Browsing by Issue Date, starting with "2014-06-03"
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- 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.
- Identification of an IRES element in the human mTOR transcript: its structural and functional featuresPublication . Marques-Ramos, Ana; Menezes, Juliane; Lacerda, Rafaela; Teixeira, Alexandre; Romão, LuísaMammalian 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. The main mTOR targets are proteins responsible for ribosome recruitment to the mRNA, thus, a specific inhibitor of mTOR, for example rapamycin, leads to global inhibition of translation. Major advances are emerging regarding the regulators and effects of mTOR signaling pathway, however, regulation of mTOR gene expression, is not well known. Knowing that in stress conditions such as hypoxia, overall protein synthesis is reduced, but synthesis of mTOR protein is not inhibited, we hypothesized that mTOR 5’UTR harbors an IRES allowing cap-independent synthesis of mTOR protein in stress conditions. By using dicistronic reporter plasmids we have tested and confirmed this hypothesis. In addition, we have shown that IRES-dependent translation of mTOR is stimulated by hypoxia with associated eIF2α phosphorylation, in a manner that is independent of HIF1α induction per se. The anti- and pro-apoptotic outcomes of the unfolded protein response induced by endoplasmic reticulum stress also stimulates mTOR IRES activity, with a more pronounced effect in the pro-apoptotic phase with associated eIF2α phosphorylation. Furthermore, we have demonstrated that mTOR IRES activity is potentiated by mTORC1 inactivation, suggesting a feedback loop in order to maintain mTOR expression. Our data point out a novel regulatory mechanism of mTOR gene expression that integrates the protein profile rearrangement triggered by global translational inhibitory conditions.
- 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.
- Translational control of the human erythropoietin via an upstream open reading frame in cardiac tissuePublication . Onofre, Claudia; Barbosa, Cristina; Romão, LuísaCellular stress activates an integrated stress response, which includes rapid changes in global and gene-specific translation. Translational regulation of specific transcripts mostly occurs at translation initiation and is mediated via different cis-acting elements present in the mRNA 5’ untranslated region (5’UTR); these elements include upstream open reading frames (uORFs). uORFs modulate translation of the main ORF by decreasing the number and/or efficiency of scanning ribosomes to reinitiate at the start codon of the main ORF. In its classical hormonal role, human erythropoietin (EPO) is a glycoprotein synthesized and released mainly from the kidney, which has a key role in hematopoiesis. However, recent studies have revealed that EPO is a multifunctional molecule produced and utilized by many tissues that rapidly responds to different cell stress stimuli and tissue injuries. The 5’ leader sequence of the human EPO mRNA has one uORF with 14 codons, which is conserved among different species, indicating its potential role in translational regulation. Indeed, we have recently shown that translation of human EPO mRNA is regulated by its uORF in response to hypoxia in HeLa cells (Barbosa & Romão, RNA 2014). In the present work, we aimed to test whether EPO is translationally regulated in response to amino acid starvation induced by exposure to the histidine a nalog Reporter constructs containing several EPO 5’ leader sequences fused to the Firefly luciferase cistron were tested in H9C2 (rat heart/myocardium myoblast) and C2C12 (mouse muscle myoblast) cells to evaluate the effect of this uORF in the translational regulation of EPO in cardiac and muscular cells, respectively. Luciferase activity was measured by luminometry and the corresponding mRNA levels, quantified by real-time RT-PCR. The results revealed that the EPO uORF represses the translation of the main ORF at about 60-70%, in H9C2 and C2C12 cell lines. Furthermore, we have observed that the translation reinitiation is the main mechanism involved in the production of EPO protein. Thus, EPO mRNA has a low leaky scanning ability that contributes to the translational repression of the main ORF. Additionally, the 3’UTR is able to increase in about 2.5-fold the protein levels of luciferase in C2C12 cells probably by stabilizing the mRNA. On the contrary, in H9C2 cells the 3’UTR does not alter the protein levels showing that this structure has no influence in the translation regulation in the cardiac tissue. A better knowledge of the mechanisms implicated in EPO gene expression may be valuable in the determination of therapies for several human disorders, including cardiac disorders.