Regulation of mitochondrial mRNA stability by RNase L is translation-dependent and controls IFNalpha-induced apoptosis

Interferons (IFNs) inhibit the growth of many different cell types by altering the expression of specific genes. IFNs activities are partly mediated by the 2'-5' oligoadenylates-RNase L RNA decay pathway. RNase L is an endoribonuclease requiring activation by 2'-5' oligoadenylates to cleave single-stranded RNA. Here, we present evidence that degradation of mitochondrial mRNA by RNase L leads to cytochrome c release and caspase 3 activation during IFNalpha-induced apoptosis. We identify and characterize the mitochondrial translation initiation factor (IF2mt) as a new partner of RNase L. Moreover, we show that specific inhibition of mitochondrial translation with chloramphenicol inhibits the IFNalpha-induced degradation of mitochondrial mRNA by RNase L. Finally, we demonstrate that overexpression of IF2mt in human H9 cells stabilizes mitochondrial mRNA, inhibits apoptosis induced by IFNalpha and partially reverses IFNalpha-cell growth inhibition. On the basis of our results, we propose a model describing how RNase L regulates mitochondrial mRNA stability through its interaction with IF2mt.     

GTP hydrolysis by eRF3 facilitates stop codon decoding during eukaryotic translation termination

Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others. As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals. To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release.     

Synthesis of (2'-5')oligoadenylate and activation of an endoribonuclease in interferon-treated HeLa cells infected with reovirus.

Treatment with interferon protected HeLa cells from infection with reovirus. This virus apparently activated an antiviral mechanism that was detected by the presence of (2'-5')oligoadenylate [(2'-5')An] in intact cells. The (2'-5')An was previously shown to activate an endoribonuclease, RNase L. We measured (2'-5')An by a sensitive competition-binding assay in cells infected at different multiplicities and for different lengths of time. Nanomolar concentrations of (2'-5')An were detected in cells infected at a multiplicity of greater than 5 after 2 h of infection, the time at which the infecting virions were uncoated. The level of (2'-5')An increased up to 6 h postinfection but declined afterward. To establish whether viral mRNAs were cleaved by RNase L, we analyzed the RNA extracted from infected cells by a highly specific hybridization assay on Northern blots. Full-sized reovirus mRNAs were detected in control infected cells, but not in interferon-treated infected cells, at 6 h postinfection. At this time, a nuclease activity could be detected in these cells by demonstration of cleavage of rRNA, degradation of cellular mRNA, and polysome breakdown in the presence of emetine. Since this inhibitor freezes ribosomes, cleavage of mRNA between ribosomes could only be accounted for by an endonuclease, presumably RNase L.     

Interaction of PABPC1 with the translation initiation complex is critical to the NMD resistance of AUG-proximal nonsense mutations

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3'-5' linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.     

A viral RNA competitively inhibits the antiviral endoribonuclease domain of RNase L

Ribonuclease L (RNase L) is a latent endoribonuclease in an evolutionarily ancient interferon-regulated dsRNA-activated antiviral pathway. 2'-5' oligoadenylate (2-5A), the product of dsRNA-activated oligoadenylate synthetases (OASes), binds to ankyrin repeats near the amino terminus of RNase L, initiating a series of conformational changes that result in the activation of the endoribonuclease. A phylogenetically conserved RNA structure within group C enteroviruses inhibits the endoribonuclease activity of RNase L. In this study we report the mechanism by which group C enterovirus RNA inhibits RNase L. Viral RNA did not affect 2-5A binding to RNase L. Rather, the viral RNA inhibited the endoribonuclease domain. We used purified RNase L, purified 2-5A, and an RNA substrate with a 5' fluorophore and 3' quencher in FRET assays to measure inhibition of RNase L activity by the viral RNA. The group C enterovirus RNA was a competitive inhibitor of the endoribonuclease with a K(i) of 34 nM. Consistent with the kinetic profile of a competitive inhibitor, the viral RNA inhibited the constitutively active endoribonuclease domain of RNase L. We call this viral RNA the RNase L competitive inhibitor RNA (RNase L ciRNA).     

Molecular basis for the recognition and cleavage of RNA by the bifunctional 5'-3' exo/endoribonuclease RNase J

RNase J is a key member of the β-CASP family of metallo-β-lactamases involved in the maturation and turnover of RNAs in prokaryotes. The B.?subtilis enzyme possesses both 5'-3' exoribonucleolytic and endonucleolytic activity, an unusual property for a ribonuclease. Here, we present the crystal structure of T.?thermophilus RNase J bound to a 4 nucleotide RNA. The structure reveals an RNA-binding channel that illustrates how the enzyme functions in 5'-3' exoribonucleolytic mode and how it can function as an endonuclease. A second, negatively charged tunnel leads from the active site, and is ideally located to evacuate the cleaved nucleotide in 5'-3' exonucleolytic mode. We show that B.?subtilis RNase J1, which shows processive behavior on long RNAs, behaves distributively for substrates less than 5 nucleotides in length. We propose a model involving the binding of the RNA to the surface of the β-CASP domain to explain the enzyme's processive action.     

The iron–sulphur protein RNase L inhibitor functions in translation termination

The iron–sulphur (Fe–S)‐containing RNase L inhibitor (Rli1) is involved in ribosomal subunit maturation, transport of both ribosomal subunits to the cytoplasm, and translation initiation through interaction with the eukaryotic initiation factor 3 (eIF3) complex. Here, we present a new function for Rli1 in translation termination. Through co‐immunoprecipitation experiments, we show that Rli1 interacts physically with the translation termination factors eukaryotic release factor 1 (eRF1)/Sup45 and eRF3/Sup35 in Saccharomyces cerevisiae. Genetic interactions were uncovered between a strain depleted for Rli1 and sup35‐21 or sup45‐2. Furthermore, we show that downregulation of RLI1 expression leads to defects in the recognition of a stop codon, as seen in mutants of other termination factors. By contrast, RLI1 overexpression partly suppresses the read‐through defects in sup45‐2. Interestingly, we find that although the Fe–S cluster is not required for the interaction of Rli1 with eRF1 or its other interacting partner, Hcr1, from the initiation complex eIF3, it is required for its activity in translation termination; an Fe–S cluster mutant of RLI1 cannot suppress the read‐through defects of sup45‐2.     

Tpa1p is part of an mRNP complex that influences translation termination, mRNA deadenylation, and mRNA turnover in Saccharomyces cerevisiae

In this report, we show that the Saccharomyces cerevisiae protein Tpa1p (for termination and polyadenylation) influences translation termination efficiency, mRNA poly(A) tail length, and mRNA stability. Tpa1p is encoded by the previously uncharacterized open reading frame YER049W. Yeast strains carrying a deletion of the TPA1 gene (tpa1Delta) exhibited increased readthrough of stop codons, and coimmunoprecipitation assays revealed that Tpa1p interacts with the translation termination factors eRF1 and eRF3. In addition, the tpa1Delta mutation led to a 1.5- to 2-fold increase in the half-lives of mRNAs degraded by the general 5'-->3' pathway or the 3'-->5' nonstop decay pathway. In contrast, this mutation did not have any affect on the nonsense-mediated mRNA decay pathway. Examination of mRNA poly(A) tail length revealed that poly(A) tails are longer than normal in a tpa1Delta strain. Consistent with a potential role in regulating poly(A) tail length, Tpa1p was also found to coimmunoprecipitate with the yeast poly(A) binding protein Pab1p. These results suggest that Tpa1p is a component of a messenger ribonucleoprotein complex bound to the 3' untranslated region of mRNAs that affects translation termination, deadenylation, and mRNA decay.     

游仆虫肽链释放因子eRF1对编程性翻译移码的影响

编程性翻译移码是mRNA翻译为多肽链时核糖体沿mRNA正向或反向滑动1个碱基才能表达出1个完整多肽链的现象. 人的肽链释放因子eRF1对HIV-1病毒的编程性-1移码有直接的影响. 而且在频繁发生编程性+1移码的单细胞真核生物游仆虫中,肽链释放因子eRF1对编程性移码也有明显的影响. 为进一步研究eRF1中影响编程性翻译移码的关键序列及调控机理,本研究将含有不同终止密码子的移码序列和已报道的游仆虫移码基因Ndr2分别插入双荧光素酶报告基因中,成功建立了可在酵母中进行研究的编程性移码报告检测体系. 利用游仆虫肽链释放因子Eo-eRF1b的N结构域和酵母肽链释放因子Sc eRF1的MC结构域构建了杂合肽链释放因子(Eo/Sc eRF1),检测Eo-eRF1b N结构域中的不同突变位点对移码效率的影响. 结果表明,游仆虫肽链释放因子eRF1b中YCF区的突变能明显促进含终止密码UAA的移码序列的移码,推测这可能是由于eRF1突变体降低了对UAA的识别所导致. 此外,杂合肽链释放因子Eo/Sc eRF1能够有效地提高移码基因Ndr2的移码效率. eRF1b中YCF区的突变同样能明显促进 Ndr2的移码. 因此, 游仆虫肽链释放因子YCF区的特殊序列可能是这种生物中发生编程性移码频率较高的原因之一. 本研究为探讨纤毛虫编程性翻译移码调控机制提供了实验数据.     

Suppression of nonsense and frameshift mutations obtained by different methods for inactivating the translation termination factor eRF3 in yeast Saccharomyces cerevisiae

Mutations in genes of omnipotent nonsense suppressors SUP35 and SUP45 in yeast Saccharomyces cerevisiae encoding translation termination factors eRF3 and eRF1, respectively, and prionization of the eRF3 protein may lead to the suppression of some frameshift mutations (CPC mutations). Partial inactivation of the translation termination factor eRF3 was studied in strains with unstable genetically modified prions and also in transgenic yeast S. cerevisiae strains with the substitution of the indigenous SUP35 gene for its homolog from Pichia methanolica or for a recombinant S. cerevisiae SUP35 gene. It was shown that this partial inactivation leads not only to nonsense suppression, but also to suppression of the frameshift lys2-90 mutation. Possible reasons for the correlation between nonsense suppression and suppression of the CPC lys2-90 mutation and mechanisms responsible for the suppression of CPC mutations during inactivation of translation termination factors are discussed.     

Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation

Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the deadenylation of the poly(A) tail at the 3' end. However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated. Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae. The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP. When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged. The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening. Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon. These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.     

The 5' UTR of HIV-1 full-length mRNA and the Tat viral protein modulate the programmed -1 ribosomal frameshift that generates HIV-1 enzymes

Translation of the full-length messenger RNA (mRNA) of the human immunodeficiency virus type 1 (HIV-1) generates the precursor of the viral enzymes via a programmed -1 ribosomal frameshift. Here, using dual-luciferase reporters, we investigated whether the highly structured 5' untranslated region (UTR) of this mRNA, which interferes with translation initiation, can modulate HIV-1 frameshift efficiency. We showed that, when the 5' UTR of HIV-1 mRNA occupies the 5' end of the reporter mRNA, HIV-1 frameshift efficiency is increased about fourfold in Jurkat T-cells, compared with a control dual-luciferase reporter with a short unstructured 5' UTR. This increase was related to an interference with cap-dependent translation initiation by the TAR-Poly(A) region at the 5' end of the messenger. HIV-1 mRNA 5' UTR also contains an internal ribosome entry site (IRES), but we showed that, when the cap-dependent initiation mode is available, the IRES is not used or is weakly used. However, when the ribosomes have to use the IRES to translate the dual-luciferase reporter, the frameshift efficiency is comparable to that of the control dual-luciferase reporter. The decrease in cap-dependent initiation and the accompanying increase in frameshift efficiency caused by the 5' UTR of HIV-1 mRNA is antagonized, in a dose-dependent way, by the Tat viral protein. Tat also stimulates the IRES-dependent initiation and decreases the corresponding frameshift efficiency. A model is presented that accounts for the variations in frameshift efficiency depending on the 5' UTR and the presence of Tat, and it is proposed that a range of frameshift efficiencies is compatible with the virus replication.     

The elongation, termination, and recycling phases of translation in eukaryotes

This work summarizes our current understanding of the elongation and termination/recycling phases of eukaryotic protein synthesis. We focus here on recent advances in the field. In addition to an overview of translation elongation, we discuss unique aspects of eukaryotic translation elongation including eEF1 recycling, eEF2 modification, and eEF3 and eIF5A function. Likewise, we highlight the function of the eukaryotic release factors eRF1 and eRF3 in translation termination, and the functions of ABCE1/Rli1, the Dom34:Hbs1 complex, and Ligatin (eIF2D) in ribosome recycling. Finally, we present some of the key questions in translation elongation, termination, and recycling that remain to be answered.     

RNase L, a crucial mediator of innate immunity and other cell functions

The 2-5A/RNase L pathway is one of the first cellular defences against viruses. RNase L is an unusual endoribonuclease which activity is strictly regulated by its binding to a small oligonucleotide, 2-5A. 2-5A itself is very unusual, consisting of a series of 5'- triphosphorylated oligoadenylates with 2'-5' bonds. But RNase L activity is not limited to viral RNA cleavage. RNase L plays a central role in innate immunity, apoptosis, cell growth and differentiation by regulating cellular RNA stability and expression. Default in its activity leads to increased susceptibility to virus infections and to tumor development. RNase L gene has been identified as HPC1 (Hereditary Prostate Cancer 1) gene. Study of RNase L variant R462Q in etiology of prostate cancer has led to the identification of the novel human retrovirus closely related to xenotropic murine leukemia viruses (MuLVs) and named XMRV.     

Inhibition of translation termination mediated by an interaction of eukaryotic release factor 1 with a nascent peptidyl-tRNA

Expression of the human cytomegalovirus UL4 gene is inhibited by translation of a 22-codon-upstream open reading frame (uORF2). The peptide product of uORF2 acts in a sequence-dependent manner to inhibit its own translation termination, resulting in persistence of the uORF2 peptidyl-tRNA linkage. Consequently, ribosomes stall at the uORF2 termination codon and obstruct downstream translation. Since termination appears to be the critical step affected by translation of uORF2, we examined the role of eukaryotic release factors 1 and 3 (eRF1 and eRF3) in the inhibitory mechanism. In support of the hypothesis that an interaction between eRF1 and uORF2 contributes to uORF2 inhibitory activity, specific residues in each protein, glycines 183 and 184 of the eRF1 GGQ motif and prolines 21 and 22 of the uORF2 peptide, were found to be necessary for full inhibition of downstream translation. Immunoblot analyses revealed that eRF1, but not eRF3, accumulated in the uORF2-stalled ribosome complex. Finally, increased puromycin sensitivity was observed after depletion of eRF1 from the stalled ribosome complex, consistent with inhibition of peptidyl-tRNA hydrolysis resulting from an eRF1-uORF2 peptidyl-tRNA interaction. These results reveal the paradoxical potential for interactions between a nascent peptide and eRF1 to obstruct the translation termination cascade.