National Institute of Health Dr. Ricardo Jorge
Repositório Científico do Instituto Nacional de SaúdeNot a member yet
9086 research outputs found
Sort by
The interplay between mRNA translation and nonsense-mediated decay in transcripts with short open reading frames
No full textMammalian nonsense-mediated mRNA decay (NMD) is a splicing- and translation-dependent surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). In addition, several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs, and examples of natural NMD targets are transcripts containing upstream short open reading frames or long 3’ untranslated regions.
The strength of the NMD response appears to reflect multiple determinants on a target mRNA. In general, the location of a PTC greater than 50 nucleotides upstream to the last exon-exon junction constitutes a major determinant of NMD. However, we have reported that human mRNAs with a PTC in close proximity to the translation initiation codon (AUG-proximal PTC), and thus, with a short open reading frame, can substantially escape NMD. Our data support a model in which cytoplasmic poly(A)-binding protein 1 (PABPC1) is brought into close proximity with an AUG-proximal PTC via interactions with the translation initiation complexes. This proximity of PABPC1 to the AUG-proximal PTC allows PABPC1 to interact with eRF3 with a consequent enhancement of the release reaction and repression of the NMD response. Here, we present strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. In addition, we dissect the biochemical interactions of the eIF3 subunits in bridging PABPC1/eIF4G complex to the 40S ribosomal subunit. Together, our data provide a framework for understanding the mechanistic details of PTC definition and translation initiation.This work was partially supported by Fundação Luso-Americana para o Desenvolvimento and Fundação para a Ciência e a Tecnologia (UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC).N/
The interaction between mRNA translation and nonsense-mediated decay in AUG-proximal nonsense-mutated transcripts
No full textNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTCs). Although NMD has been intensively studied, it is still poorly understood how NMD discriminates between PTCs and normal stop codons. The unified model for NMD proposes that the decision of NMD triggering is the outcome of the competition between the cytoplasmic poly(A)-binding protein 1 (PABPC1) and the NMD effector UPF1 for the termination complex. Consequently, PTCs located far, in a linear sense, from the poly(A) tail and associated PABPC1, in mRNAs containing residual downstream exon junction complexes (EJCs), are expected to elicit NMD. Nevertheless, we have reported that human mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD through a mechanism independent of translation re-initiation. Here, we will present the mechanistic basis for this NMD resistance and how it involves the step of mRNA translation initiation.This work was partially supported by Fundação para a Ciência e a Tecnologia (PEst-OE/BIA/UI4046/2011 and FCT/PTDC/BIM-MED/0352/2012).N/
A network integrative approach to unravel new links between NMD and mRNA processing pathways
No full textNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and
selectively degrades mRNAs carrying premature translation termination codons (PTCs).
This process has been associated with many genetic diseases and some forms of cancer
caused by nonsense or frameshift mutations that introduce PTCs. Moreover, recent
studies have shown that NMD is also involved in the regulation of a large number of
transcripts, suggesting a major role in the control of gene expression. To further
investigate the biological relevance of NMD and how this process can be modulated, we
used a network analysis approach that integrates 1) protein-protein, 2) kinasetarget,
3) phosphatase-target, 4) miRNA-target, 5) transcription factors-target, 6)
gene co-expression, 7) ubiquitination and 8) signaling interactions. The generated
network was used to find novel NMD-associated proteins, prioritizing candidates with
simultaneous interactions with different mRNA processing pathways (mRNA splicing,
mRNA transport, mRNA translation and mRNA decay). Taking in account all information
sources integrated in our network, we have created a scoring algorithm to identify
new potentially important players in NMD, which can be essential to further
understand the interplay between mRNA translation, PTC definition and NMD. Due to the
diversity of regulatory links integrated in this workflow, we propose it can be
applied to find molecular bridges between related biological processes and generate
novel hypotheses about the molecular mechanisms co-regulating these phenomena.FCT-PD/BD/130959/2017N/
Characterization of an Internal Ribosome Entry Site (IRES) in p53 mRNA
No full textThe tumour suppressor p53 gene is one of the most studied cancer-related genes. So far, many p53 isoforms have been identified either resulting from alternative splicing or from non-canonical translation mechanisms. It is known that cap-dependent translation is repressed under stress conditions to preserve energy. Therefore, other translational mechanisms are required to keep the synthesis of vital proteins. Internal Ribosome Entry Sites (IRESes) were first discovered in viruses, and then observed in eukaryotes, as secondary structures present in RNA that were capable of recruiting ribosomes to the vicinity of an initiation codon inserted in an optimal environment allowing cap-independent translation of mRNAs. Translation of Δ40p53, a p53 isoform, is one example of this non-canonical mechanism due to the presence of an IRES near an alternative initiation codon (AUG40). Here, we will present and characterize a new IRES in p53 mRNA. We present details on the localization, structure, function and regulation of this IRES under normal and stress conditions. Importantly, our data reveals that the function of this IRES is required for cell survival and proliferation under certain cell conditions. This finding can have grave implications for understanding p53 function dynamics and cancer progression in specific environments.FCT/PTDC/BIM-ONC/4890/2014N/
Study of the function of translating ribosomes within mRNA 5’ untranslated regions in colorectal cancer tumorigenesis
No full textColorectal cancer (CRC) has a high incidence and mortality rates worldwide. Its carcinogenenic process is based in a continuous accumulation of genetic alterations with concomitant variations in the profiles of gene expression. In order to study the variations in the gene expression profiles involved in cancer progression, genome-wide analyses have so far focused on the abundance of mRNA as measured either by microarray or RNA sequencing. However, neither approach provides information on the rate of protein synthesis, a step closer to the end-point of gene expression. Furthermore, the correlation between transcript and protein abundance is underestimated, as it does not account for the translational regulation mechanisms, thus limiting and masking the analysis of gene expression. To this end, ribosome profiling (Ribo-seq) emerges to monitor in vivo translation, providing global and quantitative measurements of translation by deep sequencing of ribosome-protected mRNA fragments (RPFs). The advent of this technique led to the identification of translation beyond the known annotated coding sequences. For instance, Ribo-seq analysis is informative about other start sites relative to the annotated canonical start codon leading to alternative open reading frames (AltORFs). In addition, this approach detects translating ribosomes within the 5’ untranslated regions (5’UTRs) consistent with the translation of upstream ORFs (uORFs), as well as ribosomes at the 3’UTR. Our aim is to determine the biological role of specific uORFs in the process of CRC tumorigenesis. For that, we will use already available Ribo-seq data from different cancer cell lines to get the 5’UTR translation profiles and choose potential uORFs-containing targets for further study. Then, we will analyze the role of such uORFs in translational regulation and study the biological function of those translatable uORFs at the level of cell viability and proliferation, and acquisition of malignant features to understand their involvement in CRC development. We analyzed the 5’UTR ribosome occupancy profiles obtained by available Ribo-seq data using a selection criteria based on a higher number of RPFs at the 5’UTR of cancer cells compared to the non-neoplasic cell lines as a proxy of uORFs translation. Then, those targets were characterized in terms of molecular function and biological process by gene ontology analysis in order to choose the ones with a cancer-related function. We are currently mapping the exact 5’-end of each transcript 5’UTR by circular rapid amplification of cDNA ends (cRACE) to finally clone them in a reporter plasmid and study their function in translational control.info:eu-repo/semantics/publishedVersio
G418 as a suppression therapy for beta-thalassemia disease
No full textPremature translation-termination codons (PTCs or nonsense codons) can arise from mutations in germ or somatic cells. The introduction of a PTC into an mRNA can trigger nonsense-mediated decay (NMD), an important mRNA surveillance mechanism that typically recognizes and degrades mRNAs containing PTCs to prevent the synthesis of C-terminally truncated proteins potentially toxic for the cell. The physiological relevance of NMD is manifested by the fact that about one third of genetic disease-associated mutations generate PTCs, including -thalassemia.
In recent years, a novel therapeutic approach entitled suppression therapy has been developed based on low molecular weight compounds to induce the translation machinery to recode a PTC into a sense codon, the so called “readthrough” (or suppression). Here, by using a model of constructs containing the 5’ part of the normal, or nonsense-mutated, human -globin gene fused to the firefly luciferase gene as a reporter, we intend to prove the principle that the suppression therapy can restore enough -globin protein to outweight the manifestations of -thalassemia. Our results from bioluminescence assays and Western blot analyses have shown that the aminoglycoside G418 is able to suppress a nonsense mutation at codon 15 or 39 of the human -globin mRNA, in cultured HEK293 cells. We are now interested in stablishing how NMD inhibition can increase the efficiency of suppression therapy. A depper study on the suppression therapy is crucial, as it offers a major approach to treat a wide range of inherited pathologies.This work is partially supported by Fundação para a Ciência e a Tecnologia (PTDC/BIM-MEC/3749/2014).info:eu-repo/semantics/publishedVersio
Expression of human UPF1 is regulated by a cap-independent translation initiation mechanism
No full textGene expression comprises several intertwined steps. Translation initiation, which, under normal circumstances, is mostly cap-dependent, can also occur via a cap-independent mechanism, which drives protein synthesis under stress conditions impairing canonical translation initiation. Human up-frameshift 1 (UPF1) is a key-protein involved in nonsense-mediated mRNA decay, telomere replication and homeostasis, and cell cycle progression. These crucial UPF1 functions suggest its tight gene expression regulation. To test whether UPF1 5’ untranslated region (5’UTR) mediates cap-independent translation, we cloned the UPF1 5’UTR in a bicistronic luciferase vector upstream the downstream cistron (Firefly luciferase [FLuc]), and transfected cervical and colorectal cancer cell lines with this construct. We observed a significant increase in FLuc expression levels compared to those from Renilla luciferase (upstream cistron) in cells transfected with the UPF1 5’UTR-containing constructs compared to those transfected with the empty transcript. To find which sequence segments are required for mediating cap-independent translation, we performed a deletional and mutational analysis of the sequence and verified that cap-independent translation was ceased when the first 100 nucleotides, or the last 125, were absent or altered. Also, such activity is maintained under canonical translation initiation-impairing conditions, such as hypoxia or endoplasmic reticulum stress. We also produced in vitro cap-lacking monocistronic UPF1 5’UTR-containing transcripts and observed a significant increase in relative FLuc expression levels in cells transfected with them.
These results indicate that UPF1 5’UTR mediates cap-independent translation initiation. Understanding this mechanism and its biological relevance might provide tools for developing new therapies for UPF1 deregulation-associated diseases, such as cancer.FCTN/
Analysis of translation of 5’ untranslated regions in colorectal cancer
No full textCarcinogenesis is characterized by a continuous accumulation of genetic alterations that changes the overall gene expression profiles. Those alterations have been stuied by microarray or RNA sequencing that measure the abundance of mRNA but do not provide information on protein synthesis, which is a step closer to end-point of gene expression. Ribosome profiling (Ribo-seq) emerges to monitor in vivo translation by deep sequencing of ribosome-protected mRNA fragments. This technique reveals the presence of ribosomes outside of known protein-coding regions, identifying translation of upstream open reading frames (uORFs) within 5’ untranslated regions (5’UTRs). Our aim is to determine the role of specific uORFs in cancer tumorigenesis, mainly in colorectal cancer (CRC). Thus, we will use already available Ribo-seq data from different cancer cell lines to get the 5’UTR translation profiles to choose potential uORFs-containing targets. Then, we will analyze the role of such uORFs in translational regulation and study the biological function of those translatable uORFs at the level of cell viability and proliferation, and acquisition of malignant features to understand their involvement in CRC development. Based in 5’UTR ribosome occupancy profiles from Ribo-seq analysis we chose ABCE1, PAIP2, eIF4G2 and eIF2A as our uORFs-containing mRNAs. By semi-quantitative RT-PCR ABCE1 transcript is shown down- and up-regulated in HCT116 and SW480 cells, respectively, in comparison to the non-neoplasic colorectal cell line (NCM460). To test the potential function of uORFs of our transcripts, we are now mapping the exact 5’-end of each 5’UTRs by circular rapid amplification of cDNA ends to finally clone them in a reporter plasmid and study their function in translational control.This work was partially supported by Fundação para a Ciência e a Tecnologia (UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC). Joana Silva is supported by a fellowship from Fundação para a Ciência e a Tecnologia (SFRH/BD/106081/2015).N/
Identification and characterization of Internal Ribosome Entry Sites (IRES) in cancer pathways
No full textDissertação de mestrado em Bioquímica, apresentada à Faculdade de Ciências da Universidade de Lisboa, 2018Trabalho desenvolvido no grupo de oncobiologia do Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA)In eukaryotes, most proteins are translated through a canonical translation initiation mechanism that involves recognition of the cap structure at the messenger ribonucleic acid (mRNA) 5’end in order to recruit the ribosome. Yet, during certain physiological and pathological conditions, canonical translation is impaired and protein synthesis is globally decreased, in part due to eIF2α phosphorylation. However, some mRNA that encode, among others, proteins associated with stress-response are translated through alternative cap-independent translation initiation mechanisms. Internal ribosome entry sites (IRES) consist of structures within the mRNA that can recruit the ribosome to the vicinities of, or directly to, the initiation codon, in a cap-independent manner. Overall, IRES-dependent translation initiation does not require the complete set of eukaryotic translation initiation factors (eIF) for ribosomal recruitment but additional factors named IRES trans-acting factors (ITAF) are required to modulate the IRES activity. Several cellular mRNA-containing IRES are related to stress-response, programmed cell death, cell proliferation, cell growth and angiogenesis, and their deregulation has been associated with tumor development. Nonetheless, IRES-mediated translation mechanisms are not well understood in eukaryotic cells nor is it their role in cancer. Therefore, the main goal of this work was to understand the role of IRES-dependent translation in cancer development with possible implications for cancer treatment. Here, we studied the putative IRES-mediated translation of two isoforms of proteins that were shown to be upregulated in several cancers, and whose expression was shown to be promoted during cap-dependent translation inhibition: the tumor suppressor p53 isoform, Δ160p53, and a yet-to-be described GTPase H-Ras isoform, p14H-Ras. Additionally, we evaluated the effect of cancer-related mutations in the activity of each putative IRES. Therefore, we used a bicistronic construct, which contained as the 5’ cistron the coding sequence of Renilla luciferase (Rluc)⸺cap-dependently translated⸺and as the 3’ cistron the coding sequence of firefly luciferase (Fluc)⸺cap-independently translated⸺, and immediately upstream Fluc’s initiation codon the putative IRES’ sequence. The expression of each protein was assessed by quantifying their respective luciferase activity by measuring the resulting bioluminescence from each reaction with the corresponding substrate. We studied the activity of both putative IRES in the absence and in the presence of thapsigargin, an inhibitory drug of a calcium pump from the endoplasmic reticulum (ER), which leads to ER stress, and, consequently to eIF2α phosphorylation. In previous reports, Δ160p53 was shown to be expressed in an IRES-dependent way from an IRES located within the first 432 nucleotides (nt) from Δ160p53 coding sequence. Throughout this work, we performed an in silico analysis of Δ160p53’s 432-nt sequence, which indicated that this region might be, indeed, a good IRES candidate. Although not statistically significant, our bioluminescence assays’ results suggest a putative wild-type Δ160p53 IRES activity and that Δ160p53 5’UTR represses its putative IRES activity. Regarding the effect of p53’s cancer-related missense mutations (R175H, R248Q and R273H) in the putative IRES activity, our results indicate that both R248Q and R273H are capable of inducing Δ160p53 putative IRES activity in the presence of Δ160p53 5’UTR during thapsigargin-induced ER stress, whereas R175H seems to have no effect in the IRES activity. This suggests that R248Q and R273H p53 cancer-related mutations may drive tumorigenesis by promoting IRES-dependent expression of Δ160p53, which has been shown to harbor oncogenic functions. Furthermore, according to the in silico analysis, these two mutations are located within the same loop, which corresponds to the most stable one, thus suggesting that this loop may be more important for IRES activity. Additionally, we performed initial experiments to characterize the secondary structure of Δ160p53 putative IRES by chemical probing using dimethyl sulfate (DMS) as well as to detect new IRES regulated by murine double minute 2 human homolog (Hdm2), a known ITAF of X-linked Inhibitor of Apoptosis Protein (XIAP) IRES that is also known to bind to Δ40p53 IRES and to regulate p53 expression, by RNA deep sequencing of Hdm2-bound RNA previously co-immunoprecipitated (co-IP) using anti-Hdm2 antibodies⸺we started by optimizing Hdm2 immunoprecipitation (IP). Regarding H-Ras putative IRES, preliminary experiments from our lab, showed that the expression of a yet-to-be described H-Ras short isoform, p14H-Ras, was upregulated during stress conditions, and that an H-Ras cancer-related silent mutation (T81>C), which is associated with higher risk for developing cancer, promoted its expression. Therefore, we hypothesized that H-Ras mRNA might contain an IRES within a 195-nt sequence, which corresponds to the putative sequence between the initiation codons of p21H-Ras and p14H-Ras. We started by performing an in silico analysis regarding the stability of possible structures located within the 195-nt sequence, which indicated that this region might be a good candidate, as well. Our results from the bioluminescence assays suggest that wild-type H-Ras putative IRES sequence is able to drive IRES-dependent expression under ER stress conditions, as well as the T81>C-mutated H-Ras putative IRES sequence. This suggests that T81>C mutation may induce the IRES-dependent expression of H-Ras, which may contribute for cancer development. In the future, we aim to perform a drug screening for drugs targeting both putative IRES and evaluate if we can possibly revert tumor progression using the most promising screened drugs. Additionally, we are expecting to characterize the IRES structure of both putative IRES studied throughout this work and to identify new IRES through RNA deep sequencing of samples obtained by Hdm2-bound RNA co-IP. We intend to identify proteins, whose IRES-mediated translation may be implicated in tumorigenesis, thus allowing the development of new cancer therapies.Em eucariotas, a informação genética está codificada na molécula de ácido desoxirribonucleico (DNA, do inglês deoxyribonucleic acid), sendo transcrita para ácido ribonucleico mensageiro prematuro (pre-mRNA, do inglês premature ribonucleic acid), num processo denominado transcrição, e posteriormente traduzida para proteína, num processo designado tradução. Durante a transcrição, a molécula de pre-mRNA sofre um processo de maturação durante o qual lhe são adicionadas uma estrutura cap (guanina metilada, m7G) na extremidade 5’, e uma cadeia de poliadenosinas [cauda poli(A)] na extremidade 3’, e num fenómeno designado por splicing, se dá a remoção das regiões não codificantes (intrões) que intercalam com regiões codificantes/regulatórias (exões), e a junção das últimas. Seguidamente, o mRNA maduro é translocado para o citoplasma onde é traduzido para proteína nos ribossomas. A tradução implica, normalmente, o reconhecimento da estrutura cap por factores de iniciação da tradução (eIF, do inglês eukaryotic translation initiation factor). Após este reconhecimento, o complexo de preiniciação da tradução 43 S (43S PIC, do inglês 43S preinitiation complex), que depende da formação prévia do complexo ternário [factor eucariótico de iniciação da tradução 2 (eIF2, do inglês eukaryotic translation initiation factor 2) ligado a uma guanosina trifosfato (GTP, do inglês guanosine triphosphate) e à molécula de RNA de transferência (tRNA, do inglês transfer ribonucleic acid) que transporta a primeira metionina da cadeia peptídica (Met-tRNAi, do inglês initiator tRNA methionine complex)], é recrutado para a extremidade 5’ do mRNA, de onde iniciará o rastreamento da região 5’ transcrita mas não traduzida do mRNA (5’UTR, do inglês 5’untranslated region), até que este reconheça um codão de iniciação num contexto favorável. Quando um codão de iniciação é reconhecido, inicia-se a fase de alogamento da tradução, que consiste na síntese de uma cadeia peptídica. A terminação da tradução ocorre quando um codão stop é reconhecido pelo ribossoma, o que conduz à dissociação da recentemente formada cadeia peptídica do ribossoma e à reciclagem deste. Em condições de stresse, as células reduzem globalmente a síntese proteica sobretudo através da inibição da iniciação canónica da tradução. Esta inibição pode ser mediada, entre outras vias, pela fosforilação da subunidade alfa (α) do eIF2 (eIF2α), impedindo a sua reciclagem que é necessária para a formação de um novo complexo ternário, e consequentemente, do 43S PIC. A fosforilação do eIF2α é mediada por diferentes cinases em resposta a diferentes estímulos, designadamente stresse do retículo endoplasmático (RE), escassez de nutrientes e danos no DNA. Contudo, proteínas associadas à resposta ao stresse podem continuar a ser traduzidas usando mecanismos alternativos, permitindo às células redireccionar os seus esforços para combater o stresse. Algumas dessas proteínas são codificadas por mRNA que contêm regiões estruturadas designadas por locais de entrada internos do ribossoma (IRES, do inglês internal ribosome entry sites), que recrutam o ribossoma internamente para a vizinhança de codões de iniciação. A iniciação da tradução através destas estruturas requer muitas vezes a interacção com proteínas específicas, ITAF (do inglês, IRES trans-acting factor), e elimina a necessidade de reconhecimento da estrutura cap. A transformação de células normais em células tumorais ocorre devido a um acumular de mutações que conduz à inactivação ou à ativação de proteínas, ou ainda à alteração da sua actividade biológica. Muitas das proteínas que se encontram alteradas em cancro regulam vias essenciais ao crescimento e desenvolvimento de uma célula, e estão também associadas a programas de resposta ao stresse. Deste modo, e como seria expectável, são muitas as proteínas associadas ao cancro, cujos mRNA contêm IRES, permitindo, deste modo, a sua expressão em condições em que a tradução canónica está inibida. Este mecanismo de protecção celular, é explorado por células tumorais, que estão muitas vezes sujeitas a condições de stresse (escassez de oxigénio, escassez de nutrientes, ou danos no DNA), de forma a aumentar a sua capacidade de sobrevivência e proliferação. Este projecto teve por objectivo o estudo da expressão mediada por IRES de isoformas de proteínas que se encontram alteradas em diversos tipos de cancro: a isoforma Δ160p53 do supressor de tumores p53, que parece apresentar funções oncogénicas previamente associadas a mutações missense no gene TP53; e uma isoforma ainda não descrita do GTPase H-Ras, p14H-Ras, cuja expressão parece ser induzida em condições de stresse do RE a um nível bastante superior em relação à expressão da isoforma canónica do H-Ras (p21H-Ras). Recorrendo a uma análise in silico da estabilidade da região com possível actividade de IRES para cada um dos alvos, e de acordo com o conteúdo em GC e a energia mínima livre de Gibbs previstos, concluímos que ambos se tratavam de bons candidatos. Adicionalmente, pretendíamos avaliar o efeito de mutações associadas ao desenvolvimento de cancro no funcionamento deste mecanismo alternativo. Desta forma, usámos um vector bicistrónico que contém, como cistrão a 5’, a região codificante da luciferase da medusa Renilla reniformis (Rluc, do inglês Renilla luciferase), e, como cistrão a 3’, a região codificante da luciferase do pirilampo Photynus pyralis (Fluc, do inglês firefly luciferase). Deste modo, analisámos a expressão de cada uma das proteínas através da respectiva actividade de luciferase por medição directa da luminescência resultante de cada uma das reacções com o respectivo substrato. Este vector bicistrónico contém um hairpin (estrutura em grampo) estável a jusante do codão stop da Rluc, para impedir que o ribossoma reinicie a tradução após terminação da tradução canónica da Rluc, de maneira que a tradução da Fluc ocorrerá de forma independente da estrutura cap, e apenas na presença de estruturas no mRNA localizadas na vizinhança do respectivo codão de iniciação que permitam o recrutamento interno do ribossoma. As sequências em estudo para a actividade de IRES foram inseridas imediatamente a montante do codão de iniciação da Fluc e as mutações estudadas foram inseridas nesses mesmos constructos por mutagénese dirigida. A actividade de IRES foi estudada na ausência e na presença de thapsigargina, uma droga inibidora da bomba de cálcio do RE, que induz o stresse deste organelo, promovendo assim a inibição da tradução canónica por fosforilação do eIF2α. Relativamente ao estudo da expressão mediada por IRES do Δ160p53, observações anteriores indicaram que Δ160p53 contém um IRES nos primeiros 432 nucleótidos (nt) codificantes da isoforma Δ160p53, e que parte da região codificante de outra isoforma do p53, Δ133p53, localizada a montante do correspondente codão de iniciação do Δ160p53 (5’UTR do Δ160p53), inibe a actividade de IRES. Usando o sistema bicistrónico descrito anteriormente, analisámos a actividade de IRES dos 432 nt do Δ160p53 e a sua inibição por parte da respectiva 5’UTR. No constructo bicistrónico que contém os 432 nt do Δ160p53 observámos um aumento (não estatisticamente significativo) na actividade de luciferase da Fluc, e que, na presença da 5’UTR, esta é inibida. Além disso, analisámos o efeito de três das mutações missense mais comuns do TP53 (R175H, R248Q e R273H) na actividade de IRES. De acordo com os nossos resultados, as mutações R248Q e R273H parecem induzir a actividade do IRES em condições de stresse do RE. Portanto, a função oncogénica destas mutações poderá estar relacionada com o aumento da expressão dependente de IRES do Δ160p53 em tecidos tumorais, promovendo a capacidade de proliferação, sobrevivência e invasão das células. Além de identificar IRES, pretendíamos caracterizá-los a nível da estrutura e da regulação. Deste modo, iniciámos o processo de optimização da caracterização in vivo da estrutura secundária do IRES do Δ160p53 através do método de modificação química de ácidos nucleicos usando o dimetilsulfato (DMS), bem como das condições de imunoprecipitação do Hdm2 (do inglês murine double minute 2 human homolog)⸺foi descrito como ITAF capaz de regular a expressão dependente de IRES do XIAP (do inglês X-linked inhibitor of apoptosis protein) e cuja interação com um IRES presente no mRNA do p53 foi observada⸺para identificar novos IRES regulados por esta ITAF através da sequenciação de RNA previamente co-immunoprecipitados usando anticorpos anti-Hdm2. Em diferentes condições de stresse, foi observado recentemente no nosso laboratório o aumento da expressão de uma isoforma ainda não descrita do GTPase H-Ras, p14H-Ras, indicando que um mecanismo de tradução alternativo poderá regular a sua expressão. Além disso, observou-se também que a presença da mutação silenciosa H27H (T81>C), associada a um maior risco de desenvolvimento de cancro, promovia o aumento da expressão de p14H-Ras em condições de stresse. Usando o mesmo sistema bicistrónico, analisámos a actividade de IRES de 195 nt da região codificante do p21H-Ras (região codificante limitada pelo codão de iniciação do p21H-Ras e pelo hipotético codão de initiação do p14H-Ras). Além disso, analisámos o efeito na actividade deste hipotético IRES da mutação silenciosa T81>C. Os nossos resultados sugerem que, em condições de stresse, esta região é capaz de mediar a tradução de forma independente da estrutura cap e que a mutação estimula a actividade de IRES. Um possível papel oncogénico desta mutação será promover a expressão desta isoforma, que, tal como Δ160p53, poderá apresentar funções oncogénicas. No futuro, pretendemos realizar um rastreio de drogas capazes de inibir a actividade dos IRES aqui estudados, e avaliar se estas poderão reverter o processo de tumorigénese. Além disso, pretendemos caracterizar a estrutura secundária de cada um destes IRES, identificar novos IRES e novas ITAF. Pretendemos, assim, identificar proteínas cuja expressão através de IRES possa estar implicada no desenvolvimento de cancro, e assim fornecer novas abordagens para a terapia desta doença.N/
The interface between mRNA translation and nonsense-mediated decay in AUG-proximal nonsense-mutated transcripts
No full textNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). In addition, several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs, and examples of natural NMD targets are transcripts containing upstream short open reading frames or long 3’ untranslated regions.
The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have reported that human mRNAs with a PTC in close proximity to the translation initiation codon (AUG-proximal PTC), and thus, with a short open reading frame, can substantially escape NMD. Our data support a model in which cytoplasmic poly(A)-binding protein 1 (PABPC1) is brought into close proximity with an AUG-proximal PTC via interactions with the translation initiation complexes. This proximity of PABPC1 to the AUG-proximal PTC allows PABPC1 to interact with eRF3 with a consequent enhancement of the release reaction and repression of the NMD response. Here, we provide strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. In addition, we dissect the biochemical interactions of the eIF3 subunits in bridging PABPC1/eIF4G complex to the 40S ribosomal subunit. Together, our data provide a framework for understanding the mechanistic details of PTC definition and translation initiation.This work was partially supported by Fundação para a Ciência e a Tecnologia (PEst-OE/BIA/UI4046/2011, FCT/PTDC/BIM-MED/0352/2012 and PTDC/BIM-MEC/3749/2014).N/