The 3' untranslated region of tobacco necrosis virus RNA contains a barley yellow dwarf virus-like cap-independent translation element

RNAs of many viruses are translated efficiently in the absence of a 5' cap structure. The tobacco necrosis virus (TNV) genome is an uncapped, nonpolyadenylated RNA whose translation mechanism has not been well investigated. Computational analysis predicted a cap-independent translation element (TE) within the 3' untranslated region (3' UTR) of TNV RNA that resembles the TE of barley yellow dwarf virus (BYDV), a luteovirus. Here we report that such a TE does indeed exist in the 3' UTR of TNV strain D. Like the BYDV TE, the TNV TE (i) functions both in vitro and in vivo, (ii) requires additional sequence for cap-independent translation in vivo, (iii) has a similar secondary structure and the conserved sequence CGGAUCCUGGGAAACAGG, (iv) is inactivated by a four-base duplication in this conserved sequence, (v) can function in the 5' UTR, and (vi) when located in its natural 3' location, may form long-distance base pairing with the viral 5' UTR that is conserved and probably required. The TNV TE differs from the BYDV TE by having only three helical domains instead of four. Similar structures were found in all members of the Necrovirus genus of the Tombusviridae family, except satellite tobacco necrosis virus, which harbors a different 3' cap-independent translation domain. The presence of the BYDV-like TE in select genera of different families indicates that phylogenetic distribution of TEs does not follow standard viral taxonomic relationships. We propose a new class of cap-independent TE called BYDV-like TE.     

A four-nucleotide translation enhancer in the 3'-terminal consensus sequence of the nonpolyadenylated mRNAs of rotavirus

The 5' cap and poly(A) tail of eukaryotic mRNAs work synergistically to enhance translation through a process that requires interaction of the cap-associated eukaryotic initiation factor, eIF-4G, and the poly(A)-binding protein, PABP. Because the mRNAs of rotavirus, and other members of the Reoviridae, contain caps but lack poly(A) tails, their translation may be enhanced through a unique mechanism. To identify translation-enhancement elements in the viral mRNAs that stimulate translation in vivo, chimeric RNAs were prepared that contained an open reading frame for luciferase and the 5' and 3' untranslated regions (UTRs) of a rotavirus mRNA or of a nonviral mRNA. Transfection of the chimeric RNAs into rotavirus-infected cells showed that the viral 3' UTR contained a translation-enhancement element that promoted gene expression. The element did not enhance gene expression in uninfected cells and did not affect the stability of the RNAs. Mutagenesis showed that the conserved sequence GACC located at the 3' end of rotavirus mRNAs operated as an enhancement element. The 3'-GACC element stimulated protein expression independently of the sequence of the 5' UTR, although efficient expression required the RNA to contain a cap. The results indicate that the expression of viral proteins in rotavirus-infected cells is specifically up-regulated by the activity of a novel 4-nt 3' translation enhancer (TE) common to the 11 nonpolyadenylated mRNAs of the virus. The 4-nt sequence of the rotavirus 3' TE represents by far the shortest of any of the sequence enhancers known to stimulate translation.     

Structure and function of a cap-independent translation element that functions in either the 3' or the 5' untranslated region

Barley yellow dwarf virus RNA lacks both a 5' cap and a poly(A) tail, yet it is translated efficiently. It contains a cap-independent translation element (TE), located in the 3' UTR, that confers efficient translation initiation at the AUG closest to the 5' end of the mRNA. We propose that the TE must both recruit ribosomes and facilitate 3'-5' communication. To dissect its function, we determined the secondary structure of the TE and roles of domains within it. Nuclease probing and structure-directed mutagenesis revealed that the 105-nt TE (TE105) forms a cruciform secondary structure containing four helices connected by single-stranded regions. TE105 can function in either UTR in wheat germ translation extracts. A longer viral sequence (at most 869 nt) is required for full cap-independent translation in plant cells. However, substantial translation of uncapped mRNAs can be obtained in plant cells with TE105 combined with a poly(A) tail. All secondary structural elements and most primary sequences that were mutated are required for cap-independent translation in the 3' and 5' UTR contexts. A seven-base loop sequence was needed only in the 3' UTR context. Thus, this loop sequence may be involved only in communication between the UTRs and not directly in recruiting translational machinery. This structural and functional analysis provides a framework for understanding an emerging class of cap-independent translation elements distinguished by their location in the 3' UTR.     

A potential mechanism for selective control of cap-independent translation by a viral RNA sequence in cis and in trans.

Highly efficient cap-independent translation initiation at the 5'-proximal AUG is facilitated by the 3' translation enhancer sequence (3'TE) located near the 3' end of barley yellow dwarf virus (BYDV) genomic RNA. The role of the 3'TE in regulating viral translation was examined. The 3'TE is required for translation and thus replication of the genomic RNA that lacks a 5' cap (Allen et al., 1999, Virology253:139-144). Here we show that the 3'TE also mediates translation of uncapped viral subgenomic mRNAs (sgRNA1 and sgRNA2). A 109-nt viral sequence is sufficient for 3'TE activity in vitro, but additional viral sequence is necessary for cap-independent translation in vivo. The 5' extremity of the sequence required in the 3' untranslated region (UTR) for cap-independent translation in vivo coincides with the 5' end of sgRNA2. Thus, sgRNA2 has the 3'TE in its 5' UTR. Competition studies using physiological ratios of viral RNAs showed that, in trans, the 109-nt 3'TE alone, or in the context of 869-nt sgRNA2, inhibited translation of genomic RNA much more than it inhibited translation of sgRNA1. The divergent 5' UTRs of genomic RNA and sgRNA1 contribute to this differential susceptibility to inhibition. We propose that sgRNA2 serves as a novel regulatory RNA to carry out the switch from early to late gene expression. Thus, this new mechanism for temporal control of translation control involves a sequence that stimulates translation in cis and acts in trans to selectively inhibit translation of viral mRNA.     

Identification of two critical base pairings in 5' untranslated regions affecting translation efficiency of synthetic uncapped globin mRNAs.

We observed a marked difference between the in vitro translation efficiency of two uncapped synthetic mRNAs, displaying the entire human alpha or beta globin mRNA sequences and some additional non-globin sequences in 5'. The comparison of the translation efficiencies of chimeric mRNAs indicated that the alpha 5' untranslated region (5' UTR) is responsible for a low translation efficiency that cannot be explained neither by primary sequence nor by the overall stability of 5' UTR secondary structures only. By point mutations in this alpha 5' UTR, we identified two base pairings at position -1 and -2 preceding the initiation codon which are associated with a negative effect on translation efficiency.     

Human cytoplasmic serine hydroxymethyltransferase is an mRNA binding protein

The 5' untranslated region (UTR) of the human cytoplasmic serine hydroxymethyltransferase (cSHMT) message is alternatively spliced, creating a full-length 5' UTR (LUTR) encoded within exons 1-3 and a shorter UTR (SUTR) that results from excision of exon 2. The role of the 5' UTRs in cSHMT expression was investigated by fusing the cSHMT 5' UTRs to the 5' end of the luciferase gene. Human cSHMT protein at 10 microM inhibits in vitro translation of cSHMT 5' UTR-luciferase fusion mRNA templates by more than 90%, but does not inhibit translation of the luciferase message lacking the UTR. Translation inhibition is independent of amino acid and folate substrate binding to the cSHMT enzyme. The cSHMT SUTR-luciferase mRNA binds to the cSHMT.glycine.5-formyltetrahydrofolate ternary complex with an apparent K(d) of 10 microM. Gel mobility shift assays demonstrate that the human cSHMT protein binds to the cSHMT LUTR-luciferase fusion mRNA in the presence and absence of glycine and 5-formyltetrahydrofolate pentaglutamate. The fusion cSHMT SUTR-luciferase message at 65 microM inhibits the cSHMT-catalyzed cleavage of allothreonine as a partial mixed type inhibitor, reducing both k(cat) and K(m) by 40 and 75%, respectively, while tRNA has no effect on cSHMT catalysis. These studies indicate that the cSHMT protein can bind mRNA, and displays increased affinity for the 5' untranslated region of its mRNA.     

Position and stability are determining factors for translation repression by an RNA G-quadruplex-forming sequence within the 5' UTR of the NRAS proto-oncogene

Nucleic acid secondary structures in the 5' untranslated regions (UTRs) of mRNAs have been shown to play a critical role in translation regulation. We recently demonstrated that a naturally occurring, conserved, and stable RNA G-quadruplex element (5'-GGGAGGGGCGGGUCUGGG-3'), located close to the 5' cap within the 5' UTR of the NRAS proto-oncogene mRNA, modulates gene expression at the translational level. Herein, we show that the translational effect of this G-quadruplex motif in NRAS 5' UTR is not uniform, but rather depends on the location of the G-quadruplex-forming sequence. The RNA G-quadruplex-forming sequence represses translation when situated relatively proximal to the 5' end, within the first 50 nt, in the 5' UTR of the NRAS proto-oncogene, whereas it has no significant effect on translation if located comparatively away from the 5' end. We have also demonstrated that the thermodynamic stability of the RNA G-quadruplex at its natural position within the NRAS 5' UTR is an important factor contributing toward its ability to repress translation.     

The 3'-terminal hexamer sequence of classical swine fever virus RNA plays a role in negatively regulating the IRES-mediated translation

The 3' untranslated region (UTR) is usually involved in the switch of the translation and replication for a positive-sense RNA virus. To understand the 3' UTR involved in an internal ribosome entry site (IRES)-mediated translation in Classical swine fever virus (CSFV), we first confirmed the predicted secondary structure (designated as SLI, SLII, SLIII, and SLIV) by enzymatic probing. Using a reporter assay in which the luciferase expression is under the control of CSFV 5' and 3' UTRs, we found that the 3' UTR harbors the positive and negative regulatory elements for translational control. Unlike other stem loops, SLI acts as a repressor for expression of the reporter gene. The negative cis-acting element in SLI is further mapped to the very 3'-end hexamer CGGCCC sequence. Further, the CSFV IRES-mediated translation can be enhanced by the heterologous 3'-ends such as the poly(A) or the 3' UTR of Hepatitis C virus (HCV). Interestingly, such an enhancement was repressed by flanking this hexamer to the end of poly(A) or HCV 3' UTR. After sequence comparison and alignment, we have found that this hexamer sequence could hypothetically base pair with the sequence in the IRES IIId1, the 40 S ribosomal subunit binding site for the translational initiation, located at the 5' UTR. In conclusion, we have found that the 3'-end terminal sequence can play a role in regulating the translation of CSFV.     

Characterization of the 5' and 3' untranslated regions in murine mdm2 mRNAs

The murine double minute 2 (mdm2) gene is essential for embryogenesis in mice that express the p53 tumor suppressor protein. Mdm2 levels must be regulated tightly because overexpression of mdm2 contributes to tumorigenesis. We investigated whether the 5' and 3' untranslated regions (UTRs) of murine mdm2 affect the expression of MDM2 proteins. Induction of mdm2 expression by p53 results in synthesis of an mdm2 mRNA with a short 5' UTR. The long 5' UTR increases internal initiation of translation of a minor MDM2 protein, p76(MDM2), without affecting the efficiency of translation of the full-length p90(MDM2). We discovered two alternative 3' untranslated regions in murine mdm2 mRNA expressed in the testis. The longer 3' UTR contains a consensus instability element, but mdm2 mRNAs containing the long and short 3' UTRs have comparable half-lives. The 3' UTRs do not affect either initiation codon use or translation efficiency. Thus, the murine 5' UTR, but not the 3'UTR, influences the ratio of the two MDM2 proteins but neither UTR affects MDM2 abundance significantly.     

The Shine-Dalgarno-like sequence is a negative regulatory element for translation of tobacco chloroplast rps2 mRNA: an additional mechanism for translational control in chloroplasts

Most prokaryotic mRNAs contain within the 5' untranslated region (UTR), a Shine-Dalgarno (SD) sequence, which is complementary to the 3' end of 16S rRNA and serves as a major determinant for correct translational initiation. The tobacco chloroplast rps2 mRNA possesses an SD-like sequence (GGAG) at a proper position (positions -8 to -5 from the start codon). Using an in vitro translation system from isolated tobacco chloroplasts, the role of this sequence in translation was examined. Unexpectedly, the mutation of the SD-like element resulted in a large increase in translation. Internal and external deletions within the 5' UTR revealed that the region from -20 to -5 was involved in the negative regulation of translation. Scanning mutagenesis assays confirmed the above result. Competition assays suggested the existence of a trans-acting factor(s) involved in translational regulation. In this study, we discuss a possible mechanism for the negative regulation of rps2 mRNA translation.     

人组织型纤溶酶原激活剂(t-PA)mRNA5′端非翻译区(UTR)对其表达的调控

运用反转录-PCR技术,从黑色素瘤细胞中扩增出t—PA cDNA 5′末端460bp的片段,再经重组获得含完整5′-UTR的t—PA cDNA克隆,在兔网织红细胞裂解物中翻译和COS-7细胞中表达发现,t—PA mRNA 5′—UTR对其表达有明显的抑制作用。将t—PA mRNA 5′—UTR用苜蓿病毒RNA 5′—UTR替换,使t—PA的表达水平提高3-7倍,mRNA翻译起始区二级结构分析结果表明,翻译起始区的二级结构与t-PA的表达水平有关。     

小麦花粉特异性表达的cDNA的分离及表达特性

应用抑制差示杂交和5′/3′RACE PCR方法分离了小麦(Triticum aestivum L.)花粉特异性表达的全长cDNA(TaPSG719,GenBank:AY451238)),该基因全长1172 bp,5′非编码区序列长达329 bp,包含多个上游可译框架(uORF);该基因编码188个氨基酸的蛋白质,大小约20 kD,等电点为12.1。Northern杂交和RT-PCR分析表明该基因在成熟花粉特异表达,而在小孢子、叶片、根和未成熟的种子、幼茎和子房等组织几乎检测不到。进一步研究小麦花粉发育过程的表达水平表明,TaPSG719在单核和双核小孢子阶段不表达,在开花前5d(已完成有丝分裂)开始表达并迅速增强达到高峰,但随着花粉的成熟表达水平逐渐下降。表明TaPSG719是一个花粉中晚期特异性表达基因。经BLAST同源性分析表明,与目前已登录的基因没有显著的同源性。Southern杂交表明TaPSG719可能为一个多拷贝基因。为研究TaPSG719 cDNA 5′非编码区序列的uORF对可译框架的翻译的影响,构建不同缺失或突变的表达载体,采用麦胚体外翻译系统,结果显示含uORF的5′非编码区序列能显著抑制蛋白质的翻译水平,表明TaPSG719基因表达至少部分是在翻译水平上调控。     

小麦花粉特异性表达的cDNA的分离及表达特性

应用抑制差示杂交和5′/3′RACE PCR方法分离了小麦(Triticum aestivum L.)花粉特异性表达的全长cDNA(TaPSG719,GenBank:AY451238)),该基因全长1 172 bp,5′非编码区序列长达329 bp,包含多个上游可译框架(uORF);该基因编码1 88个氨基酸的蛋白质,大小约20 kD,等电点为12.1.Northern杂交和RT-PCR分析表明该基因在成熟花粉特异表达,而在小孢子、叶片、根和未成熟的种子、幼茎和子房等组织几乎检测不到.进一步研究小麦花粉发育过程的表达水平表明,TaPSG719在单核和双核小孢子阶段不表达,在开花前5 d(已完成有丝分裂)开始表达并迅速增强达到高峰,但随着花粉的成熟表达水平逐渐下降.表明TaPSG719是一个花粉中晚期特异性表达基因.经BLAST同源性分析表明,与目前已登录的基因没有显著的同源性.Southern杂交表明TaPSG719可能为一个多拷贝基因.为研究TaPSG719 cDNA 5′非编码区序列的uORF对可译框架的翻译的影响,构建不同缺失或突变的表达载体,采用麦胚体外翻译系统,结果显示含uORF的5′非编码区序列能显著抑制蛋白质的翻译水平,表明TaPSG719基因表达至少部分是在翻译水平上调控.     

trans regulation of cap-independent translation by a viral subgenomic RNA

Many positive-strand RNA viruses generate 3'-coterminal subgenomic mRNAs to allow translation of 5'-distal open reading frames. It is unclear how viral genomic and subgenomic mRNAs compete with each other for the cellular translation machinery. Translation of the uncapped Barley yellow dwarf virus genomic RNA (gRNA) and subgenomic RNA1 (sgRNA1) is driven by the powerful cap-independent translation element (BTE) in their 3' untranslated regions (UTRs). The BTE forms a kissing stem-loop interaction with the 5' UTR to mediate translation initiation at the 5' end. Here, using reporter mRNAs that mimic gRNA and sgRNA1, we show that the abundant sgRNA2 inhibits translation of gRNA, but not sgRNA1, in vitro and in vivo. This trans inhibition requires the functional BTE in the 5' UTR of sgRNA2, but no translation of sgRNA2 itself is detectable. The efficiency of translation of the viral mRNAs in the presence of sgRNA2 is determined by proximity to the mRNA 5' end of the stem-loop that kisses the 3' BTE. Thus, the gRNA and sgRNA1 have "tuned" their expression efficiencies via the site in the 5' UTR to which the 3' BTE base pairs. We conclude that sgRNA2 is a riboregulator that switches off translation of replication genes from gRNA while permitting translation of structural genes from sgRNA1. These results reveal (i) a new level of control of subgenomic-RNA gene expression, (ii) a new role for a viral subgenomic RNA, and (iii) a new mechanism for RNA-mediated regulation of translation.