, suggesting that this step is simple enough for mechanistic dissection, yet complex enough to yield important insight into RNA-protein complex (RNP) assembly. Since ribosomes are the most conserved and ancient RNPs, these insights are expected to be of fundamental importance. This proposal addresses the role of Fap7, an ATPase, whose energy-requiring function positions it uniquely for a regulatory role, the putative endonuclease Nob1, and the helicase Rok1, in catalyzing successive pre-ribosome remodeling steps. Based on current data we hypothesize that energy-requiring conformational switches built into these steps are used to order and spatially and temporally regulate ribosome assembly, in a manner that is responsive to changes in the cellular growth environment. We will use a unique combination of yeast genetics, biochemical and biophysical experiments, and mechanistic enzymology to address Specific Aims: (1) Characterize a Conformational Change that Regulates Nob1. (2) Determine the Role of Rok1 in Promoting a Conformational Change. (3) Establish the Role of Fap7 In Ribosome Assembly. This work will lead to partial reconstitution of facilitated ribosome assembly, the discovery of novel intermediates and molecular-level information about the function of key assembly factors in orchestrating a conformational change that spatially and temporally regulates 40S assembly. In addition, knowledge of slow, regulated steps and information about the proteins catalyzing these steps will allow the ribosome assembly pathway to be targeted for drug purposes.

Public Health Relevance

Ribosomes catalyze protein synthesis in all cells. Failure to correctly assembly ribosomes or to correctly regulate assembly can result in serious human disease, suggesting the ribosome assembly pathway as an important drug target. However, knowledge of individual processing steps and their regulation is required and will be garnered in this proposal.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM086451-05
Application #
8500363
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
2009-08-10
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
5
Fiscal Year
2013
Total Cost
$355,809
Indirect Cost
$176,108
Name
Scripps Florida
Department
Type
DUNS #
148230662
City
Jupiter
State
FL
Country
United States
Zip Code
33458
Karbstein, Katrin (2015) What will the future hold: RNP quality control and degradation. RNA 21:657-8
Tyagi, Richa; Shahani, Neelam; Gorgen, Lindsay et al. (2015) Rheb Inhibits Protein Synthesis by Activating the PERK-eIF2α Signaling Cascade. Cell Rep :
de la Cruz, Jesús; Karbstein, Katrin; Woolford Jr, John L (2015) Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo. Annu Rev Biochem 84:93-129
Ghalei, Homa; Schaub, Franz X; Doherty, Joanne R et al. (2015) Hrr25/CK1δ-directed release of Ltv1 from pre-40S ribosomes is necessary for ribosome assembly and cell growth. J Cell Biol 208:745-59
Young, Crystal L; Khoshnevis, Sohail; Karbstein, Katrin (2013) Cofactor-dependent specificity of a DEAD-box protein. Proc Natl Acad Sci U S A 110:E2668-76
Karbstein, Katrin (2013) Quality control mechanisms during ribosome maturation. Trends Cell Biol 23:242-50
Young, Crystal; Karbstein, Katrin (2012) Analysis of cofactor effects on RNA helicases. Methods Enzymol 511:213-37
Strunk, Bethany S; Novak, Megan N; Young, Crystal L et al. (2012) A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits. Cell 150:111-21
Lamanna, Allison C; Karbstein, Katrin (2011) An RNA conformational switch regulates pre-18S rRNA cleavage. J Mol Biol 405:3-17
Woolls, Heather A; Lamanna, Allison C; Karbstein, Katrin (2011) Roles of Dim2 in ribosome assembly. J Biol Chem 286:2578-86

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