Browsing by Author "Rodrigues Neves, Ana Rita"
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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.