Arlen W. Johnson - US grants

DESCRIPTION (provided by applicant): Summary: Ribosomes are responsible for the rapid and accurate production of all the thousands of different proteins in cells in all forms of life on earth. The ability of these molecuar machines to do this depends upon a complex structure that is both stable and flexible, allowing it to interact with ligands in a dynamic fashion. The mature ribosome has binding sites for multiple ligands, including initiation factors, elongation factors, termination factors, tRNAs and mRNA, and it must ratchet between multiple stable conformations, moving tRNAs in and out at enormous rates. The accurate assembly of a fully functional eukaryotic ribosome involves over 200 accessory assembly factors, whose function, in many cases, is still unknown. However much recent excitement has focused on the role of some of these proteins in quality control checkpoints that occur late in the biogenesis process in the cytoplasm. We have recently proposed that the 60S subunit undergoes a cytoplasmic test drive in which newly assembled ribosomes are tested for function by biogenesis factors that structurally mimic translation factors. This proposal seeks to understand the mechanism of 60S maturation, focusing on the last steps of this pathway, the release of Tif6 and Nmd3, which, we believe, comprise the primary quality control checks on the large subunit. This proposal is directed at understanding quality control mechanisms that assess the functional and structural integrity of the large ribosomal subunit. This proposal builds on our recent results from the previous funding period suggesting that the newly assembled subunit undergoes a test drive that uses molecular mimics of translation factors prior to bona fide translation. In collaboration with Dr Jan Cools (VIB, Belgium) we have also recently shown that a defect in this quality control check is associated with ~8% of pediatric acute T-cell leukemia samples. Defects in this step are also associated with Shwachman-Diamond syndrome, an inherited disease that manifests in bone marrow failure, pancreatic dysfunction and predisposition to cancers among other conditions. These results highlight the importance of understanding the mechanism of this quality control step for its potential target for new drug therapies.

The proper spatial and temporal control of gene expression is essential for the normal growth and development of an organism. Gene expression is initiated by transcription but active degradation of mRNA is also necessary for strict temporal control. In eukaryotic cells there are two primary pathways for general mRNA degradation: a decapping-dependent pathway that utilizes 5'-exoribonucleases and a 3'-pathway that acts on mRNAs subsequent to deadenylation. Cell viability requires at least one of these pathways to be functional. In yeast, 5' decay is the major degradation pathway, but in mammalian cells evidence suggests that 3'-decay predominates. In addition to degrading mRNAs, the 3'-decay pathway blocks translation of poly(A) minus RNA that might arise as intermediates of degradation or from expression of viral messages such as the endogenous killer virus in yeast. The major components of this pathway are a heterotrimeric protein complex composed of Ski2p (a putative ATPase/RNA helicase), Ski3p and Ski8p, the Ski7 protein and a multisubunit exoribonuclease complex called the exosome. All components are required in vivo for 3'-decay. The Ski2/3/8 complex is thought to act as an adapter to facilitate mRNA 3'-degradation by the exosome. However, little is known about the molecular mechanism of 3'-decay and the interplay of the 3'-decay machinery with the translation apparatus. This project focuses on the biochemical function of the Ski2/3/8 complex and its interaction with Ski7p and the exosome in yeast.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Multiple poorly understood protein interactions are involved in the biogenesis and transport of the large ribosomal subunits. We are interested in whether or not these interactions can be visualized by FRET.

DESCRIPTION (provided by applicant): The ribosome is responsible for protein synthesis and is essential in all cells. Its faithful translation of the genetic code and the proper regulation of its activity are necessary for normal cell growth and development. Ribosome assembly is a complex, dynamic process and mechanisms must exist that ensure the correct assembly of ribosomes in order to maintain the fidelity of translation. In addition, ribosome biogenesis accounts for a large portion of the energy expenditure of a rapidly dividing cell and must be coordinated with the metabolic needs of a cell. Indeed, the rapid cell growth of many cancers requires up-regulation of ribosome biogenesis. Thus, understanding the mechanisms regulating ribosome biogenesis will provide insight for the development of new tools for controlling cell proliferation in disease states. The delineation of fundamental cellular pathways such as ribosome biogenesis is also necessary for the intelligent development of new drugs that are specific to their intended cellular targets without impinging adversely on other cellular pathways. This proposal is directed at understanding how the large ribosomal subunit in yeast is transported from its site of assembly in the nucleus to its site of function in the cytoplasm and activated for translation. Although we use yeast as a model organism, these pathways are highly conserved and findings from our work will be relevant to understanding these pathways in human cells as well. This proposal will: 1) Identify how the ribosomal protein Rpl25 and the export receptor Arx1 collaborate in nuclear export of the large (60S) ribosomal subunit. 2) Develop an ordered pathway of all the known cytoplasmic maturation events of the 60S subunit. In this work we will examine the role that assembling the ribosome stalk plays in the final maturation steps of the ribosome. 3) Determine if ribosome biogenesis is coupled with translation in controlling the final maturation step of the 60S subunit. PUBLIC HEALTH RELEVANCE: This project delineates essential and fundamental molecular pathways that are conserved throughout eukaryotes. Understanding these pathways and how they are integrated with other cellular pathways will provide the intellectual underpinning for investigators carrying out translational research.

DESCRIPTION (provided by applicant): All cells require ribosomes for translation, the process of decoding messenger RNA and synthesizing proteins. To satisfy the translational needs of a rapidly dividing eukaryotic cell, thousands of ribosomes must be synthesized per minute in a process that consumes a large portion of the cell's energy budget. Indeed, the rapid growth of many cancers requires up-regulation of ribosome biogenesis. Thus, understanding the mechanisms regulating ribosome biogenesis will provide insight for the development of new tools for controlling cell proliferationin disease states. The delineation of fundamental cellular pathways such as ribosome biogenesis is also critical for building the intellectual foundation for translational research. Many of the fundamental steps of eukaryotic ribosome biogenesis are poorly understood. Our functional genetic approach (Johnson lab) combined with the development of in vitro biochemical models (Correll lab) places us in a particularly strong position to study a fundamental step in eukaryotic ribosome biogenesis; the transition from the pre-ribosome particle (90S) to the pre-40S particle (small subunit precursor). Our overarching model is that the methyltransferase Bud23 monitors that status of 40S assembly, triggering the RNA helicase Ecm16 to promote release of the pre-40S from 90S only after completion of transcription and critical folding of the RNA. A major achievement in our preliminary results is our ability to trap an Ecm16-intermediate particle. To begin to address this model, our studies test four specific hypotheses: (i) Ecm16 is the helicase that dissociates U3 snoRNA and its associated proteins from the pre-rRNA (ii) Bud23 activates this helicase activity; (iii) Imp4 stabilizes the duplex that is the target of Ecm16; and (iv) the ribosomal protein Rps2 chaperones formation of the central pseudoknot, a key structural feature in 18S rRNA whose formation is sterically blocked until Ecm16 disrupts the U3-pre-rRNA interactions. The pre-rRNA processing pathways and the genes to be studied in the yeast Saccharomyces cerevisiae have counterparts in higher eukaryotes. Hence, S. cerevisiae is our model organism of choice because it allows us to combine powerful molecular genetic and biochemical approaches.

PI: Arlen W Johnson Summary Ribosomes are responsible for the rapid and accurate production of all proteins in cells in all forms of life on earth. The ability of these molecular machines to carry out faithful translation depends on their complex structure that allows dynamic interaction with ligands. The mature ribosome in eukaryotic cells is composed of two parts, the large 60S subunit that carries out polypeptide synthesis and the small 40S subunit that decodes mRNA. The assembly of a eukaryotic ribosome involves over 200 accessory assembly factors, whose function, in many cases, is still unknown. Considering the complexity of ribosome structure and function and its critical role in decoding our genetic information, ensuring their correct assembly would seem a necessary but daunting task for cells. Lately, considerable interest has been focused on mechanisms of quality control in the ribosome biogenesis pathway. This proposal focuses on two distinct topics within ribosome assembly; (1) completion of the peptidyl transferase center of the 60S subunit and the mechanisms for assessing its functional integrity and (2) the transition from the early 90S pre-ribosomal precursor to the pre-40S precursor. This proposal is directed at understanding the quality control mechanisms that assess the functional and structural integrity of the peptidyl transferase center of the subunit, upon insertion of ribosomal protein Rpl10. This proposal builds on our recent determination of the atomic structure of a preribosome and release of two factors, Tif6 and Nmd3. The release of Nmd3 and Tif6 is dependent on the two GTPases Efl1 and Lsg1 and constitutes the primary quality control check point in during 60S maturation. In humans, defects in the quality control step lead to T-cell acute lymphoblastic leukemia and Shwachman-Diamond syndrome. Assembly of the small ribosomal subunit involves stepwise cotranscriptional assembly of the 90S particle, a large protein-RNA complex, scaffolded on U3-snoRNA. However, the presence of U3 is mutually incompatible with the final folded structure of small subunit RNA and must be removed once transcription of the RNA is complete and the 90S particle has fully assembled. The transition from the 90S to pre-40S is poorly understood. We propose that the displacement of U3 by the RNA helicase Dhr1 is a primary event that drives the transition of the 90S into the pre-40S particle. We will determine how the activity of Dhr1 is regulated to ensure the timely release of U3.