Departamento de Genética Humana
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Browsing Departamento de Genética Humana by advisor "Candeias, Marco Marques"
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- Identification and characterization of Internal Ribosome Entry Sites (IRES) in cancer pathwaysPublication . Rodrigues Neves, Ana Rita; Loison, Luísa Romão; Candeias, Marco MarquesIn 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.
- Study on the regulation of the expression of alternative protein isoforms involved in carcinogenesisPublication . Pereira, Bruna; Loison, Luísa Romão; Candeias, Marco MarquesIn eukaryotes, gene expression is a highly complex process composed of several steps. One of those is translation, the step that converts the genetic information contained in the messenger ribonucleic acid (mRNA) into functional proteins. Under normal conditions, most proteins are translated through the canonical translation initiation mechanism, which starts with cap structure recognition at the 5’-end of mRNAs, followed by 5’ UTR (untranslated region) scanning until the appearance of an initiation codon in a favorable context. Yet, under unfavorable or energy-depriving conditions, such as endoplasmic reticulum (ER) stress, hypoxia, nutrient starvation, mitosis and cell differentiation, canonical translation is impaired and protein synthesis globally decreases. Nevertheless, some mRNAs, usually related to stress-responses, cell growth and cell death control, continue to be translated through alternative mechanisms. One of them involves internal ribosome entry sites (IRES), in which the ribosome is directly recruited to the vicinity of the initiation codon, without requiring the cap structure. This mechanism relies on mRNA secondary structures and can be assisted by some canonical factors and other auxiliary proteins named ITAFs (IRES trans-acting factors). Tumor cells take advantage of this mechanism to cope with the unfavorable conditions that characterize tumor microenvironment and proliferate. Indeed, many mRNAs containing IRES elements are found deregulated in cancer. One example is the tumor suppressor p53. The TP53 gene is the most commonly mutated gene in cancer and surprisingly, p53 mutations usually lead to the production of a mutant protein with oncogenic functions. The presence of three promoters on TP53 gene leads to the expression of different transcripts expressing different alternative translation products: the full-length transcripts allow the expression of FL-p53, Δ40p53 and Δ160p53, and a shorter transcript produces Δ133p53 and also Δ160p53. While FL-p53 and Δ40p53 protein isoforms have been widely studied in terms of internal initiation mechanisms, the fact that Δ160p53 expression is mediated through an IRES element was not known until recently. Since this shorter p53 isoform was already associated with survival, proliferation and invasion of tumor cells, the recently identified Δ160p53 IRES may have an important role in tumorigenic functions of Δ160p53. Thus, considering the aforementioned data, we proposed to study the regulation of the expression of alternative protein isoforms involved in carcinogenesis, more specifically, the regulation of Δ160p53 expression through its IRES element, aiming to understand the role of IRES-mediated translation in cancer development. Knowing that Δ160p53 IRES is located within the first 432 nucleotides of Δ160p53 coding sequence and that its activity is inhibited by Δ160p53 5’ UTR, we evaluated the effect of hotspot p53 missense mutations (R175H, R248Q, R273H e R282W) in reverting the inhibitory effect of Δ160p53 5’ UTR on Δ160p53 IRES activity. To do that, we used a bicistronic system containing two reporter genes: Renilla Luciferase (RLuc) and firefly Luciferase (FLuc). RLuc is the first cistron and its expression is driven by cap-dependent mechanisms, while FLuc is the second cistron and is only translated if there is an upstream sequence capable of promoting its translation through cap-independent mechanisms. In our experiments, we were able to see the inhibitory effect of Δ160p53 5’ UTR on Δ160p53 IRES activity, in HeLa cells, corroborating the previous reported results. Moreover, from all tested p53 missense mutations (R175H, R248Q, R273H e R282W), only R175H was capable of reverting some of the 5’ UTR inhibitory effect on Δ160p53 IRES activity. This was observed for cells under 2 μM Thapsigargin-induced ER stress, which is known to impair cap-dependent translation. The obtained results seem to indicate that R175H oncogenic functions go beyond the alteration or loss of protein function, since this mutation also appears to act through an mRNA-dependent manner by inducing the expression of Δ160p53, an isoform that has already been shown to have importance in promoting tumorigenesis. Furthermore, in this thesis, we also aimed the identification of Δ160p53 IRES auxiliary proteins, using a system that takes advantage of MS2 RNA–MS2 coat protein interaction. Cloning p53 sequences of interest, such as the Δ160p53 IRES, upstream of MS2 RNA repeats, followed by co-transfection of these constructs with that expressing the MS2 coat protein and co-immunoprecipitation, will allow the identification of p53 mRNA-interacting proteins, through mass spectrometry. Here, we describe some of the cloning strategies used, though unsuccessfully, to attempt to clone p53 sequences of interest upstream MS2 repeats as well assome possible solutions. Moreover, knowing that Hdm2 (murine double minute 2 human homolog) interacts with several mRNAs commonly deregulated in cancer cells, such as p53 and XIAP (X-linked inhibitor of apoptosis protein), regulating their non-canonical translation, we also performed Hdm2 immunoprecipitation optimizations, so that, in the future, co-immunoprecipitation of Hdm2-bound mRNAs can be performed. Then, new possible IRES-containing mRNAs regulated by Hdm2 that may also have a preponderant role in cancer progression will be identified by RNA sequencing. At the end, concluding all these lines of research, we hope to unveil new insights regarding IRES-mediated translation of cancer-related mRNAs. In fact, understanding how IRES-containing mRNAs are regulated under different stress conditions and how the switch between cell homeostasis and cell neoplastic transformation is triggered, will provide important knowledge for the development of new therapeutic strategies.
- Studying and targeting the functions of p53 mRNA in carcinogenesisPublication . Damasceno, Beatriz; Candeias, Marco Marques; Dias, DeodáliaThe majority of mRNA translation is ensured by the canonical cap-dependent translation, which represents the cellular process with the biggest expense of energy and resources. Thus, in response to various adverse conditions such as hypoxia, viral infection, nutrients and growth factors starvation, endoplasmic-reticulum stress (ER-stress), among others, the canonical cap-dependent translation mechanism is supressed. However, in these adverse situations the expression of certain proteins that mediate adaptation and damage repair is of extreme importance. Under these limiting conditions some key mRNAs ensure the translation of these pivotal proteins through alternative non-canonical translation initiation mechanisms. One well-known example of a regulatory protein that is expressed even in very unfavourable conditions is the tumour suppressor p53. The TP53 gene, containing three promoters and together with alternative internal initiation and alternative splicing, expresses at least fifteen reported isoforms. The p53 isoforms have been shown to have unique transcription targets, often linked to non-redundant functions, and demonstrate responsiveness to different specific stress signals. These findings help to better understand how p53 integrates such a variety of pathways including DNA repair, cell cycle regulation, apoptosis, metabolism and even aging. Having such a wide range of functions that deeply impact the cellular survival, p53 expression is regulated through a variety of mechanisms that include the Hdm2 (murine double minute 2 human homolog) mediated poly-ubiquitination and 26S-mediated proteasomal degradation. However, despite its tight regulation, p53 proteins are frequently reported to grant cells with carcinogenic-like features, causing particularly aggressive and treatment-resistant tumours. Indeed, TP53 gene is the most commonly mutated gene in cancer, representing a strategic target for transformed cells, as the aberrant expression of the p53 protein isoforms offers the means to overcome the extremely challenging microenvironments, characteristic in cancer onset and tumour development. Our team's research has been greatly focused on the Δ160p53 isoform, specifically the study of the recently identified Δ160p53 IRES, as this isoform promotes, to a greater extent than other p53 isoforms, nefarious carcinogenic-like functions, namely invasion and cellular proliferation. One approach we propose to better understand the Δ160p53 IRES-mediated translation initiation mechanism is the identification of small drug compounds that inhibit Δ160p53 IRES. For this purpose, we started by establishing the optimal conditions, cell line and the system for this drug screening. Simultaneously, we cloned neomycin resistant plasmid constructs containing a bicistronic system with two reporter genes (Renilla Luciferase and Firefly Luciferase), using already available Δ160p53 IRES containing sequences, in order to select stable eukaryotic cells for the drug screening assays. Even though at the time of this thesis' experiments we did not have the chance to test the cloned plasmids and evaluate their performance in the drug screening assays, we present the best cloning strategy out of the two approaches used. In parallel, our laboratory has invested in the identification of other cancer-related IRES-translated mRNAs specifically regulated by Hdm2, as it is known that Hdm2 can act as an ITAF, positively regulating commonly deregulated proteins in cancer. With this in mind, we modified the Hdm2 sequence using site directed mutagenesis to create two missense mutations in the 395th codon. The 395th codon has a Serine that is phosphorylated upon DNA damage, triggering the Hdm2's activity as an ITAF. Thus, we altered the Serine to an Alanine, which cannot be phosphorylated, therefore it will not be able to bind to mRNAs. The second mutagenesis aimed the modification of the Serine to Aspartic acid. The aspartic acid mimics the phosphorylated Serine, thereby permanently inducing the Hdm2's activity as an ITAF. These sequences were sent to the partner laboratory of the Department of Human Genetics in Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), in Lisbon, Portugal, to perform co-immunoprecipitation (co-IP) assays of Hdm2-bound mRNAs.For a long time, researchers focused on the p53 regulation at the protein level however, attentions started to turn to the p53 mRNA as more evidences of mRNA's regulatory functions, beyond the well studied role in translation, appeared. Indeed, it was demonstrated that p53 mRNA has a protective function over p53 protein by blocking its degradation under cellular stress, resembling a regulatory "noncoding"-like function. Furthermore, recent findings have correlated the mammalian mRNA post-transcriptional modification on adenine bases by a methyl group in the nitrogen-6 position (m6A) with cap-independent translation initiation. We were intrigued by reports that pointed to the existence of m6A modifications located in the vicinities of IRESs, suggesting a potential correlation of this alteration with IRES-mediated translation initiation. Therefore, we proposed to determine if the adenine of the 213th codon, a commonly mutated codon of the Δ160p53 isoform, bears the m6A modification. To identify the methylation on Δ160p53 mRNA at one-nucleotide resolution we optimized hybrid-DNA probe binding and T3 ligase experiments, using total RNA extracted from HeLa (Human cervical cancer-derived cell line) and A549 (Human lung carcinoma-derived cell line). Two sets of probes were custom made for the Δ160p53 segment of interest containing the 213th codon, and also for both MALAT1 positive control and Δ160p53 sequence containing AUG160, used as negative control. The obtained products were amplified through PCR, and separated by agarose gel separation. Even though it was not possible to determine if the adenine of the 213th codon is methylated, we were able to demonstrate the designed probes are binding to the complementary sequences, and also that the T3 ligase is successfully joining the left and right probes. With the fulfilment of the objectives defined for the present thesis, we seek to discern which are the best approaches for the drug screening, targeted to the inhibition of the Δ160p53 IRES, as well as offer essential tools for the Hdm2-coIP assays and drug screening. Additionally, by undertaking a new method in our laboratory to determine the existence of the still poorly understood m6A mRNA modification in Δ160p53, we ambition to lay the foundations for further studies in regard to this modification's function in non-canonical mRNA translation. Ultimately, with the present thesis we hope to assist the valuable investigation regarding the mechanisms of IRES-mediated translation, and to provide new insights that support the development of new therapeutic strategies.