Дисертації з теми "Large ribosomal subunit"

L'activité de traduction des ribosomes est portée directement par les ARN ribosomiques (ARNr) qui composent ses deux sous-unités. La grande sous-unité (60S) est formée par les ARNr 25S, 5.8S et 5S et la petite sous-unité (40S) est formée par l'ARNr 18S. Un des principaux buts de la biogenèse des ribosomes est de convertir les ARNr en molécules correctement repliées et donc actives. La production des sous-unités ribosomiques est le résultat d'étapes successives de maturation de particules précurseurs, les pré-60S et les pré-40S, précurseurs de la grande (60S) et la petite (40S) sous-unité ribosomique, respectivement. Des protéines ribosomiques, des facteurs d'assemblage (FA) et des petites particules ribonucléoprotéique (snoRNPs) sont impliquées dans ces étapes successives. Ces facteurs jouent des rôles importants dans l'organisation spatiale et le maintien de l'intégrité structurelle des ARNr. Les ARN hélicases forment le plus grand groupe des FA et peuvent moduler les interactions ARN-ARN et ARN-protéine. Elles forment des candidates potentielles du repliement tridimensionnelle des ARNr. Cependant, les mécanismes par lesquels ces enzymes participent à la production des ribosomes restent vagues. Dans cette étude, on se focalise sur la fonction de l'ARN hélicase à boîte DEAD Dbp6 dans la structuration précoce de l'ARNr de la grande sous-unité ribosomique (60S). Dbp6 est essentielle pour la production de la grande sous-unité ribosomique. En son absence la production de la première particule pré-60S est inhibée. Néanmoins, l'importance des activités enzymatiques de Dbp6 pour la production de la première particule pré-60S n'ont pas été évalué et ses substrats ARN n'ont pas été déterminé. Dans notre étude, nous avons démontré que Dbp6 montre les activités biochimiques attendues, tels que l'hydrolyse d'ATP et la liaison à l'ARN. Dbp6 n'a pas montré une activité de dissociation de brins d'ARN (hélicase) dans les conditions testées dans le laboratoire. Nous avons pu identifier et étudier une activité d'association de brins (annealing) qui est contrôlée par l'ATP. En étudiant des mutants de Dbp6 qui ciblent les motifs conservés du cœur hélicase, nous avons établi que l'hydrolyse ATP est importante mais pas essentielle pour la survie cellulaire. Cependant, l'activité d'annealing semble jouer un rôle clé dans la fonction moléculaire de l'enzyme. Nous avons ensuite identifié les substrats in vivo de Dbp6 par l'expérience de pontage aux UV et analyse de l'ADNc (CRAC). Cela a montré que Dbp6 interagie principalement avec des snoARN qui se lient dans la région 5' de l'ARNr 25S et parmi lesquels certains sont des snoARN orphelins qui ne guident pas de modifications chimiques de nucléotides. Ces résultats soutiennent la notion que Dbp6 pourrait participer à l'organisation spatiale de cette région de l'ARNr de la grande sous-unité par l'intermédiaire des snoARN chaperons
The translation activity of ribosomes is directly held by the ribosomal RNAs (rRNAs) composing its two subunits. The large ribosomal subunit (60S) is formed of the 25S, 5.8S and 5S rRNAs and the small ribosomal subunit (40S) of the 18S rRNA. One of the main goals of ribosome biogenesis is to turn the rRNAs into correctly folded and active molecules. The production of the ribosomal subunits is the result of successive processing and maturation steps of precursor particles, the pre-60S and the pre-40S particles, precursors of the large (60S) and small (40S) ribosomal subunits, respectively. Ribosomal proteins (RPs), assembly factors (AFs) and small ribonucleoprotein particles (snoRNPs) are implicated in these successive steps. These factors play important roles in the spatial organization and in maintaining the structural integrity of the rRNAs. RNA helicases form the largest group of AFs and can modulate RNA-RNA and RNA-protein interactions. They form potential candidates for the tridimensional folding of the rRNAs. However, the mechanisms by which these enzymes participate in ribosomal particles production remain vague. In this study, we focus on the DEAD-box RNA helicase Dbp6's function in the early structuring of rRNAs of the large ribosomal subunit (60S). Dbp6 is essential for the production of the large ribosomal subunits. In its absence the production of the first pre-60S particle is impaired. Nevertheless, Dbp6 enzymatic activities' importance for the first pre-60S particle production has not been assessed nor have its RNA substrates been determined. In our study, we demonstrated that Dbp6 displays expected biochemical activities, such as ATP hydrolysis and RNA binding. Dbp6 did not show any RNA strands dissociation activity (helicase activity) in the conditions tested in the laboratory. We were able to identify and study a strand association activity (annealing activity) that is controlled by ATP. By studying Dbp6's mutants targeting the conserved helicase core motifs, we established that ATP hydrolysis is important but not essential for cell survival. However, the annealing activity seems to play a key role in the molecular function of the enzyme. We then identified Dbp6 in vivo substrates by in vivo cross-linking and analysis of cDNA experiment (CRAC). This showed that Dbp6 mostly interacts with snoRNAs that bind the 5' region of the 25S rRNA of which several are orphan snoRNA that do not guide the chemical modification of nucleotides. These findings support the notion that Dbp6 might participate in the spatial organization of this region of the large subunit rRNA by the intermediate of chaperoning snoRNAs

The Crepidotaceae (Imai) Singer (Basidiomycetes: Agaricales) represents a proposed family of saprophytic fungi containing five agaricoid (Crepidotus, Tubaria, Melanomphalia, Simocybe, Pleurotellus) and four cyphelloid (Episphaeria, Phaeosolenia, Pellidiscus, Chromocyphella) genera. Several contemporary classification systems exist that delegate some or all of these genera to other agaric families. Phylogenetic relationships for the most prevalent genera in the Crepidotaceae were investigated using nuclear large subunit ribosomal DNA (LSU rDNA) sequences. Parsimony analysis of the molecular data supports the Singer classification of Crepidotus, Melanomphalia, and Simocybe as a single monophyletic unit within the Agaricales. The affinities of the genus Tubaria remain uncertain. Crepidotus (Fr.) Staude is the largest and most phenotypically variable genus in the Crepidotaceae. Sequencing of the LSU rDNA region for a cross-section of morphologically diverse species suggests that Crepidotus is not a monophyletic genus. Analysis of morphological characters for 23 Crepidotus taxa shows that characters traditionally applied for infrageneric classification of Crepidotus are homoplasic in origin, but that less commonly emphasized characters such as spore shape and ultrastructure of spore wall ornamentation may be indicative of monophyletic clades for this complex. A unique pattern of basidiospore dormancy and germination, unknown in any other species of agaric, is reported for 11 species of Crepidotus. Similar patterns were also encountered in species of Simocybe and Melanomphalia. In these species an endogenous period of spore dormancy of four to six months is followed by an activation period where the factors necessary for subsequent germination appear to involve a minimal nutritional component, water, and exposure to light.
Master of Science

Ribosome biogenesis in eukaryotes involves the transcription, folding, and processing of ribosomal RNA (rRNA), as well as the concomitant assembly of ribosomal proteins. Several hundred trans-acting assembly factors also play a role in the complex process of ribosome biogenesis. Investigations of the construction of ribosomes have focused primarily on the roles of these assembly factors. Little is understood about how ribosomal proteins (r-proteins) function in ribosomal subunit biogenesis in vivo, in either prokaryotes or eukaryotes. I began by focusing on a subset of r-proteins surrounding the polypeptide exit tunnel of the large ribosomal subunit in yeast. R-proteins in this neighborhood, namely L17, L26, L35, and L37, are of importance because they fail to assemble with preribosomes when early pre-rRNA processing steps are blocked. I showed that these rproteins are important for the next pre-rRNA processing, cleavage of the ITS2 spacer sequence in 27SB pre-rRNA. Interestingly, I showed that this biogenesis defect is not due to changes in structure of ITS2. Instead, these r-proteins are required for stable recruitment of key assembly factors that function in this event. I then carried out a global survey of the majority of r-proteins in the 60S subunit. I found that co-transcriptional binding of r-proteins influences post-transcriptional stabilization of 60S subunit structural neighborhoods. This led to a model wherein structural domains of eukaryotic large ribosomal subunits are constructed in a hierarchical fashion. Assembly begins at the convex solvent side, followed by the polypeptide exit tunnel, the intersubunit side, and finally the central protuberance. This hierarchy serves as an initial framework to further understand 60S assembly in vivo. I also showed that pre-ribosomes become more stable as assembly proceeds and that the final steps in 60S maturation occur around regions important for ribosome function. My results also support the hypothesis that the formation of the 3’ end of 27S pre-rRNA is important for early steps of 60S assembly occurring near the 5’ end of pre-rRNA. I also studied the functions of conserved and eukaryote-specific extensions of rproteins that are intrinsically disordered. This revealed distinct roles of extensions in 60S subunit biogenesis and supported a model for the sequential binding of globular and then extended domains of r-proteins during ribosome assembly. My surprising finding for a eukaryote-specific r-protein tail highlights the importance of understanding why several yeast r-proteins have evolved extra sequences that are conserved in higher eukaryotes. Together, these investigations revealed important principles governing ribosome assembly. Furthermore, striking similarities and differences between assembly of bacterial and eukaryotic large ribosomal subunits also emerged, providing insights into how these RNA–protein particles evolved.

Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC.
Bizkaia:Talent and the European Union's Seventh Framework Program (Marie Curie Actions; COFUND; to S.C., A.S., T.K.); Marie Curie Actions Career Integration Grant (PCIG14-GA-2013-632072 to P.F.); Ministerio de Economía Y Competitividad (CTQ2014-55907-R to P.F., S.C.); FIRB Futuro in Ricerca from the Italian Ministero dell'Istruzione, dell'Universitá e della Ricerca (RBFR130VS5_001 to A.F.); Peruvian Programa Nacional de Innovación para la Competitividad y Productividad (382-PNICP-PIBA-2014 (to P.M. and A.F.)). Funding for open access charge: Institutional funding.
Revisión por pares

In all life ribosomes are the ribonucloprotein machines in charge of decoding the genetic code and synthesizing proteins. In eukaryotes, ribosomes are pre-assembled in the nucleus and exported to the cytoplasm where the final maturation steps occur prior to their partaking in translation. My dissertation work focused on aspects of the last two known steps of the pre-60S subunit cytoplasmic maturation. In the penultimate step, the anti-association factor Tif6 is released from 60S by the concerted action of the translocase-like GTPase Efl1 and Sdo1. The release of Tif6 is necessary for the ultimate maturation step, which involves release of the export adaptor Nmd3 by the ribosomal protein Rpl10 and the putative GTPase Lsg1. Nmd3 is an essential export adaptor of the 60S subunit. Nmd3 binds to the ribosome in the nucleolus and is the last known trans-acting factor to be released from the subunit in the cytoplasm. In order to gain a better understanding of the molecular events leading to the release of Nmd3 from the 60S subunit I set out to identify the binding site of Nmd3 on 60S. In a collaboration with Dr Joachim Frank’s laboratory, we obtained a cryo-EM model of Nmd3 in a complex with 60S showing Nmd3 binding to the subunit joining face of the ribosome. This work provided the first visualization of an export factor on a ribosomal subunit. The release of the anti-association factor Tif6 requires the translocase-like GTPase Efl1. Mutations in a loop of Rpl10 which embraces the P site tRNA trapped Tif6 on the subunit. These Rpl10 mutants could be suppressed by Tif6 mutants which have weakened affinity for the subunit. Mutations in Efl1 which suppress these Rpl10 mutants were also obtained. These suppressing mutations in Efl1 mapped to regions on the translocases eEF2 and EF-G important for conformational changes during translation. These results highlight molecular signaling between the P site, involving a loop of Rpl10, and Tif6, 90Å away. I propose that Efl1 promotes a translocation-like event during biogenesis of the 60S subunit prior to its first round of bona fide translation.
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The work in this thesis addresses the general problem of how ribosomal subunits are exported from the nucleus to mature in the cytoplasm. There are three parts in this dissertation. In the first part, I asked questions about the specificity for export receptors in the nuclear export of the large (60S) ribosomal subunit in yeast. In principle, I tethered different export receptors that are known to work in various unrelated export pathways to the ribosome by fusing them to the trans-acting factor Nmd3. Interestingly, all the chimeric receptors were able to support export, although to different degrees. Moreover, 60S export driven by these chimeric receptors was independent of Crm1, an export receptor that is essential for 60S export in wild-type cells. The second question I addressed in this project was whether or not a nuclear export signal could be provided in cis on ribosomal proteins (Rpls) rather than in trans by a transacting factor. The nuclear export signal (NES) of Nmd3 was fused to different ribosomal proteins and tested for support of 60S export. Several Rpl-NES fusion constructs worked to promote 60S export. Rpl3 gave the best efficiency. In conclusion, these results imply unexpected flexibility in the 60S export pathway. This may help explain how different export receptors could have evolved in different eukaryotic lineages. In the second part of my thesis, I identified the assembly pathway for the base of the ribosome stalk. The stalk is an important functional domain of the large ribosomal subunit because of its requirement for interaction with translation factors. Mrt4 is a nuclear paralog of P0, which is an essential part of the stalk. Here, I identified Yvh1 a novel ribosome biogenesis factor that is required for the release of Mrt4. Yvh1 is a conserved dual phosphatase, but the C-terminal zinc-binding domain rather than the phosphatase function was required for its activity to release Mrt4. Mrt4 localizes in the nucleus and nucleolus in the wild-type cells, but was persistent on cytoplasmic 60S subunits in yvh1[Delta] cells. The persistence of Mrt4 on the 60S subunits blocked the loading of P0 and assembly of the stalk. I also found the binding of Yvh1 depended on Rpl12, a protein that binds together with P0 to form the base of the stalk. Deletion of Rpl12 phenocopied yvh1[Delta]. These data identified the function of Yvh1 as a release factor of Mrt4. I also showed that the function of Yvh1 is conserved in human cells. In my final project, I analyzed the interdependence and order of the known cytoplasmic maturation events of the 60S subunit. 60S subunits require several maturation steps in the cytoplasm before they become competent in translation. There are four major steps involving two ATPases, Drg1 and Ssa1, and two GTPases, Efl1 and Lsg1. In my study, I ordered these steps into one serial pathway. Drg1 releases Rlp24 in the earliest step of 60S maturation in the cytoplasm. Truncation of the C-terminus of Rlp24 blocked cytoplasmic maturation of the large subunit by preventing the recruitment of Drg1 and led to a secondary defect in the release of Arx1 because of a failure to recruit Rei1. Deletion of REI1 mislocalized Tif6 from the nucleus and nucleolus to the cytoplasm and deletion of ARX1 suppressed the Tif6 mislocalization, indicating that the release of Arx1 was required for Tif6 release downstream. I found that mutation of efl1 or sdo1, the known release factors for Tif6, also blocked Nmd3 release. Tif6-V192F, which could bypass the growth defects of efl1 or sdo1 mutants, suppressed the defect of Nmd3 recycling. These results showed that the release of Tif6 was a prerequisite for Nmd3 release. Thus, the release of Nmd3 is downstream of the Tif6 release step. In conclusion, I have ordered the events of cytoplasmic maturation with Drg1 as the first step after ribosome export, followed by Rei1/Jji1 and then Sdo1/Efl1. The release of Nmd3 by Lsg1 appears to be the last step of ribosome maturation in the cytoplasm. Thus, the two ATPases Drg1 and Ssa work first and then the two GTPases Efl1 and Lsg1 work in a linear pathway of 60S maturation in the cytoplasm.
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Ramaria is a genus of epigeous fungi common to the coniferous forests of the Pacific Northwest of North America. The extensively branched basidiocarps and the positive chemical reaction of the context in ferric sulfate are distinguishing characteristics of the genus. The genus is estimated to contain between 200-300 species and is divided into four subgenera, i.) R. subgenus Ramaria, ii.) R. subgenus Laeticolora, iii.) R. subgenus Lentoramaria and iv.) R. subgenus Echinoramaria, according to macroscopic, microscopic and macrochemical characters. The systematics of Ramaria is problematic and confounded by intraspecific and possibly ontogenetic variation in several morphological traits. To test generic and intrageneric taxonomic classifications, two gene regions were sequenced and subjected to maximum parsimony analyses. The nuclear large subunit ribosomal DNA (nuc LSU rDNA) was used to test and refine generic, subgeneric and selected species concepts of Ramaria and the mitochondrial small subunit ribosomal DNA (mt SSU rDNA) was used as an independent locus to test the monophyly of Ramaria. Cladistic analyses of both loci indicated that Ramaria is paraphyletic due to several non-ramarioid taxa nested within the genus including Clavariadelphus, Gautieria, Gomphus and Kavinia. In the nuc LSU rDNA analyses, R. subgenus Ramaria species formed a monophyletic Glade and were indicated for the first time to be a sister group to Gautieria. Ramaria subgenus Ramaria and Gautieria were derived from R. subgenus Laeticolora, which formed a paraphyletic grade that included Gomphus. Ramaria subgenus Lentoramaria species also formed a paraphyletic grade in the nuc LSU rDNA analyses. The Phallales and Clavariadelphus were indicated as sister taxa to the R. stricta complex and Kavinia and R. abietina of R. subgenus Echinoramaria grouped with the basal species, R. pinicola, of R. subgenus Lentoramaria. In the mt SSU rDNA analyses, Gautieria and Gomphus again nested within Ramaria; however, the Phallales were indicated as a sister taxon to the Gomphales. A single evolutionary origin of the terrestrial habit was inferred for Ramaria with the terrestrial species, R. rainierensis, bridging the gap between the lignicolous R. subgenus Lentoramaria and the terrestrial R. subgenus Laeticolora. Species concepts tested included R. amyloidea and R. celerivirescens both of R. subgenus Laeticolora that differ primarily in the presence of clamp connections. The results supported these two taxa as distinct, sister species. These analyses were consistent with the ramarioid morphology as ancestral for the Gomphales with unique derivations of the club, false truffle and gomphoid morphologies.
Graduation date: 2000

碩士
國立陽明大學
遺傳學研究所
91
During the process of protein translocation, the nascent polypeptide chain is recognized by signal recognition particles (SRP) which carry the ribosome-nascent polypeptide complex (RNC) to reside on the ER membrane. In the previous studies, our laboratory has found that human large subunit ribosomal protein L7 specifically bound to the ER membrane. This thesis is focused on the finding of another contact point from ribosome to the ER. Based on the 2.4-angstrom high-resolution structure of Archaea (Haloarcula marismortui) 50S ribosome, and the data of cryo-EM reconstitution of eukaryotic RNC-Sec61 complex structure, human ribosomal protein L19 has tentatively been suggested as one of the contact points from ribosome to ER. Thus, in this thesis, L19 was cloned and expressed in both prokaryotic and eukaryotic expression vectors. As the result shown, we have confirmed that L19 also has the ability to bind ER. We also found that recombinant L19 has made into ribosome assembly in 293T cell. During the process, we observed that L19 was co-localized with L7 in forming of the nuclear body-like microbodies outside the nucleoli. The observations predicted that the nuclear body-like microbodies may be a precursor of ribosome assembly. In the last part of this thesis, we have made a recombinant ribosome-containing modified L19 protein which carried a heart muscle kinase (HMK) recognition sequence (RRASV) at the C-terminal end of L19 protein. We examined whether this recombinant ribosome is able to be detected by kinase reaction or not, and the result was negative, suggesting that the C-terminal end of L19 is not exposed on surface.

碩士
國立陽明大學
遺傳學研究所
92
An early cryo-EM data has proposed that ribosome has four connecting sites to the translocon of the ER. These sites are ribosomal proteins L19, L25, L26 and L35. In this study, I have focused on the study of the interaction between L35 and the ER. Firstly, I use recombinant protein L35 as a ligand to perform microsome binding assay, and the result shows that ribosomal protein L35 alone is able to bind the ER, but L35 lack of the last 11 residues at carboxyl terminal end is not, suggesting that the C-terminal end is essential for the ER-binding. Secondary, I have made a recombinant ribosome by overexpressing a phosphorylation-tagged ribosomal protein in cell. A phosphorylation peptide, RRASV was used as the tag and being inserted at C-terminal end of recombinant proteins. With this insertion the recombinant ribosome is distinguishable from native ribosome in cell. By examine the function of these recombinant ribosomes, the role of L35 protein in ribosome is defined. Using this approach, L35 and it’s truncated L35 recombinant ribosomes carried the ER-binding property. I also found that defection on one of the four connecting sites did not affect the binding ability of ribosome to the ER. Third, in the kinase labeling in vivo and in vitro experiments, I also observed that the interaction between L35 in ribosome and the translocon is very tightly because the association has prevented kinase labeling on the RRASV site of L35 protein. Finally, using cross-linking reagent I was able to find that a 10kD molecule, a possible Sec61β, is associated with L35.

Rhizopogon is a hypogeous fungal genus that forms ectomycorrhizae with genera of the Pinaceae. The greatest number and species of Rhizopogon are found in coniferous forests of the Pacific Northwestern United States, where members of the Pinaceae are also concentrated. Rhizopogon spp. are host-specific primarily with Pinus spp. and Pseudotsuga spp. and thus are an important component of these forest ecosystems. Rhizopogon includes over 100 species; however, the systematics of Rhizopogon have not been well understood. Currently the genus is placed in the Boletales, an order of ectomycorrhizal fungi that are primarily epigeous and have a tubular hymenium. Suillus is a stipitate genus closely related to Rhizopogon that is also in the Boletales and host specific with Pinaceae. I examined the relationship of Rhizopogon to Suillus and other genera in the Boletales. Infrageneric relationships in Rhizopogon were also investigated to test current taxonomic hypotheses and species concepts. Through phylogenetic analyses of large subunit and internal transcribed spacer nuclear ribosomal DNA sequences, I found that Rhizopogon and Suillus formed distinct monophyletic groups. Rhizopogon was composed of four distinct groups; sections Amylopogon and Villosuli were strongly supported monophyletic groups. Section Rhizopogon was not monophyletic, and formed two distinct clades. Section Fulviglebae formed a strongly supported group within section Villosuli. Taxonomic revisions were proposed. Suillus, Truncocolumella, and the Gomphidiaceae were transferred to the Rhizopogonaceae. In Rhizopogon, sections Amylopogon, Rhizopogon, and Villosuli were elevated to subgenera. Subgenus Roseoli was erected to accommodate the second section Rhizopogon Glade. In section Fulviglebae, Stirps Vinicolor, Rhizopogon ochraceisporus, R. subclavitisporus, and R. clavitisporus were transferred to subgenus Villosuli while the remaining species in section Fulviglebae were transferred to subgenus Rhizopogon.
Graduation date: 1999

碩士
國立陽明大學
遺傳學研究所
92
The interaction between the ribosome and ER (endoplasmic reticulum) has been revealed by cryo-electron microscopy at 15.4Å resolution. The information predicts that four contact points, namely L19, L25, L26 and L35 of ribosome, provide the major attachments to the ER. So far, no direct biochemical evidence has been given in respect to the attachments. This thesis intends to investigate the binding of human ribosomal protein L17 and L26 to the ER. In this study I have demonstrated that the phosphorylation- tagged L17 and that of L26 can be assembled into recombinant ribosomes, and maintain a normal functions in translation. In addition I have detected that the using phosphorylation probing C-terminal regions of L17 and that of L26 expose on the surface of the ribosome. In test of purified ribosomal proteins binding the rough ER it shows that the L17 and L26 can bind to ER by the microsome floating assay. Furthermore, in a cross-linking experiment I have observed that L17 does not crosslink to the components of translocon protein complex, but rather associates with other high molecular weight proteins of ER. This result of this study has provided a useful information in understanding the mechanism of protein translocation.