Browsing by Author "Marques-Ramos, Ana"
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- Analysis of human Argonaute 1 5’ untranslated region shows internal ribosome entry site activityPublication . Lacerda, Rafaela; Teixeira, Alexandre; Marques-Ramos, Ana; Romão, Luísa
- Analysis of the 5’ untranslated region of human UPF1 mRNA indicates both cryptic promoter and internal ribosome entry site activityPublication . Lacerda, Rafaela; Marques-Ramos, Ana; Teixeira, Alexandre; Romão, LuísaApart from its role in nonsense-mediated mRNA decay, a mechanism that promotes rapid degradation of transcripts carrying premature translation termination codons, the human up-frameshift 1 (UPF1) DNA and RNA helicase protein plays a crucial role in telomere replication and homeostasis, and in cell cycle progression. Due to its relevance for several physiological roles, and to the fact that it is expressed during G2/M phase, in which overall protein synthesis is reduced, we hypothesized that its translation may occur via an internal ribosome entry site (IRES). IRESs can occur at the 5’ untranslated region (UTR) of transcripts and allow the direct recruitment of the ribosome to the vicinity of the main AUG, therefore bypassing the need of scanning the entire UTR. To test this hypothesis, we cloned the human UPF1 5’UTR in the dicistronic vector p_Renilla_Firefly and transfected HeLa cells with either this construct or the control counterparts. We observed a 15- to 25-fold increase in relative luciferase activity of the UPF1 5’UTR-containing construct compared to the levels obtained from the empty counterpart, which suggests the presence of an IRES. However, these levels of luciferase activity could be due to the presence of a cryptic promoter. Hence, we transfected cells with promoterless plasmids and observed a 20-fold increase in relative luciferase activity levels. These data demonstrate that UPF1 5’UTR contains a cryptic promoter, whose activity may be masking IRES activity. To check the IRES activity alone, we have transfected cells with in vitro transcribed, capped and polyadenylated mRNAs and observed a 2-fold increase in protein levels. This is also observed in two other cell lines. Besides, UPF1 IRES activity is maintained under conditions of global protein synthesis inhibition. Deletional analysis of UPF1 5’UTR revealed that the first 50 nucleotides at the 5’ end of this region are essential for both cryptic promoter and IRES activity. These results evidence, for the first time, the existence of both a cryptic promoter and an IRES element within UPF1 5’UTR and provide new insights on the regulation of UPF1 expression in human cells.
- Cap-independent translation ensures mTOR expression and function upon protein synthesis inhibitionPublication . Marques-Ramos, Ana; Romão, Luísa; Candeias, Marco; Menezes, Juliane; Lacerda, Rafaela; Willcocks, M.; Teixeira, Alexandre; Locker, NicolasThe mechanistic/mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates cellular signals from the nutrient and energy status to act, namely, on the protein synthesis machinery. While major advances have emerged regarding the regulators and effects of the mTOR signaling pathway, little is known about the regulation of mTOR gene expression. Here, we show that the human mTOR transcript can be translated in a cap-independent manner, and that its 5' untranslated region (UTR) is a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that mTOR is able to bypass the cap requirement for translation both in normal and hypoxic conditions. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell-cycle progression into S phase. These results suggest a novel regulatory mechanism for mTOR gene expression that integrates the global protein synthesis changes induced by translational inhibitory conditions.
- Cap-independent translation regulation of mammalian target of rapamycin (mTOR)Publication . Marques-Ramos, Ana; Teixeira, Alexandre; Lacerda, Rafaela; Romão, Luísa
- Characterization of an Internal Ribosome Entry Site (IRES) in p53 mRNAPublication . Neves, Ana Rita; Lacerda, Rafaela; Marques-Ramos, Ana; Romão, Luísa; Matsuda, M.; Candeias, MarcoThe 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.
- Different Bcl2 transcripts coding the same protein have different posttranscriptional control mechanismsPublication . Marques-Ramos, Ana; Teixeira, Alexandre; Romão, Luísa
- Human Argonaute 1 5’ untranslated region can mediate cap-independent translation initiation via an internal ribosome entry sitePublication . Lacerda, Rafaela; Marques-Ramos, Ana; Teixeira, Alexandre; Romão, LuísaArgonaute proteins (AGOs) are essential effectors in RNA-mediated gene silencing pathways. There are eight AGO-like proteins in human cells, grouped in two families: the eIF2C/AGO subfamily and the PIWI subfamily. The eIF2C1 gene encodes AGO1, a member of the former subfamily, that is ubiquitously expressed at low to medium levels and it is highly conserved during evolution reflecting its important physiological roles. Moreover, recent studies concluded that AGO1 protein is overexpressed in colorectal cancer, relative to adjacent non-cancer tissue, without a concomitant increase in mRNA levels. These pieces of evidence lead us to suspect that high AGO1 protein levels may be due to internal ribosome entry site (IRES)-mediated translation. IRESs are structures that can mediate cap-independent translation initiation by directly recruiting ribosomes to the AUG vicinity, thus skipping the scanning of the whole 5’ untranslated region (UTR), in response to stress. To confirm this hypothesis, we transiently transfected colorectal cancer HCT116 and cervical cancer HeLa cells with an AGO1 5’UTR-containing dicistronic vector, and luciferase activity was measured by luminometry assays. Results have shown a 2-fold increase in relative luciferase activity in both cell lines, when compared to the cells transfected with the empty counterpart (P<0.05). Transfection of the corresponding promoterless plasmids ruled out the hypothesis of this fold to be due to the existence of a cryptic promoter. In addition, RT-PCR analyses of the dicistronic mRNAs confirmed that no cryptic splicing occurs. Besides, the knock-down of the eIF4E subunit induced a significant 2- to 4-fold increase in IRES activity. Taken together, these data suggest the presence of an IRES in the AGO1 5’UTR whose biological relevance is still under thorough investigation.
- Human mTOR mRNA allows cap-independent translation that insures its expression and function during inhibition of global translationPublication . Marques-Ramos, Ana; Candeias, Marco; Menezes, Juliane; Lacerda, Rafaela; Willcocks, M.; Locker, Nicolas; Romão, LuísaThe mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates signals from the cellular nutrient- and energy-status, acting namely on the protein synthesis machinery. Deregulation of mTOR signaling is implicated in major diseases, such as cancer, mainly due to its role in regulating protein synthesis. Major advances are emerging regarding the regulators and effects of mTOR signaling pathway; however, regulation of mTOR gene expression is not well known. Here, we show that the 5’ untranslated region of the human mTOR transcript forms a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that this cis-acting RNA regulon is active both in normal and stress conditions, and that its activation status in response to translational adverse conditions parallels mTOR protein levels. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell cycle progression into S-phase. These results suggest a novel regulatory mechanism of mTOR gene expression that integrates the protein profile rearrangement triggered by global translation inhibitory conditions.
- Human UPF1 translation initiation is regulated by a cap-independent mechanismPublication . Lacerda, Rafaela; Menezes, Juliane; Marques-Ramos, Ana; Teixeira, Alexandre; Romão, LuísaGene expression is a very intricate process comprising several tightly regulated steps. One of those is translation initiation that, under normal circumstances, is mostly cap-dependent. However, some proteins can initiate translation via a cap-independent mechanism. This allows the maintenance of protein synthesis under conditions that reduce global protein synthesis. Human up-frameshift 1 (UPF1) has a key role in several cellular processes such as nonsense-mediated mRNA decay, telomere replication and homeostasis, and cell cycle progression, suggesting a tight regulation in order to prevent abnormal proliferation. These data suggest UPF1 might initiate translation in a cap-independent way, allowing the cell to overcome stress conditions that impair cap-dependent translation. To test this hypothesis, we cloned the UPF1 5’UTR in a dicistronic vector and transfected cervical and colorectal cancer cell lines with either this construct or the control counterparts. We observed a 15- to 25-fold increase in relative luciferase activity of the UPF1 5’UTR-containing construct compared to the levels obtained from the empty counterpart in all tested cell lines, suggesting a cap-independent translation initiation. Cells transfection with in vitro transcribed mRNAs resulted in a 2-fold increase in protein levels, confirming translation can occur in a cap-independent way. This is maintained under conditions of global protein synthesis inhibition. Deletional analysis of the UPF1 5’UTR revealed that the minimal core required for cap-independent activity is present either within the first 100 nucleotides or within the last 125. Further experiments are being undertaken to understand the biological role of a cap-independent mechanism for the translation of UPF1 and how it contributes to the roles UPF1 plays in the cell.
- Human UPF1: a cap-independent translation initiation mechanism and a cryptic promoter regulate its gene expression in cancer cellsPublication . Lacerda, Rafaela; Marques-Ramos, Ana; Teixeira, Alexandre; Romão, LuísaCervical (CC) and colorectal (CRC) cancers are among the leading causes of death worldwide and their development and progression is dependent on regulation of gene expression. Eukaryotic cells possess a variety of post-transcriptional mechanisms by which they regulate gene expression, namely at translation initiation level. Although in most cases it occurs via the cap-dependent mechanism, several oncogenes, growth factors or proteins involved in the regulation of programmed cell death, among others, have a different translation initiation mechanism. It involves the direct recruitment of the ribosome to the vicinity of the initiation codon without the involvement of the cap structure. This allows the maintenance of protein synthesis under several cellular stresses and promotes tumourigenesis. Apart from its role in nonsense-mediated decay, the human up-frameshift 1 (UPF1) DNA and RNA helicase protein plays a crucial role in telomere replication and homeostasis, and in cell cycle progression. In addition, its expression levels are maintained in every phase of the cell cycle, thus indicating that its translation may occur via a cap-independent mechanism. To test this hypothesis, we cloned the human UPF1 5’UTR in a dicistronic vector and transfected CC and CRC cell lines with either this construct or the control counterparts. We observed a 15- to 25-fold increase in relative luciferase activity of the UPF1 5’UTR-containing construct compared to the levels obtained from the empty counterpart in all tested cell lines, suggesting a cap-independent translation initiation. To control whether these levels of luciferase activity could be due to the presence of a cryptic promoter, we transfected cells with promoterless plasmids and observed the same result, demonstrating that UPF1 5’UTR contains a cryptic promoter. To check the cap-independent translation activity alone, we transfected cells with in vitro transcribed, capped and polyadenylated mRNAs and observed a 2-fold increase in protein levels, which shows that translation can occur in a cap-independent way. This is maintained under conditions of global protein synthesis inhibition. Deletional analysis of UPF1 5’UTR revealed that first 50 nucleotides are essential for both cryptic promoter and cap-independent activities. These results provide new insights on the regulation of UPF1 expression in human cancer cells.
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