In-stem molecular beacon targeted to a 5'-region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity.

Cellular functions are regulated by the up- and down-regulation and localization of RNA molecules. Therefore, many RNA detection methods have been developed to analyze RNA levels and localization. Molecular beacon (MB) is one of the major methods for quantitative RNA detection and analysis of RNA localization. Most oligonucleotide-based probes, including MB, are designed to target a long flexible region on the target RNA molecule, e.g., a single-stranded region. Recently, analyses of tRNA localization and levels became important, as it has been shown that environmental stresses and chemical reagents induce nuclear accumulation of tRNA and tRNA degradation in mammalian cells. However, tRNA is highly structured and does not harbor any long flexible regions. Hence, only a few methods are currently available for detecting tRNA. In the present study, we attempted to detect elongator tRNAMet (eMet) and initiator tRNAMet (iMet) by using an in-stem molecular beacon (ISMB), characterized by more effective quenching and significantly higher sensitivity than those of conventional MB. We found that ISMB1 targeted a 5'- region that includes the D arm of tRNA and that it detected eMet and iMet transcripts as well as mature eMet with high sensitivity. Moreover, the analysis revealed that the formation of the ISMB/tRNA transcript complex required more time than the formation of an ISMB/unstructured short RNA complex. These results suggest that ISMB-based tRNA detection can be a useful tool for various biological and medical studies.

Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL.

The tRNA 3'-processing endoribonuclease (tRNase Z or 3' tRNase; EC 3.1.26.11) is an essential enzyme that removes the 3' trailer from pre-tRNA. The long form (tRNase ZL) can cleave a target RNA in vitro at the site directed by an appropriate small-guide RNA (sgRNA). Here, we investigated whether this sgRNA/tRNase ZL strategy could be applied to gene therapy for AIDS. We tested the ability of four sgRNA-expression plasmids to inhibit HIV-1 gene expression in COS cells, using a transient-expression assay. The three sgRNAs guide inhibition of HIV-1 gene expression in cultured COS cells. Analysis of the HIV-1 mRNA levels suggested that sgRNA directed the tRNase ZL to mediate the degradation of target RNA. The observation that sgRNA was localized primarily in nuclei suggests that tRNase ZL cleaves the HIV-1 mRNA when complexed with sgRNA in this location. We also examined the ability of two retroviral vectors expressing sgRNA to suppress HIV-1 expression in HIV-1-infected Jurkat T cells. sgRNA-SL4 suppressed HIV-1 expression almost completely in infected cells for up to 18 days. These results suggest that the sgRNA/tRNase ZL approach is effective in downregulating HIV-1 gene expression.

Small stable RNA level depends on the physiological state of the cell.

The level of 5S rRNA and tRNAi(Met)1 synthesized by RNA polymerase III was investigated in human epidermoid carcinoma cells A431 at different physiological states: low and high proliferation and apoptosis. The real-time RT-PCR method using SYBR Green I was applied to measure certain RNA species in total cellular RNA. The share of 5S rRNA was practically the same in slowly and actively proliferating A431 cells, but increased about 2.5-fold in apoptotic cells. The share of initiator tRNAi(Met)1 in actively proliferating and apoptotic cells was 1.5-2.0 times higher than in slowly proliferating cells. Our results suggest a possible existence of special mechanisms regulating RNA polymerase III-directed transcription from different type promoters in accordance with the physiological state of the cell.

Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules.

Environmental stress-induced phosphorylation of eIF2alpha inhibits protein translation by reducing the availability of eIF2-GTP-tRNA(i)Met, the ternary complex that joins initiator tRNA(Met) to the 43S preinitiation complex. The resulting untranslated mRNA is dynamically routed to discrete cytoplasmic foci known as stress granules (SGs), a process requiring the related RNA-binding proteins TIA-1 and TIAR. SGs appear to be in equilibrium with polysomes, but the nature of this relationship is obscure. We now show that most components of the 48S preinitiation complex (i.e., small, but not large, ribosomal subunits, eIF3, eIF4E, eIF4G) are coordinately recruited to SGs in arsenite-stressed cells. In contrast, eIF2 is not a component of newly assembled SGs. Cells expressing a phosphomimetic mutant (S51D) of eIF2alpha assemble SGs of similar composition, confirming that the recruitment of these factors is a direct consequence of blocked translational initiation and not due to other effects of arsenite. Surprisingly, phospho-eIF2alpha is recruited to SGs that are disassembling in cells recovering from arsenite-induced stress. We discuss these results in the context of a translational checkpoint model wherein TIA and eIF2 are functional antagonists of translational initiation, and in which lack of ternary complex drives SG assembly.

Characterization of the initiator tRNA gene locus and identification of a strong promoter from Mycobacterium tuberculosis.

An initiator tRNA gene, metA, and a closely linked fragment of a second initiator-tRNA-like sequence, metB, from Mycobacterium tuberculosis H37Ra have been cloned and characterized. The promoter region of metA shows the presence of conserved sequence elements, TAGCCT and TTGGCG, with resemblance to -10 and -35 promoter regions. The deduced sequence of the mature tRNA contains the three unique features of the eubacterial initiator tRNAs represented by (i) a C:U mismatch at position 1:72, (ii) three consecutive base pairs, 29-31G:C39-41 in the anticodon stem, and (iii) a purine:pyrimidine (A:U) base pair at position 11:24 in the dihydrouridine stem. A putative hairpin structure consisting of an 11 bp stem and a three-base loop found in the 3' flanking region is followed by a stretch of T residues and may serve as a transcription terminator. Analysis of the expression of metA and of its promoter using chloramphenicol acetyltransferase fusion constructs in Mycobacterium smegmatis shows that metA is a functional gene driven by a strong promoter. Furthermore, the overexpressed transcripts are fully processed and formylated in vivo. The metB clone shows the presence of sequences corresponding to those downstream of position 30 of the tRNA. However, the CCA sequence at the 3' end has been mutated to CCG. Interestingly, the 3' flanking sequences of both the genes are rich in GCT repeats. The metB locus also harbours a repeat element, IS6110. A method to prepare total RNA from mycobacteria (under acidic conditions) to analyse in vivo status of tRNAs is described.

Escherichia coli B lacks one of the two initiator tRNA species present in E. coli K-12.

We show that the metY locus which specifies tRNA(2fMet) in Escherichia coli K-12 specifies tRNA(1fMet) in E. coli B. This conclusion is based on results of Southern blot analysis of E. coli B and K-12 DNAs and on polymerase chain reaction amplification, cloning, and sequencing of an approximately 200-bp region of DNA corresponding to the metY loci of E. coli B and E. coli K-12. We also show that the metY locus of E. coli B is transcriptionally active. E. coli strains transformed with the multicopy plasmid vector pUC19 carrying the metY locus of E. coli B overproduce tRNA(1fMet) in E. coli B and E. coli K-12 in contrast to strains transformed with pUC19 carrying the corresponding locus from E. coli K-12, which overproduce tRNA(2fMet).

Analysis of magnesium, europium and lead binding sites in methionine initiator and elongator tRNAs by specific metal-ion-induced cleavages.

The specificity of cleavages in yeast and lupin initiator and elongator methionine tRNAs induced by magnesium, europium and lead has been analysed and compared with known patterns of yeast tRNA(Phe) hydrolysis. The strong D-loop cleavages occur in methionine elongator tRNAs at similar positions and with comparable efficiency to those found in tRNA(Phe), while the sites of weak anticodon loop cuts, identical in methionine elongator tRNAs, differ from those found in tRNA(Phe). Methionine initiator tRNAs differ from their elongator counterparts: (a) they are cleaved in the D-loop with much lower efficiency; (b) they are cleaved in the variable loop which is completely resistant to hydrolysis in elongator tRNAs; (c) cleavages in the anticodon loop are stronger in initiator tRNAs and they are located mostly at the 5' side of the loop whereas in elongator tRNAs they occur mostly at the opposite, 3' side of the loop. The distinct pattern of the anticodon loop cleavages is considered to be related to different conformations of the anticodon loop in the two types of methionine tRNAs.

Transfer-RNA, magnesium and the formation of ternary complexes by eukaryotic initiation factor eIF-2.

The likely concentrations of free magnesium ions in assay systems measuring ternary complex formation with the eukaryotic initiation factor eIF-2 and the exchange of bound GDP have been calculated. Contrary to the suggestion of Roy et al. (Biochem. Biophys. Res. Commun. 146, 114-120) amounts of added tRNA are unlikely to sequester sufficient magnesium ions to affect significantly their role in the assays. There seems little correlation between methionyl-tRNA added and the extent of ternary complex formation in published data other than that expected from mass action. In vivo the concentration of methionyl-tRNA is probably greater than or equal to that of eIF-2 which is shown to be necessary for efficient functioning of the initiation factor.