The role of ribosomal proteins in activation of tumor suppressor p53

Abstract

Ciljevi istraživanja: Na temelju rezultata istraživanja u zadnjih desetak godina predložen je model u kojem inhibicija sinteze ribosoma rezultira oštecenjem jezgrice i oslobaanjem ribosomskih proteina (RP) RPL5, RPL11, RPL23, RPL26 i RPS7 iz jezgrice u jezgru, gdje vežu negativni regulator p53, Mdm2 (engl. mouse double minute 2 homolog), inhibiraju njegovu ubikvitin-ligaznu aktivnost, te posljedicno aktiviraju supresor tumora p53. Ciljevi su : 1. Odrediti specificnu ulogu RPL5, RPL11, RPL23, RPL26 i RPS7 u aktivaciji p53 nakon inhibicije razlicitih koraka u sintezi ribosoma. 2. Razjasniti precizne molekularne mehanizme putem kojih ti RP aktiviraju p53. Materijali i metode: U stanicama A549 i U-2 OS sinteza ribosoma inhibirana je aktinomicinom D (AktD), 5-fluorouracilom (5-FU) ili primjenom specificnih malih molekula RNA, siRNA (utišavajucih RNA, engl. silencing RNA) protiv komponenti koje sudjeluju u sintezi ribosoma. Podrijetlo, stabilnost i unutarstanicni smještaj novosintetiziranih RP te njihova udruženost s drugim proteinima analizirana je brojnim metodama molekularne i stanicne biologije te biokemije (tretman stanica inhibitorima sinteze proteina i proteasoma, izolacija stanicnih odjeljaka, metoda Western blot, imunotaloženje, lancana reakcija polimerazom i konfokalna mikroskopija). Rezultati: Dokazano je da su RPL5 i RPL11, a ne RPL23, RPL26 i RPS7, kljucni pozitivni regulatori p53 nakon inhibicije sinteze ribosoma. Vecina RP sintetizira se i nakon inhibicije sinteze ribosoma, ali se vrlo brzo razgrauju u proteasomima. Novosintetizirani RPL5 i RPL11 ne razgrauju se u proteasomima, vec se nakupljaju u izvanribosomskim odjeljcima gdje vežu negativni regulator p53, Mdm2. Sklop RPL5-RPL11-Mdm2-p53 transportira se u oštecenu jezgricu i udružuje s aktivatorom p53, proteinom PML (engl. promyelocytic leucemia protein), što rezultira potpunom aktivacijom p53. Zakljucci: Dokazano je da su RPL5 i RPL11 kljucni aktivatori p53 nakon inhibicije sinteze ribosoma te su razjašnjeni precizni molekularni mehanizmi ove regulacije. Naši rezultati mogu pomoci u razumijevanju patogeneze bolesti uzrokovanih poremacajima sinteze ribosoma.Objectives: Overexpression experiments suggested that perturbation of ribosome biogenesis causes nucleolar disruption and translocation of a number of ectopically expressed ribosomal proteins (RPs), including RPL5, RPL11, RPL23, RPL26, and RPS7, from the nucleolus to the nucleoplasm, where they bind to Mdm2 and inhibit its ubiquitin ligase function toward p53, leading to p53 up-regulation. Our objectives are: 1. To determine the role of specific endogenous RPs in p53 activation upon impairments of various steps of ribosome biogenesis. 2. To elucidate the molecular mechanisms by which specific RPs activate p53 under these conditions. Material and Methods: Ribosome biogenesis was inhibited with actinomycin D, 5- fluorouracil or siRNAmediated depletion of RPs. A number of biochemical as well as molecular and cellular biological methods were used to analyze the source, stability and intracellular localization of newly synthesized RPL5 and RPL11 upon impairment of ribosome biogenesis, including treatment with protein synthesis or proteasome inhibitors, Western blot analysis, qRT-PCR, immunoprecipitation, cellular fractionations and live-cell imaging microscopy. Results: We showed that RPL5 and RPL11, but not RPL23, RPL26 and RPS7, are required for p53 activation upon inhibition of ribosome biogenesis. Whereas several other newly synthesized RP are degraded by proteasomes upon inhibition of Pol I activity by actinomycin D, RPL5 and RPL11 accumulate in the ribosome-free fraction where they bind to Mdm2. This selective accumulation of free RPL5 and RPL11 is due to their mutual protection from proteasomal degradation. Furthermore, the endogenous, newly synthesized RPL5 and RPL11 continue to be imported into nucleoli even after nucleolar disruption and colocalize with Mdm2, p53 and PML. Conclusions: We demonstrate the key role of RPL5 and RPL11 in p53 activation by impairments of ribosome biogenesis and uncover the molecular mechanisms of this regulation. Our findings may have important implications with respect to understanding the pathogenesis of diseases caused by impaired ribosome biogenesis