RESUMO
Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs) Ubp2 and Ubp14, and E3 ligases Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the intranuclear quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with ribosomopathies.
Assuntos
Poliubiquitina , Proteínas Ribossômicas , Ribossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Poliubiquitina/metabolismo , Poliubiquitina/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteostase , Núcleo Celular/metabolismoRESUMO
During the last ten years, developments in cryo-electron microscopy have transformed our understanding of eukaryotic ribosome assembly. As a result, the field has advanced from a list of the vast array of ribosome assembly factors toward an emerging molecular movie in which individual frames are represented by structures of stable ribosome assembly intermediates with complementary biochemical and genetic data. In this review, we discuss the mechanisms driving the assembly of yeast and human small and large ribosomal subunits. A particular emphasis is placed on the most recent findings that illustrate key concepts of ribosome assembly, such as folding of preribosomal RNA, the enforced chronology of assembly, enzyme-mediated irreversible transitions, and proofreading of preribosomal particles.
Assuntos
Microscopia Crioeletrônica , Proteínas Ribossômicas , Ribossomos , Humanos , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Ribossomos/química , Ribossomos/genética , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , RNA Ribossômico/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Modelos Moleculares , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Dobramento de RNA , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , AnimaisRESUMO
The production of new ribosomes requires proper folding of the rRNA and the addition of more than 50 ribosomal proteins. The structures of some assembly intermediates have been determined by cryo-electron microscopy, yet these structures do not provide information on the folding dynamics of the rRNA. To visualize the changes in rRNA structure during ribosome assembly in E. coli cells, transcripts were pulse-labeled with 4-thiouridine and the structure of newly made rRNA probed at various times by dimethyl sulfate modification and mutational profiling sequencing (4U-DMS-MaPseq). The in-cell DMS modification patterns revealed that many long-range rRNA tertiary interactions and protein binding sites through the 16S and 23S rRNA remain partially unfolded 1.5 min after transcription. By contrast, the active sites were continually shielded from DMS modification, suggesting that these critical regions are guarded by cellular factors throughout assembly. Later, bases near the peptidyl tRNA site exhibited specific rearrangements consistent with the binding and release of assembly factors. Time-dependent structure-probing in cells suggests that many tertiary interactions throughout the new ribosomal subunits remain mobile or unfolded until the late stages of subunit maturation.
RESUMO
Mitochondrial ribosome assembly is a complex multi-step process involving many additional factors. Ribosome formation differs in various groups of organisms. However, there are universal steps of assembly and conservative factors that have been retained in evolutionarily distant taxa. METTL17, the object of the current study, is one of these conservative factors involved in mitochondrial ribosome assembly. It is present in both bacteria and the mitochondria of eukaryotes, in particular mice and humans. In this study, we tested a hypothesis of putative METTL17 methyltransferase activity. MALDI-TOF mass spectrometry was used to evaluate the methylation of a putative METTL17 target - a 12S rRNA region interacting with METTL17 during mitochondrial ribosome assembly. The investigation of METTL17 and other mitochondrial ribosome assembly factors is of both fundamental and practical significance, because defects in mitochondrial ribosome assembly are often associated with human mitochondrial diseases.