Suppression of Cellular Transformation by Poly (A) Binding Protein Interacting Protein 2 (Paip2)

Controlling translation is crucial for the homeostasis of a cell. Its deregulation can facilitate the development and progression of many diseases including cancer. Poly (A) binding protein interacting protein 2 (Paip2) inhibits efficient initiation of translation by impairing formation of the necessary closed loop of mRNA. The over production of Paip2 in the presence of a constitutively active form of hRasV12 can reduce colony formation in a semi-solid matrix and focus formation on a cell monolayer. The ability of Paip2 to bind to Pabp is required to suppress the transformed phenotype mediated by hRasV12. These observations indicate that Paip2 is able to function as a tumor suppressor

Time-dependent increase in ribosome processivity

We created a novel tripartite reporter RNA to separately and simultaneously examine ribosome translation rates at the 5′- and 3′-ends of a large open reading frame (ORF) in vitro in HeLa cell lysates. The construct contained Renilla luciferase (RLuc), β-galactosidase and firefly luciferase (FLuc) ORFs linked in frame and separated by a viral peptide sequence that causes cotranslational scission of emerging peptide chains. The length of the ORF contributed to low ribosome processivity, a low number of initiating ribosomes completing translation of the entire ORF. We observed a time-dependent increase in FLuc production rate that was dependent on a poly(A) tail and poly(A)-binding protein, but was independent of eIF4F function. Stimulation of FLuc production occurred earlier on shorter RNA templates. Cleavage of eIF4G at times after ribosome loading on templates occurred did not cause immediate cessation of 5′-RLuc translation; rather, a delay was observed that shortened when shorter templates were translated. Electron microscopic analysis of polysome structures in translation lysates revealed a time-dependent increase in ribosome packing and contact that correlated with increased processivity on the FLuc ORF. The results suggest that ORF transit combined with PABP function contribute to interactions between ribosomes that increase or sustain processivity on long ORFs

Unidirectional constant rate motion of the ribosomal scanning particle during eukaryotic translation initiation

According to the model of translation initiation in eukaryotes, the 40S ribosomal subunit binds to capped 5′-end of mRNA and subsequently migrates along 5′-UTR in searching for initiation codon. However, it remains unclear whether the migration is the result of a random one-dimensional diffusion, or it is an energy-driven unidirectional movement. To address this issue, the method of continuous monitoring of protein synthesis in situ was used for high precision measurements of the times required for translation of mRNA with 5′-UTRs of different lengths and structures in mammalian and plant cell-free systems. For the first time, the relationship between the scanning time and the 5′-UTR length was determined and their linear correlation was experimentally demonstrated. The conclusion is made that the ribosome migration is an unidirectional motion with the rate being virtually independent of a particular mRNA sequence and secondary structure

Alternatively spliced isoforms of the human elk-1 mRNA within the 5′ UTR: implications for ELK-1 expression

The expression of cellular proteins that play central roles in the regulation of cell growth and differentiation is frequently tightly controlled at the level of translation initiation. In this article, we provide evidence that the ETS domain transcription factor ELK-1 forms part of this class of genes. Its mRNA 5′ UTR is composed of a complexed mosaic of elements, including uAUGs, uORFs and RNA structure, that interplay to modulate ribosomal access to the ELK-1 AUG start codon. Superimposed upon this is the generation of two different 5′ UTRs via alternative splicing. The two spliced isoforms show altered cellular and tissue distributions and behave differently in polysomal recruitment assays in the presence of the drug rapamycin. We propose that repression is therefore the sum of a series of interplaying negative elements within the 5′ UTRs, a situation which may reflect the need for tight translational control of ELK-1 in different tissues and under changing physiological conditions

Coordinated Destruction of Cellular Messages in Translation Complexes by the Gammaherpesvirus Host Shutoff Factor and the Mammalian Exonuclease Xrn1

Several viruses encode factors that promote host mRNA degradation to silence gene expression. It is unclear, however, whether cellular mRNA turnover pathways are engaged to assist in this process. In Kaposi's sarcoma-associated herpesvirus this phenotype is enacted by the host shutoff factor SOX. Here we show that SOX-induced mRNA turnover is a two-step process, in which mRNAs are first cleaved internally by SOX itself then degraded by the cellular exonuclease Xrn1. SOX therefore bypasses the regulatory steps of deadenylation and decapping normally required for Xrn1 activation. SOX is likely recruited to translating mRNAs, as it cosediments with translation initiation complexes and depletes polysomes. Cleaved mRNA intermediates accumulate in the 40S fraction, indicating that recognition occurs at an early stage of translation. This is the first example of a viral protein commandeering cellular mRNA turnover pathways to destroy host mRNAs, and suggests that Xrn1 is poised to deplete messages undergoing translation in mammalian cells

Epigenetic Activation of a Subset of mRNAs by eIF4E Explains Its Effects on Cell Proliferation

BACKGROUND: Translation deregulation is an important mechanism that causes aberrant cell growth, proliferation and survival. eIF4E, the mRNA 5′ cap-binding protein, plays a major role in translational control. To understand how eIF4E affects cell proliferation and survival, we studied mRNA targets that are translationally responsive to eIF4E. METHODOLOGY/PRINCIPAL FINDINGS: Microarray analysis of polysomal mRNA from an eIF4E-inducible NIH 3T3 cell line was performed. Inducible expression of eIF4E resulted in increased translation of defined sets of mRNAs. Many of the mRNAs are novel targets, including those that encode large- and small-subunit ribosomal proteins and cell growth-related factors. In addition, there was augmented translation of mRNAs encoding anti-apoptotic proteins, which conferred resistance to endoplasmic reticulum-mediated apoptosis. CONCLUSIONS/SIGNIFICANCE: Our results shed new light on the mechanisms by which eIF4E prevents apoptosis and transforms cells. Downregulation of eIF4E and its downstream targets is a potential therapeutic option for the development of novel anti-cancer drugs

The translational landscape of the splicing factor SRSF1 and its role in mitosis

The shuttling serine/arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene network that is translationally regulated by SRSF1, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified approximately 1500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding proteins involved in cell cycle regulation, such as spindle, kinetochore, and M phase proteins, which are essential for accurate chromosome segregation. Indeed, we show that translational activity of SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets, strongly suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on the complex role of SRSF1 in the control of gene expression at multiple levels and its implications in cancer.Fil: Maslon, Magdalena M. . Institute of Genetics and Molecular Medicine, University of Edinburgh; Reino UnidoFil: Heras, Sara R.. Institute of Genetics and Molecular Medicine, University of Edinburgh; Reino Unido. Universidad de Granada; EspañaFil: Bellora, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación En Biodiversidad y Medioambiente; Argentina. Universitat Pompeu Fabra; EspañaFil: Eyras, Eduardo. Universitat Pompeu Fabra; España. Catalan Institution for Research and Advanced Studies (ICREA), Barcelona; EspañaFil: Cáceres, Javier F.. University Of Edinburgh; Reino Unid

Translational control by the mRNA 5' cap and 3'poly (A) tail interacting proteins

An important aspect of the regulation of eukaryotic gene expression is the modulation of translation rates, which is most often controlled at the level of initiation. The interaction of proteins of the translational machinery with the mRNA cis-acting elements, governs mRNA translatability. Although both the 5' cap structure and 3' poly(A) tail act independently to stimulate translation, together they synergistically enhance translation. Therefore, an in-depth study of translation initiation would require examining the role of regulatory proteins acting on the 5' and 3' untranslated region (UTR) of the mRNA, and their influence on translation rates. A study of proteins interacting with the mRNA 5' UTR, reveals that translational homeostasis is induced by eIF4E through control of the 4E-BP1 and p70 S6 kinase activities. In an eIF4E tetracycline inducible system, overexpression of eIF4E leads to dephosphorylation of 4E-BP1 and p70 S6 kinase (but not Akt), with the extent of dephosphorylation proportional to the expression level of eIF4E. Therefore, a negative feedback loop is engendered by eIF4E expression that targets a downstream component of PI 3-kinase. The mechanisms of translational control imparted via the 3' UTR of the mRNA was studied by cloning a novel PABP interacting protein-2 (Paip2). Paip2 inhibited translation both in vivo and in vitro, in a dose dependent manner. The dual mechanism by which Paip2 inhibits the stimulatory role of PABP on translation is through (i) preventing the interaction of PABP with the poly(A) and disrupting the repeating structure of the poly(A) ribonucleoprotein structure and (ii) through direct competition with the translational co-activator Paip1 for PABP binding. BIACore data indicates that Paip2 binds PABP using a two site simple fit model. Consistent with this, two Paip2 molecules may be associated with one PABP molecule in vivo . Thus, the functional significance of interaction of Paip2 with PABP acting at the 3' UTR is

Creating a Training Package to Reduce Self-Injurious Behaviors, Based on the Lived Experiences of Teenagers Suffering From Self-Injurious Behaviors

The present study aimed to develop and validate an educational program designed to reduce self-injurious behavior (SIB) among adolescents. This program was developed based on the real-life experiences of adolescents who engage in self-harming behaviors. Employing a qualitative research approach, specifically the phenomenological method, the study encompassed all adolescents displaying self-harming behaviors in Shiraz city. The sampling process was deliberate, with the sample size determined by reaching a point of theoretical saturation. Ultimately, the perspectives of 15 adolescents (comprising 8 girls and 7 boys) were actively incorporated into the development of this educational and therapeutic package. This process involved the formulation of a model based on their lived experiences, which consisted of three core themes and 13 sub-themes. The end result was an educational-therapeutic package specifically designed to mitigate self-harming behaviors. The content validity of this educational-therapeutic package was assessed and confirmed using the content validity (Lawshe method). Importantly, this package holds significant potential for use in implementing educational and therapeutic interventions aimed at addressing self-harming behaviors among adolescents. These interventions can be effectively delivered within the context of schools, counseling centers, and educational institutions