Preferential translation of Hsp83 in Leishmania requires a thermosensitive polypyrimidine-rich element in the 3′ UTR and involves scanning of the 5′ UTR

Heat shock proteins (HSPs) provide a useful system for studying developmental patterns in the digenetic Leishmania parasites, since their expression is induced in the mammalian life form. Translation regulation plays a key role in control of protein coding genes in trypanosomatids, and is directed exclusively by elements in the 3′ untranslated region (UTR). Using sequential deletions of the Leishmania Hsp83 3′ UTR (888 nucleotides [nt]), we mapped a region of 150 nt that was required, but not sufficient for preferential translation of a reporter gene at mammalian-like temperatures, suggesting that changes in RNA structure could be involved. An advanced bioinformatics package for prediction of RNA folding (UNAfold) marked the regulatory region on a highly probable structural arm that includes a polypyrimidine tract (PPT). Mutagenesis of this PPT abrogated completely preferential translation of the fused reporter gene. Furthermore, temperature elevation caused the regulatory region to melt more extensively than the same region that lacked the PPT. We propose that at elevated temperatures the regulatory element in the 3′ UTR is more accessible to mediators that promote its interaction with the basal translation components at the 5′ end during mRNA circularization. Translation initiation of Hsp83 at all temperatures appears to proceed via scanning of the 5′ UTR, since a hairpin structure abolishes expression of a fused reporter gene.     

The 3′-Untranslated Region of Hepatitis C Virus RNA Enhances Translation from an Internal Ribosomal Entry Site

Translation of most eukaryotic mRNAs and many viral RNAs is enhanced by their poly(A) tails. Hepatitis C virus (HCV) contains a positive-stranded RNA genome which does not have a poly(A) tail but has a stretch of 98 nucleotides (X region) at the 3′-untranslated region (UTR), which assumes a highly conserved stem-loop structure. This X region binds a polypyrimidine tract-binding protein (PTB), which also binds to the internal ribosome entry site (IRES) in HCV 5′-UTR. These RNA-protein interactions may regulate its translation. We generated a set of HCV RNAs differing only in their 3′-UTRs and compared their translation efficiencies. HCV RNA containing the X region was translated three- to fivefold more than the corresponding RNAs without this region. Mutations that abolished PTB binding in the X region reduced, but did not completely abolish, enhancement in translation. The X region also enhanced translation from another unrelated IRES (from encephalomyocarditis virus RNA), but did not affect the 5′-end-dependent translation of globin mRNA in either monocistronic or bicistronic RNAs. It did not appear to affect RNA stability. The free X region added in trans, however, did not enhance translation, indicating that the translational enhancement by the X region occurs only in cis. These results demonstrate that the highly conserved 3′ end of HCV RNA provides a novel mechanism for enhancement of HCV translation and may offer a target for antiviral agents.     

The hepatitis C virus 3'-untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase

Enhancement of eukaryotic messenger RNA (mRNA) translation initiation by the 3′ poly(A) tail is mediated through interaction of poly(A)-binding protein with eukaryotic initiation factor (eIF) 4G, bridging the 5′ terminal cap structure. In contrast to cellular mRNA, translation of the uncapped, non-polyadenylated hepatitis C virus (HCV) genome occurs independently of eIF4G and a role for 3′-untranslated sequences in modifying HCV gene expression is controversial. Utilizing cell-based and in vitro translation assays, we show that the HCV 3′-untranslated region (UTR) or a 3′ poly(A) tract of sufficient length interchangeably stimulate translation dependent upon the HCV internal ribosomal entry site (IRES). However, in contrast to cap-dependent translation, the rate of initiation at the HCV IRES was unaffected by 3′-untranslated sequences. Analysis of post-initiation events revealed that the 3′ poly(A) tract and HCV 3′-UTR improve translation efficiency by enabling termination and possibly ribosome recycling for successive rounds of translation.     

Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3′UTR

Requiem (REQ/DPF2) was originally identified as an apoptosis-inducing protein in mouse myeloid cells and belongs to the novel Krüppel-type zinc finger d4-protein family of proteins, which includes neuro-d4 (DPF1) and cer-d4 (DPF3). Interestingly, when a portion of the REQ messenger ribonucleic acid (mRNA) 3′ untranslated region (3′UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3′UTR of REQ mRNA plays a physiological role. Here, we present evidence that the REQ mRNA 3′UTR, along with its trans-acting factor, Staufen1 (STAU1), is able to reduce the level of REQ mRNA via STAU1-mediated mRNA decay (SMD). By screening a complementary deoxyribonucleic acid (cDNA) expression library with an RNA–ligand binding assay, we identified STAU1 as an interactor of the REQ mRNA 3′UTR. Specifically, we provide evidence that STAU1 binds to putative 30-nucleotide stem–loop-structured RNA sequences within the G8 region, which we term the protein binding site core; this binding triggers the degradation of REQ mRNA and thus regulates translation. Furthermore, we demonstrate that siRNA-mediated silencing of either STAU1 or UPF1 increases the abundance of cellular REQ mRNA and, consequently, the REQ protein, indicating that REQ mRNA is a target of SMD.     

Hsp27 binding to the 3′UTR of bim mRNA prevents neuronal death during oxidative stress–induced injury: a novel cytoprotective mechanism

Neurons face a changeable microenvironment and therefore need mechanisms that allow rapid switch on/off of their cytoprotective and apoptosis-inducing signaling pathways. Cellular mechanisms that control apoptosis activation include the regulation of pro/antiapoptotic mRNAs through their 3′-untranslated region (UTR). This region holds binding elements for RNA-binding proteins, which can control mRNA translation. Here we demonstrate that heat shock protein 27 (Hsp27) prevents oxidative stress–induced cell death in cerebellar granule neurons by specific regulation of the mRNA for the proapoptotic BH3-only protein, Bim. Hsp27 depletion induced by oxidative stress using hydrogen peroxide (H₂O₂) correlated with bim gene activation and subsequent neuronal death, whereas enhanced Hsp27 expression prevented these. This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27. Finally, we determined that enhanced Hsp27 expression in neurons exposed to H₂O₂ or glutamate prevented the translation of a reporter plasmid where bim-3′UTR mRNA sequence was cloned downstream of a luciferase gene. These results suggest that repression of bim mRNA translation through binding to the 3′UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons.     

Novel 5′ Untranslated Region Directed Blockers of Iron-Regulatory Protein-1 Dependent Amyloid Precursor Protein Translation: Implications for Down Syndrome and Alzheimer's Disease

We reported that iron influx drives the translational expression of the neuronal amyloid precursor protein (APP), which has a role in iron efflux. This is via a classic release of repressor interaction of APP mRNA with iron-regulatory protein-1 (IRP1) whereas IRP2 controls the mRNAs encoding the L- and H-subunits of the iron storage protein, ferritin. Here, we identified thirteen potent APP translation blockers that acted selectively towards the uniquely configured iron-responsive element (IRE) RNA stem loop in the 5′ untranslated region (UTR) of APP mRNA. These agents were 10-fold less inhibitory of 5′UTR sequences of the related prion protein (PrP) mRNA. Western blotting confirmed that the ‘ninth’ small molecule in the series selectively reduced neural APP production in SH-SY5Y cells at picomolar concentrations without affecting viability or the expression of α-synuclein and ferritin. APP blocker-9 (JTR-009), a benzimidazole, reduced the production of toxic Aβ in SH-SY5Y neuronal cells to a greater extent than other well tolerated APP 5′UTR-directed translation blockers, including posiphen, that were shown to limit amyloid burden in mouse models of Alzheimer''s disease (AD). RNA binding assays demonstrated that JTR-009 operated by preventing IRP1 from binding to the IRE in APP mRNA, while maintaining IRP1 interaction with the H-ferritin IRE RNA stem loop. Thus, JTR-009 constitutively repressed translation driven by APP 5′UTR sequences. Calcein staining showed that JTR-009 did not indirectly change iron uptake in neuronal cells suggesting a direct interaction with the APP 5′UTR. These studies provide key data to develop small molecules that selectively reduce neural APP and Aβ production at 10-fold lower concentrations than related previously characterized translation blockers. Our data evidenced a novel therapeutic strategy of potential impact for people with trisomy of the APP gene on chromosome 21, which is a phenotype long associated with Down syndrome (DS) that can also cause familial Alzheimer''s disease.     

Hepatitis C virus 3′UTR regulates viral translation through direct interactions with the host translation machinery

The 3′ untranslated region (3′UTR) of hepatitis C virus (HCV) messenger RNA stimulates viral translation by an undetermined mechanism. We identified a high affinity interaction, conserved among different HCV genotypes, between the HCV 3′UTR and the host ribosome. The 3′UTR interacts with 40S ribosomal subunit proteins residing primarily in a localized region on the 40S solvent-accessible surface near the messenger RNA entry and exit sites. This region partially overlaps with the site where the HCV internal ribosome entry site was found to bind, with the internal ribosome entry site-40S subunit interaction being dominant. Despite its ability to bind to 40S subunits independently, the HCV 3′UTR only stimulates translation in cis, without affecting the first round translation rate. These observations support a model in which the HCV 3′UTR retains ribosome complexes during translation termination to facilitate efficient initiation of subsequent rounds of translation.     

The 3' untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1gamma and HAX-1

Previously, we have shown that the vimentin 3′ untranslated region (3′UTR) contains a highly conserved region, which is sufficient for the perinuclear localization of a reporter mRNA. This region was shown to specifically bind protein(s) by band shift analyses. UV-cross-linking studies suggest these proteins are 46- and 35-kDa in mass. Here, we have used this sequence as ‘bait’ to isolate RNA binding proteins using the yeast three-hybrid method. This technique relies on a functional assay detecting bona fide RNA–protein interaction in vivo. Three cDNA isolates, HAX-1, eEF-1γ and hRIP, code for proteins of a size consistent with in vitro cross- linking studies. In all cases, recombinant proteins were capable of binding RNA in vitro. Although hRIP is thought to be a general mRNA binding protein, this represents an unreported activity for eEF-1γ and HAX-1. Moreover, HAX-1 binding appears to be specific to vimentin’s 3′UTR. Both in vivo synthesized eEF-1γ and HAX-1 proteins were ‘pulled out’ of HeLa whole cell extracts by binding to a RNA affinity column comprised of vimentin’s 3′UTR. Moreover, size-fractionation of extracts results in the separation of large complexes containing either eEF-1γ or HAX-1. Thus, in addition to their known functions, both eEF-1γ and HAX-1 are RNA binding proteins, which suggests new roles in mRNA translation and/or perinuclear localization.     

Regulation of the Stability of Heat-Stable Antigen mRNA by Interplay between Two Novel cis Elements in the 3′ Untranslated Region

The heat-stable antigen (HSA) is a costimulatory molecule for T-cell activation. Its expression is strictly regulated during lymphocyte development and differentiation. Recent studies using HSA-transgenic mice have demonstrated that this regulated expression is critical for normal development of T and B lymphocytes. However, the mechanisms that control the expression of HSA are largely unknown. HSA mRNA is comprised of a 0.23-kb open reading frame and a 1.5-kb 3′ untranslated region (3′UTR). The function of the long 3′UTR has not been addressed. Here we investigate the role of the 3′UTR of HSA mRNA. We show that a 160-bp element, located in the region of nucleotides 1465 to 1625 in the 3′UTR of HSA mRNA, promotes RNA degradation and that this effect is neutralized by a 43-bp fragment approximately 1 kb upstream of the negative cis element. Both positive and negative cis elements in the HSA mRNA are distinct from other sequences that are known to modulate mRNA stability. These results provide direct evidence that the interplay between two novel cis elements in the 3′UTR of HSA mRNA determines cell surface HSA expression by modulating its RNA stability.     

IGF2BP1 enhances HCV IRES-mediated translation initiation via the 3′UTR

The positive-strand RNA genome of the Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) in the 5′untranslated region (5′UTR) and structured sequence elements within the 3′UTR, but no poly(A) tail. Employing a limited set of initiation factors, the HCV IRES coordinates the 5′cap-independent assembly of the 43S pre-initiation complex at an internal initiation codon located in the IRES sequence. We have established a Huh7 cell-derived in vitro translation system that shows a 3′UTR-dependent enhancement of 43S pre-initiation complex formation at the HCV IRES. Through the use of tobramycin (Tob)-aptamer affinity chromatography, we identified the Insulin-like growth factor-II mRNA-binding protein 1 (IGF2BP1) as a factor that interacts with both, the HCV 5′UTR and 3′UTR. We report that IGF2BP1 specifically enhances translation at the HCV IRES, but it does not affect 5′cap-dependent translation. RNA interference against IGF2BP1 in HCV replicon RNA-containing Huh7 cells reduces HCV IRES-mediated translation, whereas replication remains unaffected. Interestingly, we found that endogenous IGF2BP1 specifically co-immunoprecipitates with HCV replicon RNA, the ribosomal 40S subunit, and eIF3. Furthermore eIF3 comigrates with IGF2BP1 in 80S ribosomal complexes when a reporter mRNA bearing both the HCV 5′UTR and HCV 3′UTR is translated. Our data suggest that IGF2BP1, by binding to the HCV 5′UTR and/or HCV 3′UTR, recruits eIF3 and enhances HCV IRES-mediated translation.     

Antisense oligonucleotides targeted to the domain IIId of the hepatitis C virus IRES compete with 40S ribosomal subunit binding and prevent in vitro translation

Initiation of protein synthesis on the hepatitis C virus (HCV) mRNA involves a structured element corresponding to the 5′ untranslated region and constituting an internal ribosome entry site (IRES). The domain IIId of the HCV IRES, an imperfect RNA hairpin extending from nucleotides 253 to 279 of the viral mRNA, has been shown to be essential for translation and for the binding of the 40S ribosomal subunit. We investigated the properties of a series of antisense 2′-O-methyloligoribonucleotides targeted to various portions of the domain IIId. Several oligomers, 14–17 nt in length, selectively inhibited in vitro translation of a bicistronic RNA construct in rabbit reticulocyte lysate with IC₅₀s <10 nM. The effect was restricted to the second cistron (the Renilla luciferase) located downstream of the HCV IRES; no effect was observed on the expression of the first cistron (the firefly luciferase) which was translated in a cap-dependent manner. Moreover, antisense 2′-O-methyloligoribonucleotides specifically competed with the 40S ribosomal subunit for binding to the IRES RNA in a filter- retention assay. The antisense efficiency of the oligonucleotides was nicely correlated to their affinity for the IIId subdomain and to their ability to displace 40S ribosomal subunit, making this process a likely explanation for in vitro inhibition of HCV-IRES-dependent translation.