RESUMO
In eukaryotes, ribosome biogenesis requires folding and assembly of the precursor rRNA (pre-rRNA) with a large number of proteins and snoRNPs into huge RNA-protein complexes. In spite of intense genetic, biochemical and high-resolution cryo-EM studies in Saccharomyces cerevisiae, information about the structure of the 35S pre-rRNA is limited. To overcome this, we performed high-throughput SHAPE chemical probing on the 35S pre-rRNA within 90S pre-ribosomes. We focused our analyses on external (5'ETS) and internal (ITS1) transcribed spacers as well as the 18S rRNA region. We show that in the 35S pre-rRNA, the central pseudoknot is not formed and the central core of the 18S rRNA is in an open configuration but becomes more constrained in 20S pre-rRNA. The essential ribosome biogenesis protein Mrd1 influences the structure of the 18S rRNA region locally and is involved in organizing the central pseudoknot and surrounding structures. We demonstrate that U3 snoRNA dynamically interacts with the 35S pre-rRNA and that Mrd1 is required for disrupting U3 snoRNA base pairing interactions in the 5'ETS. We propose that the dynamic U3 snoRNA interactions and Mrd1 are essential for establishing the structure of the central core of 18S rRNA that is required for processing and 40S subunit function.
Assuntos
Conformação de Ácido Nucleico , RNA Nucleolar Pequeno/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Sítios de Ligação , Nucléolo Celular/química , Nucléolo Celular/genética , Precursores de RNA/genética , RNA Ribossômico 18S/genética , RNA Nucleolar Pequeno/química , Ribossomos/química , Saccharomyces cerevisiae/genéticaRESUMO
While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a high-throughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein-rRNA interactions, providing intriguing insights into the temporal order of protein-rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.Ribosome biogenesis is a dynamic process that involves the ordered assembly of ribosomal proteins and numerous RNA structural rearrangements. Here the authors apply ChemModSeq, a high-throughput RNA structure probing method, to quantitatively measure changes in RNA flexibility during the nucleolar stages of 60S assembly in yeast.
Assuntos
Sondas RNA/genética , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Subunidades Ribossômicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Conformação de Ácido Nucleico , Dobramento de RNA , Sondas RNA/química , Sondas RNA/metabolismo , RNA Fúngico/genética , RNA Ribossômico/genética , RNA Ribossômico 5,8S/química , RNA Ribossômico 5,8S/genética , RNA Ribossômico 5,8S/metabolismo , Subunidades Ribossômicas/química , Subunidades Ribossômicas/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Address correspondence to Sergey Belikov or Lars Wieslander, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden. E-mail: sergey.belikov@su.se or lars.wieslander@su.se.
Assuntos
Pegada de DNA , Primers do DNA/química , Sondas de DNA/química , Sondas de DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Nucleotídeos de Desoxiguanina/metabolismo , Inosina Trifosfato/análogos & derivados , Inosina Trifosfato/metabolismo , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , Transcrição Reversa , Análise de Sequência de DNARESUMO
Ribosome synthesis is an essential process in all cells. In Sacharomyces cerevisiae, the precursor rRNA, 35S pre-rRNA, is folded and assembled into a 90S pre-ribosomal complex. The 40S ribosomal subunit is processed from the pre-ribosomal complex. This requires concerted action of small nucleolar RNAs, such as U3 snoRNA, and a large number of trans-acting factors. Mrd1p, one of the essential small ribosomal subunit synthesis factors is required for cleavage of the 35S pre-rRNA to generate 18S rRNA of the small ribosomal subunit. Mrd1p is evolutionary conserved in all eukaryotes and in yeast it contains five RNA Binding Domains (RBDs) separated by linker regions. One of these linkers, Linker 2 between RBD2 and RBD3, is conserved in length, predicted to be structured and contains conserved clusters of amino acid residues. In this report, we have analysed Linker 2 mutations and demonstrate that it is essential for Mrd1p function during pre-ribosomal processing. Extensive changes of amino acid residues as well as specific changes of conserved clusters of amino acid residues were found to be incompatible with synthesis of pre-40S ribosomes and cell growth. In addition, gross changes in primary sequence of Linker 2 resulted in Mrd1p instability, leading to degradation of the N-terminal part of the protein. Our data indicates that Linker 2 is functionally coupled to RBD2 and argues for that these domains constitute a functional module in Mrd1p. We conclude that Linker 2 has an essential role for Mrd1p beyond just providing a defined length between RBD2 and RBD3.
Assuntos
Peptídeo Hidrolases/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Divisão Celular , Mutação , Ligação Proteica , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , Ribossomos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismoRESUMO
Ribosome biogenesis in eukaryotes requires coordinated folding and assembly of a pre-rRNA into sequential pre-rRNA-protein complexes in which chemical modifications and RNA cleavages occur. These processes require many small nucleolar RNAs (snoRNAs) and proteins. Rbm19/Mrd1 is one such protein that is built from multiple RNA-binding domains (RBDs). We find that Rbm19/Mrd1 with five RBDs is present in all branches of the eukaryotic phylogenetic tree, except in animals and Choanoflagellates, that instead have a version with six RBDs and Microsporidia which have a minimal Rbm19/Mrd1 protein with four RBDs. Rbm19/Mrd1 therefore evolved as a multi-RBD protein very early in eukaryotes. The linkers between the RBDs have conserved properties; they are disordered, except for linker 3, and position the RBDs at conserved relative distances from each other. All but one of the RBDs have conserved properties for RNA-binding and each RBD has a specific consensus sequence and a conserved position in the protein, suggesting a functionally important modular design. The patterns of evolutionary conservation provide information for experimental analyses of the function of Rbm19/Mrd1. In vivo mutational analysis confirmed that a highly conserved loop 5-ß4-strand in RBD6 is essential for function.
