IRES

Il lungo dibattito giurisprudenziale riferito agli accertamenti di maggiori utili extrabilancio in capo ai soci di società di capitali a ristretta base partecipativa si è attestato su un orientamento che ammette la legittimità di tali atti impositivi considerando che non vi sarebbe violazione del divieto di doppia presunzione derivante dall’art. 2729 c.c. Invero, osservando la lunga gestazione di questa interpretazione, partendo dalla giurisprudenza degli anni ’90, si è partiti da posizioni pro contribuente, con le quali si riteneva, appunto, che un simile accertamento violasse il divieto di praesumptio de presumpto (la prima presunzione è quella ordinariamente utilizzata per accertare maggiori redditi in capo alla società, ex art. 39, comma 1, lett. d), d.P.R. n. 600/1973, la seconda è quella basata sull’affectio che sussiste tra i “pochi” soci della persona giuridica) per approdare su soluzioni di segno opposto, mitigate dalla necessità di sospendere il processo relativo all’accertamento in capo ai soci in attesa della definitività di quello relativo alla società, onde fondare il recupero in capo ai soci stessi su un atto dotato di certezza (la sentenza che legittima, appunto, l’operato dell’Ufficio relativamente alla società). Va poi riconosciuto il pregio dei risultati raggiunti recentemente dalla giurisprudenza di merito, i quali sembrano poter riaprire il dibattito osservando il tema dalla prospettiva costituzionale, ossia ritenendo che l’accertamento sui soci contrasterebbe con il principio di legalità posto dall’art. 23 Cost. a base del sistema tributario.

Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1) uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms. Moreover, HIV-1 modulates the host translation machinery by targeting key translation factors and overcomes different cellular obstacles that affect protein translation. In this review, we describe how HIV-1 proteins target several components of the eukaryotic translation machinery, which consequently improves viral translation and replication.

Polypyrimidine Tract Binding Protein (PTB) is an intensely studied RNA binding protein involved in several post-transcriptional regulatory events of gene expression. Initially described as a pre-mRNA splicing regulator, PTB is now widely accepted as a multifunctional protein shuttling between nucleus and cytoplasm. Accordingly, PTB can interact with selected RNA targets, structural elements and proteins. There is increasing evidence that PTB and its paralog PTBP2 play a major role as repressors of alternatively spliced exons, whose transcription is tissue-regulated. In addition to alternative splicing, PTB is involved in almost all steps of mRNA metabolism, including polyadenylation, mRNA stability and initiation of protein translation. Furthermore, it is well established that PTB recruitment in internal ribosome entry site (IRES) activates the translation of picornaviral and cellular proteins. Detailed studies of the structural properties of PTB have contributed to our understandin...

Resistance of translation of some eukaryotic messenger RNAs (mRNAs) to inactivation of the cap-binding factor eIF4E under unfavorable conditions is well documented. To date, it is the mechanism of internal ribosome entry that is predominantly thought to underlay this stress tolerance. However, many cellular mRNAs that had been considered to contain internal ribosome entry sites (IRESs) failed to pass stringent control tests for internal initiation, thus raising the question of how they are translated under stress conditions. Here, we show that inserting an eIF4G-binding element from a virus IRES into 5 0-UTRs of strongly cap-dependent mRNAs dramatically reduces their requirement for the 5 0-terminal m 7 G-cap, though such cap-independent translation remains dependent on a vacant 5 0-terminus of these mRNAs. Importantly, direct binding of eIF4G to the 5 0-UTR of mRNA makes its translation resistant to eIF4F inactivation both in vitro and in vivo. These data may substantiate a new paradigm of transla-tional control under stress to complement IRES-driven mechanism of translation.

A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5'-UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5'-UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5'-UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5'-terminal half of the 5'-UTR and rather strong for the 3'-terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (>50 nt) by truncation of the 5'-UTR from the 5'-end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5'-UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5'-UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.

Hepatitis C virus (HCV) core (C) protein is thought to bind to viral RNA before it undergoes oligomerization leading to RNA encapsidation. Details of these events are so far unknown. The 5ʹ-terminal C protein coding sequence that includes an adenine (A)-rich tract is a part of an internal ribosome entry site (IRES). This nucleotide sequence but not the corresponding protein sequence is needed for proper initiation of translation of viral RNA by an IRES-dependent mechanism. In this study, we examined the importance of this sequence for the ability of the C protein to bind to viral RNA. Serially truncated C proteins with deletions from 10 up to 45 N-terminal amino acids were expressed in Escherichia coli, purified and tested for binding to viral RNA by a gel shift assay. The results showed that truncation of the C protein from its N-terminus by more than 10 amino acids abolished almost completely its expression in E. coli. The latter could be restored by adding a tag to the N-terminus of the protein. The tagged proteins truncated by 15 or more amino acids showed an anomalous migration in SDS-PAGE. Truncation by more than 20 amino acids resulted in a complete loss of ability of tagged C protein to bind to viral RNA. These results provide clues to the early events in the C protein - RNA interactions leading to C protein oligomerization, RNA encapsidation and virion assembly.

Tethered hydroxyl-radical probing has been used to deter-mine the orientation of binding of polypyrimidine tract-binding protein (PTB) to the poliovirus type 1 (Mahoney) (PV-1(M)) internal ribosome entry site/segment (IRES)— the question of which RNA-binding domain (RBD) binds to which sites on the IRES. The results show that under conditions in which PTB strongly stimulates IRES activity, a single PTB is binding to the IRES, a finding which was confirmed by mass spectrometry of PTB/IRES complexes. RBDs1 and 2 interact with the basal part of the Domain V irregular stem loop, very close to the binding site of eIF4G, and RBDs3 and 4 interact with the single-stranded regions flanking Domain V. The binding of PTB is subtly altered in the presence of the central domain (p50) of eIF4G, and p50 binding is likewise modified if PTB is present. This suggests that PTB stimulates PV-1(M) IRES activity by inducing eIF4G to bind in the optimal position and orientation to promote internal ribosome...

The purpose of this article is to analyse the experiences of probation officers who supervise women in the community in Catalonia. To this end, qualitative research involving 15 semi-structured interviews with probation officers in Barcelona and Girona was carried out. The results show that professionals agree that there are important gender differences in relation to: (a) personal and penal characteristics and (b) supervision style. The perceptions of practitioners and existing empirical data are broadly consistent. Women who serve community sentences have a wide range of problems, needs and responsibilities compared to men, which is often reflected in the fact that women’s attendance is much more irregular, an issue that presents multiple challenges for practitioners. Probation officers already carry out different gender-responsive practises, however, they face different challenges in a gender-insensitive probation system. Furthermore, given the great heterogeneity of the female service users compared to the men, service provision for women in the community also needs to be intersectionally-responsive. Based on these disparities, the elements of probation that the professionals believe work better with women and those that could be improved are discussed.

In this paper, we proposed a methodology to model, verify, and generate IEC standard PLC code using Timed-MPSG (an extended version of finite state automata). It can reduce PLC development time and the errors that generally occur when PLC programs are manually programmed. For this purpose, we used Timed-MPSG to model the formal specification of the targeted shop floor controller system, after that, the graphical representation of Timed-MPSG is converted to the textual format automatically. Thereafter, ...

Platelet-derived growth factor B (PDGF-B) overexpression induces gliomas of different grades from murine embryonic neural progenitors. For the first time, we formally demonstrated that PDGF-B-induced neoplasms undergo progression from nontumorigenic low-grade tumors toward highly malignant forms. This result, showing that PDGF-B signaling alone is insufficient to confer malignancy to cells, entails the requirement for further molecular lesions in this process. Our results indicate that one of these lesions is represented by the down-regulation of the oncosuppressor Btg2. By in vivo transplantation assays, we further demonstrate that fully progressed tumors are PDGF-B-addicted because their tumor-propagating ability is lost when the PDGF-B transgene is silenced, whereas it is promptly reacquired after its reactivation. We provide evidence that this oncogene addiction is not caused by the need for PDGF-B as a mitogen but, rather, to the fact that PDGF-B is required to overcome cell-ce...

Many mammalian mRNAs possess long 5' UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5' UTRs reported to have IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested 5' UTRs with so-called 'cellular IRESes' demonstrate only background activities when placed in the intercistronic position of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5' UTRs. The cap positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual 5' UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated.

During the last decade the concept of cellular IRES-elements has become predominant to explain the continued expression of specific proteins in eukaryotic cells under conditions when the cap-dependent translation initiation is inhibited. However, many cellular IRESs regarded as cornerstones of the concept, have been compromised by several recent works using a number of modern techniques. This review analyzes the sources of artifacts associated with identification of IRESs and describes a set of control experiments, which should be performed before concluding that a 5′ UTR of eukaryotic mRNA does contain an IRES. Hallmarks of true IRES-elements as exemplified by well-documented IRESs of viral origin are presented. Analysis of existing reports allows us to conclude that there is a constant confusion of the cap-independent with the IRES-directed translation initiation. In fact, these two modes of translation initiation are not synonymous. We discuss here not numerous reports pointing to the existence of a cap-and IRES-independent scanning mechanism of translation initiation based on utilization of special RNA structures called cap-independent translational en-hancers (CITE). We describe this mechanism and suggest it as an alternative to the concept of cellular IRESs.

Utrophin, the autosomal homologue of dystrophin can functionally compensate for dystrophin deficiency. Utrophin upregulation could therefore be a therapeutic strategy in Duchenne Muscular Dystrophy (DMD) that arises from mutation in dystrophin gene. In contrast to its transcriptional regulation, mechanisms operating at post-transcriptional level of utrophin expression have not been well documented. We have previously reported translational repression of mouse utrophin-A mRNA through its 5’ and 3’UTRs. However, utrophin-A 5’UTR has been reported with internal ribosome entry site (IRES). In the present study we aimed to compare relative contribution of cap-independent and cap-dependent translation with mouse utrophin-A 5’UTR through m7G-capped and A-capped mRNA transfection based reporter assay. Our results suggest that although weak, cap-independent translation becomes crucial as a result of severe repression of cap-dependent translation with utrophin-A 5’UTR. With capped in vitro transcribed RNA we identified two sequence elements and an upstream open reading frame in utrophin 5’UTR responsible for repression. The key repressor elements may be targeted for utrophin upregulation.

PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG)n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed ...