The NIP7 protein is required for accurate pre-rRNA processing in human cells

Eukaryotic ribosome biogenesis requires the function of a large number of trans-acting factors which interact transiently with the nascent pre-rRNA and dissociate as the ribosomal subunits proceed to maturation and export to the cytoplasm. Loss-of-function mutations in human trans-acting factors or ribosome components may lead to genetic syndromes. In a previous study, we have shown association between the SBDS (Shwachman–Bodian–Diamond syndrome) and NIP7 proteins and that downregulation of SBDS in HEK293 affects gene expression at the transcriptional and translational levels. In this study, we show that downregulation of NIP7 affects pre-rRNA processing, causing an imbalance of the 40S/60S subunit ratio. We also identified defects at the pre-rRNA processing level with a decrease of the 34S pre-rRNA concentration and an increase of the 26S and 21S pre-rRNA concentrations, indicating that processing at site 2 is particularly slower in NIP7-depleted cells and showing that NIP7 is required for maturation of the 18S rRNA. The NIP7 protein is restricted to the nuclear compartment and co-sediments with complexes with molecular masses in the range of 40S–80S, suggesting an association to nucleolar pre-ribosomal particles. Downregulation of NIP7 affects cell proliferation, consistently with an important role for NIP7 in rRNA biosynthesis in human cells

The NIP7 protein is required for accurate pre-rRNA processing in human cells

Eukaryotic ribosome biogenesis requires the function of a large number of trans-acting factors which interact transiently with the nascent pre-rRNA and dissociate as the ribosomal subunits proceed to maturation and export to the cytoplasm. Loss-of-function mutations in human trans-acting factors or ribosome components may lead to genetic syndromes. In a previous study, we have shown association between the SBDS (Shwachman–Bodian–Diamond syndrome) and NIP7 proteins and that downregulation of SBDS in HEK293 affects gene expression at the transcriptional and translational levels. In this study, we show that downregulation of NIP7 affects pre-rRNA processing, causing an imbalance of the 40S/60S subunit ratio. We also identified defects at the pre-rRNA processing level with a decrease of the 34S pre-rRNA concentration and an increase of the 26S and 21S pre-rRNA concentrations, indicating that processing at site 2 is particularly slower in NIP7-depleted cells and showing that NIP7 is required for maturation of the 18S rRNA. The NIP7 protein is restricted to the nuclear compartment and co-sediments with complexes with molecular masses in the range of 40S–80S, suggesting an association to nucleolar pre-ribosomal particles. Downregulation of NIP7 affects cell proliferation, consistently with an important role for NIP7 in rRNA biosynthesis in human cells

Functional characterization of proteins NIP7 and FTSJ3 in ribosomal RNA processing in human cells

Orientador: Nilson Ivo Tonin ZanchinTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Estudos prévios realizados em nosso laboratório demonstraram a interação entre as proteínas humanas SBDS e NIP7. SBDS participa da biogênese de ribossomos e sua deficiência está associada à síndrome de Shwachman- Bodian-Diamond. NIP7 é uma proteína conservada e já foi caracterizada em levedura, onde participa da formação da subunidade ribossomal 60S. Neste trabalho, nós investigamos o papel de NIP7 na síntese de ribossomos em células humanas. A depleção de NIP7 revelou defeitos no processamento do pré-rRNA associado à produção do rRNA 18S, causando déficit na formação da subunidade ribossomal 40S. Essa divergência de resultados entre a função de NIP7 em levedura e células humanas é consistente com o fato de que NIP7 humana não complementa levedura deficiente em Nip7p. Ainda, um rastreamento em sistema de duplo-híbrido tendo NIP7 humana como isca revelou parceiros de interação diferentes daqueles reportados para Nip7p em levedura. FTSJ3 foi a parceira isolada com maior frequência. FTSJ3 é a provável ortóloga de Spb1p em levedura, a qual está envolvida na formação da subunidade ribossomal 60S. A associação entre FTSJ3 e NIP7 foi demonstrada por ensaios de pull-down e imunoprecipitação, como sendo dependente de RNA. A co-localização nucleolar e co-sedimentação dessas proteínas em fracionamento em gradiente de sacarose corroboram a associação. Além disso, células humanas deficientes em FTSJ3 revelaram defeitos na via de maturação do rRNA 18S, mesma via afetada pela depleção de NIP7. Em adição, a caracterização proteômica de complexos contendo FTSJ3 e NIP7 revelaram que essas proteínas co-purificam complexos pré-ribossomais. Uma comparação entre o conjunto de proteínas que interagem com Spb1p e as proteínas identificadas nos ensaios de pull-down com FLAG-FTSJ3 revelou que elas apresentam apenas um ortólogo em comum, o qual, incrivelmente, é Nip7/NIP7. Essas observações revelaram diferenças significativas na função desses fatores durante a síntese de ribossomos em levedura e células humanas, adicionando NIP7 e FTSJ3 na lista crescente de fatores com funções divergentes nas vias de processamento do rRNA em levedura e humanosAbstract: Previous studies from our laboratory have demonstrated the interaction between the SBDS and NIP7 human proteins. SBDS play a role in ribosome biogenesis and its deficiency is associated to the Shwachman-Bodian-Diamond syndrome. NIP7 is a conserved protein and has already been characterized in yeast, where it participates in the 60S ribosomal subunit formation. In this work, we investigated the role of NIP7 in ribosome biogenesis in human cells. NIP7 knockdown caused pre-rRNA processing defects associated to the 18S rRNA maturation, leading to deficiency in 40S ribosomal subunit synthesis. The divergence between NIP7 function in yeast and human cells is further supported by the fact that human NIP7 does not complement yeast deficient in Nip7p. In addition, a two-hybrid screen using human NIP7 as bait revealed interaction partners different from those reported for yeast Nip7p. FTSJ3 was isolated as one of the most frequent human NIP7-interacting candidates. FTSJ3 is a putative ortholog of yeast Spb1p, which has been implicated in 60S ribosomal subunit synthesis. The association between FTSJ3 and NIP7 was showed by pull-down and immunoprecipitation assays as an RNA-dependent interaction. Nucleolar colocalization and co-sedimentation on a sucrose gradiente fractionation corroborate this association. Furthermore, RNAi-mediated knockdown revealed that depletion of FTSJ3 causes pre-rRNA processing defects in the pathway leading to 18S rRNA maturation, the same pathway affected by NIP7 downregulation. In additon, proteomic characterization of FTSJ3- and NIP7- containing complexes showed that these proteins copurify pre-ribosomal complexes. A comparison of the set of Spb1p-interacting proteins with the proteins identified in the pulldown with FLAG-FTSJ3 showed that they share only one ortholog which, incredibly, is Nip7/NIP7. These observations revealed significant differences in the function of these factors during the synthesis of ribosomes in yeast and human cells, adding NIP7 and FTSJ3 to the growing list of factors with different functions in yeast and human rRNA processing pathwaysDoutoradoGenetica Animal e EvoluçãoDoutor em Genetica e Biologia Molecula

The Human Nucleolar Protein FTSJ3 Associates with NIP7 and Functions in Pre-rRNA Processing

NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A′ to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells

60S ribosomal subunit assembly dynamics defined by semi-quantitative mass spectrometry of purified complexes

During the highly conserved process of eukaryotic ribosome formation, RNA follows a maturation path with well-defined, successive intermediates that dynamically associate with many pre-ribosomal proteins. A comprehensive description of the assembly process is still lacking. To obtain data on the timing and order of association of the different pre-ribosomal factors, a strategy consists in the use of pre-ribsomal particles isolated from mutants that block ribosome formation at different steps. Immunoblots, inherently limited to only a few factors, have been applied to evaluate the accumulation or decrease of pre-ribosomal intermediates under mutant conditions. For a global protein-level description of different 60S ribosomal subunit maturation intermediates in yeast, we have adapted a method of in vivo isotopic labelling and mass spectrometry to study pre-60S complexes isolated from strains in which rRNA processing was affected by individual depletion of five factors: Ebp2, Nog1, Nsa2, Nog2 or Pop3. We obtained quantitative data for 45 distinct pre-60S proteins and detected coordinated changes for over 30 pre-60S factors in the analysed mutants. These results led to the characterisation of the composition of early, intermediate and late pre-ribosomal complexes, specific for crucial maturation steps during 60S assembly in eukaryotes

Structural studies of eukaryotic ribosome biogenesis and the sec and Bcs1 protein translocation systems

Three publications of this cumulative dissertation use cryo-electron microscopy (cryo-EM) to dissect the assembly pathway of the eukaryotic large ribosomal subunit (LSU). This pathway commences with freshly transcribed and initially unfolded rRNA in the nucleolus, which folds and incorporates ribosomal proteins while traveling to the cytoplasm, ultimately culminating in the mature LSU. During this highly complex pathway, the yeast cell must assemble four rRNAs and 79 ribosomal proteins with the help of over 200 assembly factors (AFs). Using cryo-EM, structures of nucleo\-plasmic and cytoplasmic assembly intermediates of the LSU could be solved in recent years, thus shedding light on the later stages of LSU formation. Early assembly steps remain enigmatic, as nucleolar LSU assembly intermediates have been biochemically but not structurally characterized. Taken together, we solved the structure of seven nucleolar or early nucleoplasmic intermediates at resolutions ranging from 3.3 to 4.5 Å, showing a linear assembly sequence. The first five structures show how the rRNA of the LSU is incorporated stepwise, in a non-transcriptional sequence, first forming the solvent exposed back side, and later the peptide exit tunnel and parts of the intersubunit surface (ISS). At the late nucleolar stage, the L1-stalk rRNA of domain V blocks the site of central protuberance (CP) assembly and is stabilized in a premature conformation by a range of AFs associated with the meandering, long N-terminal tail of Erb1. Two further structures show progression from this stage after release of the Erb1-Ytm1 complex by the Rea1 remodeling machinery. These intermediates, purified via Nop53, show dissociation of many early AFs from the premature ISS and destabilization of the L1-stalk. After subsequent release of the Spb1 methyltransferase, the L1-stalk rRNA can be accommodated in its mature conformation. This allows the premature CP to form, leading to a previously characterized nucleoplasmic intermediate, with a formed but premature CP. This particle is the substrate for the second Rea1 mediated structural remodeling, an intermediate of which we resolved to molecular resolution revealing Ipi1 as a central integrator for the Rix1-Ipi1-Ipi3 complex on this pre-60S particle. The binding of the Rix1-Rea1 remodeling machinery at this nucleoplasmic stage progresses maturation by inducing a 180$^{\circ}$ rotation of the 5S ribonucleoprotein particle (5S RNP) and CP. Using a combination of yeast genetics and cryo-EM we investigated the function of the AF Cgr1 in this maturation step. We showed that Cgr1 is required for CP rotation to take place, likely by stabilizing the rotated conformation. The Cgr1 function can be bypassed by introducing suppressor mutations in Rpf2 and Rrs1, two factors stabilizing the CP prior to rotation. Apart from ribosome biogenesis, two additional publications of this dissertation address protein translocation machinery, required for transport of proteins across or into membranes. The Sec translocon allows co- and posttranslational translocation of mostly unfolded substrates across the bacterial plasma and the eukaryotic endoplasmic reticulum (ER) membrane. We solved the structure of a stalled 70S ribosome-nascent chain complex (RNC) bound to the SecYEG translocon in a native like environment provided by a large lipid nanodisc. The structure shows all three subunits of the bacterial SecYEG complex and displays the lateral gate at a defined, early stage of opening or unzipping on the cytoplasmic side upon insertion of the signal anchor domain of the nascent chain. Specific pathways, such as the assembly of the mitochondrial bc1 respiratory chain complex, require folding of proteins in one compartment before translocation across a membrane to allow the protein to be active in another compartment. The bc1-complex component Rip1 folds in the mitochondrial matrix and assembles a 2Fe-2S cluster before being translocated across the inner mitochondrial membrane (IM) by the AAA-protein Bcs1. We solved the structure of Bcs1 in an ADP-bound state and two apo states, displaying a heptameric ring of Bcs1 protomers. Bcs1 forms two large aqueous vestibules separated by a seal forming middle domain. One vestibule is accessible from the matrix side and one lies within the inner mitochondrial membrane. The architecture and structural dynamics between the three states suggest an airlock like mechanism, allowing transport of folded Rip1 while maintaining the permeability barrier of the membrane

Structural studies of eukaryotic ribosome biogenesis and the sec and Bcs1 protein translocation systems

Three publications of this cumulative dissertation use cryo-electron microscopy (cryo-EM) to dissect the assembly pathway of the eukaryotic large ribosomal subunit (LSU). This pathway commences with freshly transcribed and initially unfolded rRNA in the nucleolus, which folds and incorporates ribosomal proteins while traveling to the cytoplasm, ultimately culminating in the mature LSU. During this highly complex pathway, the yeast cell must assemble four rRNAs and 79 ribosomal proteins with the help of over 200 assembly factors (AFs). Using cryo-EM, structures of nucleo\-plasmic and cytoplasmic assembly intermediates of the LSU could be solved in recent years, thus shedding light on the later stages of LSU formation. Early assembly steps remain enigmatic, as nucleolar LSU assembly intermediates have been biochemically but not structurally characterized. Taken together, we solved the structure of seven nucleolar or early nucleoplasmic intermediates at resolutions ranging from 3.3 to 4.5 Å, showing a linear assembly sequence. The first five structures show how the rRNA of the LSU is incorporated stepwise, in a non-transcriptional sequence, first forming the solvent exposed back side, and later the peptide exit tunnel and parts of the intersubunit surface (ISS). At the late nucleolar stage, the L1-stalk rRNA of domain V blocks the site of central protuberance (CP) assembly and is stabilized in a premature conformation by a range of AFs associated with the meandering, long N-terminal tail of Erb1. Two further structures show progression from this stage after release of the Erb1-Ytm1 complex by the Rea1 remodeling machinery. These intermediates, purified via Nop53, show dissociation of many early AFs from the premature ISS and destabilization of the L1-stalk. After subsequent release of the Spb1 methyltransferase, the L1-stalk rRNA can be accommodated in its mature conformation. This allows the premature CP to form, leading to a previously characterized nucleoplasmic intermediate, with a formed but premature CP. This particle is the substrate for the second Rea1 mediated structural remodeling, an intermediate of which we resolved to molecular resolution revealing Ipi1 as a central integrator for the Rix1-Ipi1-Ipi3 complex on this pre-60S particle. The binding of the Rix1-Rea1 remodeling machinery at this nucleoplasmic stage progresses maturation by inducing a 180$^{\circ}$ rotation of the 5S ribonucleoprotein particle (5S RNP) and CP. Using a combination of yeast genetics and cryo-EM we investigated the function of the AF Cgr1 in this maturation step. We showed that Cgr1 is required for CP rotation to take place, likely by stabilizing the rotated conformation. The Cgr1 function can be bypassed by introducing suppressor mutations in Rpf2 and Rrs1, two factors stabilizing the CP prior to rotation. Apart from ribosome biogenesis, two additional publications of this dissertation address protein translocation machinery, required for transport of proteins across or into membranes. The Sec translocon allows co- and posttranslational translocation of mostly unfolded substrates across the bacterial plasma and the eukaryotic endoplasmic reticulum (ER) membrane. We solved the structure of a stalled 70S ribosome-nascent chain complex (RNC) bound to the SecYEG translocon in a native like environment provided by a large lipid nanodisc. The structure shows all three subunits of the bacterial SecYEG complex and displays the lateral gate at a defined, early stage of opening or unzipping on the cytoplasmic side upon insertion of the signal anchor domain of the nascent chain. Specific pathways, such as the assembly of the mitochondrial bc1 respiratory chain complex, require folding of proteins in one compartment before translocation across a membrane to allow the protein to be active in another compartment. The bc1-complex component Rip1 folds in the mitochondrial matrix and assembles a 2Fe-2S cluster before being translocated across the inner mitochondrial membrane (IM) by the AAA-protein Bcs1. We solved the structure of Bcs1 in an ADP-bound state and two apo states, displaying a heptameric ring of Bcs1 protomers. Bcs1 forms two large aqueous vestibules separated by a seal forming middle domain. One vestibule is accessible from the matrix side and one lies within the inner mitochondrial membrane. The architecture and structural dynamics between the three states suggest an airlock like mechanism, allowing transport of folded Rip1 while maintaining the permeability barrier of the membrane

Ebp2 and Brx1 function cooperatively in 60S ribosomal subunit assembly in Saccharomyces cerevisiae

The yeast protein Ebp2 is required for early steps in production of 60S ribosomal subunits. To search for cofactors with which Ebp2 functions, or substrates on which it acts, we screened for mutants that were synthetically lethal (sl) with the ebp2-14 mutation. Four different mutant alleles of the 60S ribosomal subunit assembly factor Brx1 were found. To investigate defects of the double mutant, we constructed strains conditional for the ebp2-14 brx1- synthetic lethal phenotype. These ebp2-14 brx1 mutants were defective in processing of 27S pre-rRNA and production of 60S subunits, under conditions where each single mutant was not. Ebp2 and Brx1 exhibit a strong two-hybrid interaction, which is eliminated by some combinations of brx1 and ebp2 mutations. In one such mutant, Ebp2 and Brx1 can still associate with pre-ribosomes, but subunit maturation is perturbed. Depletion of either Ebp2 or Brx1 revealed that Brx1 requires Ebp2 for its stable association with pre-ribosomes, but Ebp2 does not depend on the presence of Brx1 to enter pre-ribosomes. These results suggest that assembly of 60S ribosomal subunits requires cooperation of Ebp2 with Brx1, together with other molecules present in pre-ribosomes, potentially including several found in assembly subcomplexes with Brx1 and Ebp2

The RNA helicase Dbp7 promotes domain V/VI compaction and stabilization of inter-domain interactions during early 60S assembly

Early pre-60S ribosomal particles are poorly characterized, highly dynamic complexes that undergo extensive rRNA folding and compaction concomitant with assembly of ribosomal proteins and exchange of assembly factors. Pre-60S particles contain numerous RNA helicases, which are likely regulators of accurate and efficient formation of appropriate rRNA structures. Here we reveal binding of the RNA helicase Dbp7 to domain V/VI of early pre- 60S particles in yeast and show that in the absence of this protein, dissociation of the Npa1 scaffolding complex, release of the snR190 folding chaperone, recruitment of the A3 cluster factors and binding of the ribosomal protein uL3 are impaired. uL3 is critical for formation of the peptidyltransferase center (PTC) and is responsible for stabilizing interac- tions between the 5′ and 3′ ends of the 25S, an essential pre-requisite for subsequent pre- 60S maturation events. Highlighting the importance of pre-ribosome remodeling by Dbp7, our data suggest that in the absence of Dbp7 or its catalytic activity, early pre-ribosomal particles are targeted for degradation

多細胞生物におけるrRNAのm1A修飾に関する研究

この博士論文は内容の要約のみの公開（または一部非公開）になっています筑波大学 (University of Tsukuba)201