Mark J. Coldwell

The Apaf-1 Internal Ribosome Entry Segment Attains the Correct Structural Conformation for Function via Interactions with PTB and unr

We have shown previously that polypyrimidine tract binding protein 1 (PTB) binds and activates the Apaf-1 internal ribosome entry segment (IRES) when the protein upstream of N-ras (unr) is prebound. Here we show that the Apaf-1 IRES is highly active in neuronal-derived cell lines due to the presence of the neuronal-enhanced version of PTB, nPTB. The unr and PTB/nPTB binding sites have been located on the Apaf-1 IRES RNA, and a structural model for the IRES bound to these proteins has been derived. The ribosome landing site has been located to a single-stranded region, and this is generated by the binding of the nPTB and unr to the RNA. These data suggest that unr and nPTB act as RNA chaperones by changing the structure of the IRES into one that permits translation initiation.

Initiation of Apaf-1 translation by internal ribosome entry

The apoptotic protease activating factor (Apaf-1) plays a central role in apoptosis: interaction of this protein with procaspase-9 leads to cleavage and activation of this initiator caspase. In common with other mRNAs whose protein products have a major regulatory function, the 5′ untranslated region (UTR) of Apaf-1 is long, G-C rich and has the potential to form secondary structure. We have shown that the 5′ UTR of Apaf-1 contains an internal ribosome entry segment, located in a 233 nucleotide region towards the 3′ end of the leader, and that the translation initiation of this mRNA occurs only by internal ribosome entry. The Apaf-1 IRES is active in almost all human cell types tested, including Human cervical carcinoma (HeLa), Human liver carcinoma (HepG2), Human breast carcinoma (MCF7), Human embryonic kidney (HK293), African Green Monkey kidney (COS7) and Human lung (MRC5). The Apaf-1 IRES initiates translation as efficiently as the HRV IRES, but is less active than the c-myc IRES. We propose that the Apaf-1 IRES ensures that a constant cellular level of Apaf-1 protein is maintained even under conditions where cap-dependent translation is compromised.

Protein Factor Requirements of the Apaf-1 Internal Ribosome Entry Segment: Roles of Polypyrimidine Tract Binding Protein and upstream of N-ras

ABSTRACT It has been reported previously that the 5′ untranslated region of the mRNA encoding Apaf-1 (apoptotic protease-activating factor 1) has an internal ribosome entry site (IRES), whose activity varies widely among different cell types. Here it is shown that the Apaf-1 IRES is active in rabbit reticulocyte lysates, provided that the system is supplemented with polypyrimidine tract binding protein (PTB) and upstream of N-ras (unr), two cellular RNA binding proteins previously identified to be required for rhinovirus IRES activity. In UV cross-linking assays and electrophoretic mobility shift assays with individual recombinant proteins, the Apaf-1 IRES binds unr but not PTB; however, PTB binding occurs if unr is present. Over a range of different cell types there is a broad correlation between the activity of the Apaf-1 IRES and their content of PTB and unr. In cell lines deficient in these proteins, overexpression of PTB and unr stimulated Apaf-1 IRES function. This is the first example where an IRES in a cellular mRNA has been shown to be functionally dependent, both in vitro and in vivo, on specific cellular RNA binding proteins. Given the critical role of Apaf-1 in apoptosis, these results have important implications for the control of the apoptotic cascade.

The p36 isoform of BAG-1 is translated by internal ribosome entry following heat shock.

BAG-1 (also known as RAP46/HAP46) was originally identified as a 46 kDa protein that bound to and enhanced the anti-apoptotic properties of Bcl-2. BAG-1 exists as three major isoforms (designated p50, p46 and p36 or BAG-1L, BAG-1M and BAG-1S respectively) and one minor isoform (p29), which are translated from a common transcript. The differing amino terminus determines both the intracellular location and the repertoire of binding partners of the isoforms which play different roles in a variety of cellular processes including signal transduction, heat shock, apoptosis and transcription. Although in vitro data suggest that the four BAG-1 isoforms are translated by leaky scanning, the patterns of isoform expression in vivo, especially in transformed cells, do not support this hypothesis. We have performed in vivo analysis of the BAG-1 5′ untranslated region and shown that translation initiation of the most highly expressed isoform (p36/BAG-1S) can occur by both internal ribosome entry and cap-dependent scanning. Following heat shock, when there is a downregulation of cap-dependent translation, the expression of the p36 isoform of BAG-1 is maintained by internal ribosome entry.

Functional Analysis of Individual Binding Activities of the Scaffold Protein eIF4G

Eukaryotic initiation factor (eIF) 4G is an integral member of the translation initiation machinery. The molecule serves as a scaffold for several other initiation factors, including eIF4E, eIF4AI, the eIF3 complex, and poly(A)-binding protein (PABP). Previous work indicates that complexes between these proteins exhibit enhanced mRNA cap-binding and RNA helicase activities relative to the respective individual proteins, eIF4E and eIF4A. The eIF4G-PABP interaction has been implicated in enhancing the formation of 48 S and 80 S initiation complexes and ribosome recycling through mRNA circularization. The eIF3-eIF4GI interaction is believed to forge the link between the 40 S subunit and the mRNA. Here we have investigated the behavior in vitro and in intact cells of eIF4GIf molecules lacking either the PABP-binding site, the eIF3-binding site, the middle domain eIF4A-binding site, or the C-terminal segment that includes the second eIF4A-binding site. Although in some cases the mutant forms were recruited more slowly, all of these eIF4G variants could form complexes with eIF4E, enter 48 S complexes and polysomes in vivo and in vitro, and partially rescue translation in cells targeted with eIF4GI short interfering RNA. In the reticulocyte lysate, eIF4G unable to interact directly with PABP showed little impairment in its ability to support translation, whereas loss of either of the eIF4A-binding sites or the eIF3-binding site resulted in a marked decrease in activity. We conclude that there is considerable redundancy in the mechanisms forming initiation complexes in mammalian cells, such that many individual interactions have regulatory rather than essential roles.

Canonical Initiation Factor Requirements of the Myc Family of Internal Ribosome Entry Segments

ABSTRACT Initiation of protein synthesis in eukaryotes requires recruitment of the ribosome to the mRNA and its translocation to the start codon. There are at least two distinct mechanisms by which this process can be achieved; the ribosome can be recruited either to the cap structure at the 5′ end of the message or to an internal ribosome entry segment (IRES), a complex RNA structural element located in the 5′ untranslated region (5′-UTR) of the mRNA. However, it is not well understood how cellular IRESs function to recruit the ribosome or how the 40S ribosomal subunits translocate from the initial recruitment site on the mRNA to the AUG initiation codon. We have investigated the canonical factors that are required by the IRESs found in the 5′-UTRs of c-, L-, and N-myc, using specific inhibitors and a tissue culture-based assay system, and have shown that they differ considerably in their requirements. The L-myc IRES requires the eIF4F complex and the association of PABP and eIF3 with eIF4G for activity. The minimum requirements of the N- and c-myc IRESs are the C-terminal domain of eIF4G to which eIF4A is bound and eIF3, although interestingly this protein does not appear to be recruited to the IRES RNA via eIF4G. Finally, our data show that all three IRESs require a ternary complex, although in contrast to c- and L-myc IRESs, the N-myc IRES has a lesser requirement for a ternary complex.

Specific Isoforms of Translation Initiation Factor 4GI Show Differences in Translational Activity

ABSTRACT The eukaryotic initiation factor (eIF) 4GI gene locus (eIF4GI) contains three identified promoters, generating alternately spliced mRNAs, yielding a total of five eIF4GI protein isoforms. Although eIF4GI plays a critical role in mRNA recruitment to the ribosomes, little is known about the functions of the different isoforms, their partner binding capacities, or the role of the homolog, eIF4GII, in translation initiation. To directly address this, we have used short interfering RNAs (siRNAs) expressed from DNA vectors to silence the expression of eIF4GI in HeLa cells. Here we show that reduced levels of specific mRNA and eIF4GI isoforms in HeLa cells promoted aberrant morphology and a partial inhibition of translation. The latter reflected dephosphorylation of 4E-BP1 and decreased eIF4F complex levels, with no change in eIF2α phosphorylation. Expression of siRNA-resistant Myc-tagged eIF4GI isoforms has allowed us to show that the different isoforms exhibit significant differences in their ability to restore translation rates. Here we quantify the efficiency of eIF4GI promoter usage in mammalian cells and demonstrate that even though the longest isoform of eIF4GI (eIF4GIf) was relatively poorly expressed when reintroduced, it was more efficient at promoting the translation of cellular mRNAs than the more highly expressed shorter isoforms used in previous functional studies.

SLiMPrints: conservation-based discovery of functional motif fingerprints in intrinsically disordered protein regions

Large portions of higher eukaryotic proteomes are intrinsically disordered, and abundant evidence suggests that these unstructured regions of proteins are rich in regulatory interaction interfaces. A major class of disordered interaction interfaces are the compact and degenerate modules known as short linear motifs (SLiMs). As a result of the difficulties associated with the experimental identification and validation of SLiMs, our understanding of these modules is limited, advocating the use of computational methods to focus experimental discovery. This article evaluates the use of evolutionary conservation as a discriminatory technique for motif discovery. A statistical framework is introduced to assess the significance of relatively conserved residues, quantifying the likelihood a residue will have a particular level of conservation given the conservation of the surrounding residues. The framework is expanded to assess the significance of groupings of conserved residues, a metric that forms the basis of SLiMPrints (short linear motif fingerprints), a de novo motif discovery tool. SLiMPrints identifies relatively overconstrained proximal groupings of residues within intrinsically disordered regions, indicative of putatively functional motifs. Finally, the human proteome is analysed to create a set of highly conserved putative motif instances, including a novel site on translation initiation factor eIF2A that may regulate translation through binding of eIF4E.

Regulation of BAG-1 IRES-mediated translation following chemotoxic stress

There are three major isoforms of BAG-1 in mammalian cells, termed BAG-1L (p50), BAG-1M (p46) and BAG-1S (p36) that function as pro-survival proteins and are associated with tumorigenesis and chemoresistance. Initiation of BAG-1 protein synthesis can occur by both cap-dependent and cap-independent mechanisms and it has been shown that synthesis of BAG-1S is dependent upon the presence of an internal ribosome entry segment (IRES) in the 5′-UTR of BAG-1 mRNA. We have shown previously that BAG-1 IRES–meditated initiation of translation requires two trans-acting factors poly (rC) binding protein 1 (PCBP1) and polypyrimidine tract binding protein (PTB) for function. The former protein allows BAG-1 IRES RNA to attain a structure that permits binding of the ribosome, while the latter protein appears to be involved in ribosome recruitment. Here, we show that the BAG-1 IRES maintains synthesis of BAG-1 protein following exposure of cells to the chemotoxic drug vincristine but not to cisplatin and that this is brought about, in part, by the relocalization of PTB and PCBP1 from the nucleus to the cytoplasm.

The human insulin receptor mRNA contains a functional internal ribosome entry segment

Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5′-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective.