Analysis of two human pre-ribosomal factors, bystin and hTsr1, highlights differences in evolution of ribosome biogenesis between yeast and mammals

Recent studies reveal that maturation of the 40S ribosomal subunit precursors in mammals includes an additional step during processing of the internal transcribed spacer 1 (ITS1), when compared with yeast Saccharomyces cerevisiae, even though the protein content of the pre-40S particle appears to be the same. Here, we examine by depletion with siRNA treatment the function of human orthologs of two essential yeast pre-ribosomal factors, hEnp1/bystin and hTsr1. Like their yeast orthologs, bystin is required for efficient cleavage of the ITS1 and further processing of this domain within the pre-40S particles, whereas hTsr1 is necessary for the final maturation steps. However, bystin depletion leads to accumulation of an unusual 18S rRNA precursor, revealing a new step in ITS1 processing that potentially involves an exonuclease. In addition, pre-40S particles lacking hTsr1 are partially retained in the nucleus, whereas depletion of Tsr1p in yeast results in strong cytoplasmic accumulation of pre-40S particles. These data indicate that ITS1 processing in human cells may be more complex than currently envisioned and that coordination between maturation and nuclear export of pre-40S particles has evolved differently in yeast and mammalian cells

Maturation des pré-ribosomes humains et nucléologenèse post-mitotique

Principalement étudiée chez la levure Saccharomyces cerevisiae, la biogenèse des ribosomes est un processus ubiquitaire encore peu décrit chez les mammifères. Les études menées ces dernières années dans ces organismes ont décrit de nouvelles étapes de maturation des ARNr au sein des métazoaires. Les différences dans les étapes de ce processus entre homme et levure ainsi que le nombre croissant de maladies liées à des défaut de biogenèse des ribosomes soulèvent l'intérêt de l'étude de ces mécanismes dans des organismes pluricellulaires, en particulier chez l'homme. Le but de ma thèse consistait à préciser les étapes de la maturation et de l'assemblage de petite sous-unité ribosomique, dont la production est plus simple que celle de la grande sous-unité. Pour cela, je me suis intéressée à deux orthologues humains de facteurs pré-ribosomiques de S. cerevisiae, Enp1p et Tsr1p, intervenant à des étapes distinctes, i.e. précoce et nucléaire pour Enp1p, et tardive et cytoplasmique pour Tsr1p. Les résultats obtenus ont montré la conservation de ces protéines en tant que facteurs pré-ribosomiques requis pour la maturation de la petite sous-unité chez l'homme. Nous avons ainsi décrit une nouvelle étape de maturation de l'ARNr 18S dans les cellules humaines, le pré-ARNr 21S-C, ainsi qu'un couplage export-maturation de la particule pré-40S spécifique à l'homme. Lors de la perte d'expression de la bystine et de certaines protéines ribosomiques, nous avons observé de manière inattendue un défaut de résorption des corps pré-nucléolaires en sortie de mitose. Les corps pré-nucléolaires (ou PNBs) sont des structures transitoires, décrites jusque là comme des plates-formes d'assemblage pour la reformation des nucléoles. Nos résultats mettent en évidence pour la première fois que la maturation des ribosomes reprend au sein même des PNBs. Ceci nous amène à proposer que ce soit ce mécanisme qui oriente la résorption ordonnée et progressive des PNBs en début de phase G1. De manière intéressante, la perte d'expression de certaines protéines ribosomiques impliquées dans la DBA entraîne des défauts de résorption des PNBs ainsi qu'un délai dans la progression en phase G1, ce qui soulève la question de la conséquence des défauts de la réorganisation post-mitotique du noyau dans les mécanismes physiopathologiques. L'ensemble de ces résultats mettent donc en exergue des spécificités de la biogenèse des ribosomes chez les mammifères et précisent une partie des mécanismes mis en œuvre lors de la nucléologenèse post-mitotique. Ils ouvrent ainsi un nouvel axe de recherche pour explorer des liens entre des défauts de biogenèse des ribosomes et le développement de pathologies.Mostly studied in the yeast S. cerevisiae, ribosome biogenesis is a ubiquitous process, which is still poorly described in mammals. Recent studies performed in these organisms have revealed new maturation steps in mammals. These differences between yeast and human pre-rRNA processing together with the increasing number of diseases linked to ribosome biogenesis defects have fueled interest for these mechanisms in pluricellular organisms, especially Human. The objective of this thesis was to better define the mechanism underlying assembly and maturation of the small subunit, the production of which is simpler than that of the large subunit. This study was focused in two human orthologs of yeast pre-ribosomal factors, Enp1p and Tsr1p, known to be required at distinct steps of 40S particle maturation, i.e. early and nuclear step for Enp1p and late and cytoplasmic step for Tsr1p. Our results show that these two proteins have conserved functions in mammalian small subunit biogenesis compared to yeast. However, we also find differences in the coordination between the export and the maturation of the pre-40S particle, and we describe a new 18S rRNA processing intermediate in human cells, the 21S-C pre-rRNA. After depletion of bystin or ribosomal protein, we unexpectedly observed a defect in pre-nucleolar bodies resorption at the end of the mitosis. Pre-nucleolar bodies (PNBs) are transient structures, described as assembly platforms for nucleoli reformation. Our results show for the first time that ribosome maturation is reactivated in PNBs. This leads us to propose that the ordered and progressive resorption of PNBs at the unset of the G1 phase is directed by pre-rRNA processing. Interestingly, depletion of some ribosomal proteins involved in DBA prevents resorption of PNBs and delays progression through G1 phase, which raises the issue of the possible involvement of post-mitotic nuclear organization defects in pathophysiological mechanisms. These results highlight ribosome biogenesis specificities in mammals and define part of post-mitotic nucleologenesis mechanisms

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

Human RioK3 is a novel component of cytoplasmic pre-40S pre-ribosomal particles

Maturation of the 40S ribosomal subunit precursors in mammals mobilizes several non-ribosomal proteins, including the atypical protein kinase RioK2. Here, we have investigated the involvement of another member of the RIO kinase family, RioK3, in human ribosome biogenesis. RioK3 is a cytoplasmic protein that does not seem to shuttle between nucleus and cytoplasm via a Crm1-dependent mechanism as does RioK2 and which sediments with cytoplasmic 40S ribosomal particles in a sucrose gradient. When the small ribosomal subunit biogenesis is impaired by depletion of either rpS15, rpS19 or RioK2, a concomitant decrease in the amount of RioK3 is observed. Surprisingly, we observed a dramatic and specific increase in the levels of RioK3 when the biogenesis of the large ribosomal subunit is impaired. A fraction of RioK3 is associated with the non ribosomal pre-40S particle components hLtv1 and hEnp1 as well as with the 18S-E pre-rRNA indicating that it belongs to a bona fide cytoplasmic pre-40S particle. Finally, RioK3 depletion leads to an increase in the levels of the 21S rRNA precursor in the 18S rRNA production pathway. Altogether, our results strongly suggest that RioK3 is a novel cytoplasmic component of pre-40S pre-ribosomal particle(s) in human cells, required for normal processing of the 21S pre-rRNA

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

Williams-Beureni sündroomi kromosoomiregiooni valk WBSCR22 kui ribosoomi biogeneesifaktor

Väitekirja elektrooniline versioon ei sisalda publikatsiooneRibosoomide biogeneesil osaleb ligi 300 valku, millest ligi kolmandik on seotud genoomsete haiguste ja kasvajate tekkega. Williams-Beureni sündroom (WBS) on arenguhäire, mida põhjustab umbes 30 geeni sisaldava regiooni puudumine seitsmendast kromosoomist. WBS patsientidel esineb rida erivaid probleeme – südame-veresoonkonna haigused, sidekoe arenguhäired, neuroloogilised probleemid, spetsiifiline kognitiivne profiil, kasvuprobleemid ja iseloomulikud näojooned. Ümberkorraldused WBS kromosoomiregioonis tekivad madalakoopiaarvuliste kordusjärjestuste esinemise tõttu, kuid hetkel pole teada, kuidas üksikute geenide koopiaarvu muutus põhjustab haigustunnuseid. Arvatakse, et paljud geenid on olulised WBS-i tekkel, mistõttu on vajalik nendelt geenidelt ekspresseeritavate valkude funktsioonide uurimine. Üheks WBS lookusest avalduvaks valguks, mille funktsioon enne antud töö raames sooritatud katsete tegemist oli teadmata, on WBSCR22. WBSCR22 iseloomustamine on oluline ka seetõttu, et tema avaldumistase on tõusnud mitmete kasvajate korral ja on teada, et ta mõjutab kasvajarakkude elulemust ja metastaaside teket. Antud doktoritöö esimese osa eesmärgiks oli WBSCR22 funktsiooni uurimine imetaja rakkudes. Leiti, et WBSCR22 valk asub hajusalt rakutuumas, kuid esineb ka valgu kogunemist tuumakesse – piirkonda, kus toimub ribosomaalse RNA (rRNA) süntees ja algab ribosoomide biogenees. Doktoritöös leiti, et WBSCR22 on oluline rakkude kasvuks, ribosoomi väikese subühiku biogeneesiks ja pre-rRNA protsessinguks. Täpsemalt, WBSCR22 ekspressiooni allareguleerimine põhjustab küpse 18S rRNA viimase eellase, 18S-E pre-rRNA, kuhjumist raku tuumas. See toob kaasa 18S rRNA ja ribosoomi väikese subühiku hulga vähenemise tsütoplasmas, mis võib viia rakkude aeglase jagunemiseni. Doktoritöös leiti, et üheks WBSCR22-ga seonduvaks valguks on TRMT112. TRMT112 paiknemine rakus on määratud WBSCR22 poolt: WBSCR22-TRMT112 kompleks asub raku tuumas ning kompleksi teke on vajalik WBSCR22 stabiilsuseks. TRMT112 hulga vähenemisel suunatakse WBSCR22 lagundamisele. Doktoritöö teises osas analüüsiti retroviiruste Gag valgu poolt indutseeritud viiruslaadsete partiklite (VLP-de) valgulist koostist ning avastati, et nendes VLP-des esineb suur hulk rakulisi valke, sealhulgas ribosoomivalke, kuid nende roll VLP-des ei ole teada.In human cells, almost 300 trans-acting factors are required for the production of ribosomes. Investigating the functions of these proteins is of biomedical importance, as one third of the factors are related to genetic diseases and cancer. Williams-Beuren syndrome is a developmental disorder caused by the contiguous deletion of 26–28 genes from chromosome 7. This genomic disorder is characterized by cardiovascular abnormalities, connective tissue anomalies, a characteristic neurocognitive and behavioural profile, growth delay, and a subtle but distinctive facial dysmorphology. The low copy repeat blocks of the seventh chromosome predispose to genomic rearrangements. However, it is not known how the alterations in the copy number of individual genes contribute to the disease phenotypes. Several genes probably contribute to the phenotype and therefore, it is important to study the cellular functions of the proteins expressed from the WBS locus. The gene encoding for WBSCR22 protein is deleted in WBS, and studying the function of WBSCR22 is also relevant for cancer biology, since several works have demonstrated that WBSCR22 is upregulated in various types of cancers and regulates the survival and metastatic potential of cancer cells. The first part of this dissertation is focused on studying the function of the WBSCR22 protein in mammalian cells, and revealed that WBSCR22 is important for cell growth, ribosome biogenesis and pre-rRNA processing. More precisely, silencing of WBSCR22 expression causes the accumulation of 18S-E pre-rRNA, the final precursor of 18S rRNA, in the cell nucleus, leading to a reduced amount of mature 18S rRNA and ribosome small subunit. In addition, it was found that WBSCR22 interacts with TRMT112 protein and that this interaction is important for the stability of WBSCR22 protein. In conditions where the amount of TRMT112 is limited, WBSCR22 is degraded by the proteasome. The second part of this thesis is focused on analysing the protein content of virus-like particles (VLPs) induced by the expression of murine leukaemia virus (MLV) Gag protein. The results revealed that these VLPs contain various cellular proteins, including ribosomal proteins of both subunits

The exosome and human ribosome biogenesis

Exoribonucleases have many important functions in the cell including RNA processing, turnover and quality control. One of the key 3’-5’ exonucleases is the exosome, a multiprotein complex that has been extensively characterised in yeast. Many substrates that undergo maturation and/or degradation involving the yeast exosome have been identified and these include tRNAs, mRNAs, snRNAs, snoRNAs and rRNAs. By comparison, the human exosome is poorly understood and it is not clear whether functions of the yeast exosome are conserved in higher eukaryotes. We show that the human exosome has degradation functions including the turnover, but not the processing, of snoRNAs and the recycling of excised pre-rRNA fragments. We and others have shown that the human exosome also participates in pre-rRNA processing to form the mature 3’ end of 5.8S rRNA. Here we identify a novel role for the exosome in the processing of the pre-rRNA internal transcribed spacer 1 (ITS1). The small (18S) and large (5.8S and 28S) subunit rRNAs are co-transcribed as a single precursor. Processing of ITS1 is a key step in ribosome biogenesis as it separates 18S from the large subunit rRNAs and in higher eukaryotes it involves an additional processing step compared to yeast. We define alternative ITS1 processing pathways in human cells. In the major pathway, following an endonucleolytic cleavage to separate the small and large subunit rRNAs, the exosome, which is not involved in ITS1 processing in yeast, processes to within 25 nucleotides of the 3’ end of 18S. Our data highlight significant differences between the nucleases involved in ITS1 processing in yeast and humans. However, it appears that the roles of several yeast biogenesis factors are conserved in higher eukaryotes. Further, we have investigated mechanisms by which exonucleolytic processing of ITS1 may be regulated and suggest how this could be coordinated with the final maturation steps of the pre-40S complex.EThOS - Electronic Theses Online ServiceBBSRCWellcome TrustGBUnited Kingdo

The exosome and human ribosome biogenesis

Exoribonucleases have many important functions in the cell including RNA processing, turnover and quality control. One of the key 3’-5’ exonucleases is the exosome, a multiprotein complex that has been extensively characterised in yeast. Many substrates that undergo maturation and/or degradation involving the yeast exosome have been identified and these include tRNAs, mRNAs, snRNAs, snoRNAs and rRNAs. By comparison, the human exosome is poorly understood and it is not clear whether functions of the yeast exosome are conserved in higher eukaryotes. We show that the human exosome has degradation functions including the turnover, but not the processing, of snoRNAs and the recycling of excised pre-rRNA fragments. We and others have shown that the human exosome also participates in pre-rRNA processing to form the mature 3’ end of 5.8S rRNA. Here we identify a novel role for the exosome in the processing of the pre-rRNA internal transcribed spacer 1 (ITS1). The small (18S) and large (5.8S and 28S) subunit rRNAs are co-transcribed as a single precursor. Processing of ITS1 is a key step in ribosome biogenesis as it separates 18S from the large subunit rRNAs and in higher eukaryotes it involves an additional processing step compared to yeast. We define alternative ITS1 processing pathways in human cells. In the major pathway, following an endonucleolytic cleavage to separate the small and large subunit rRNAs, the exosome, which is not involved in ITS1 processing in yeast, processes to within 25 nucleotides of the 3’ end of 18S. Our data highlight significant differences between the nucleases involved in ITS1 processing in yeast and humans. However, it appears that the roles of several yeast biogenesis factors are conserved in higher eukaryotes. Further, we have investigated mechanisms by which exonucleolytic processing of ITS1 may be regulated and suggest how this could be coordinated with the final maturation steps of the pre-40S complex.EThOS - Electronic Theses Online ServiceBBSRCWellcome TrustGBUnited Kingdo