Multiple elements required for translation of plastid atpB mRNA lacking the Shine-Dalgarno sequence

The mechanism of translational initiation differs between prokaryotes and eukaryotes. Prokaryotic mRNAs generally contain within their 5′-untranslated region (5′-UTR) a Shine-Dalgarno (SD) sequence that serves as a ribosome-binding site. Chloroplasts possess prokaryotic-like translation machinery, and many chloroplast mRNAs have an SD-like sequence, but its position is variable. Tobacco chloroplast atpB mRNAs contain no SD-like sequence and are U-rich in the 5′-UTR (−20 to −1 with respect to the start codon). In vitro translation assays with mutated mRNAs revealed that an unstructured sequence encompassing the start codon, the AUG codon and its context are required for translation. UV crosslinking experiments showed that a 50 kDa protein (p50) binds to the 5′-UTR. Insertion of an additional initiation region (SD-sequence and AUG) in the 5′-UTR, but not downstream, arrested translation from the authentic site; however, no inhibition was observed by inserting only an AUG triplet. We hypothesize for translational initiation of the atpB mRNA that the ribosome enters an upstream region, slides to the start codon and forms an initiation complex with p50 and other components.     

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.     

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.     

5'-untranslated regions with multiple upstream AUG codons can support low-level translation via leaky scanning and reinitiation

Upstream AUGs (uAUGs) and upstream open reading frames (uORFs) are common features of mRNAs that encode regulatory proteins and have been shown to profoundly influence translation of the main ORF. In this study, we employed a series of artificial 5′-untranslated regions (5′-UTRs) containing one or more uAUGs/uORFs to systematically assess translation initiation at the main AUG by leaky scanning and reinitiation mechanisms. Constructs containing either one or two uAUGs in varying contexts but without an in-frame stop codon upstream of the main AUG were used to analyse the leaky scanning mechanism. This analysis largely confirmed the ranking of different AUG contextual sequences that was determined previously by Kozak. In addition, this ranking was the same for both the first and second uAUGs, although the magnitude of initiation efficiency differed. Moreover, ~10% of ribosomes exhibited leaky scanning at uAUGs in the most favourable context and initiated at a downstream AUG. A second group of constructs containing different numbers of uORFs, each with optimal uAUGs, were used to measure the capacity for reinitiation. We found significant levels of initiation at the main ORF even in constructs containing four uORFs, with nearly 10% of ribosomes capable of reinitiating five times. This study shows that for mRNAs containing multiple uORFs/uAUGs, ribosome reinitiation and leaky scanning are efficient mechanisms for initiation at their main AUGs.     

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.     

Messenger RNA processing in Methanocaldococcus (Methanococcus) jannaschii

Messenger RNA (mRNA) processing plays important roles in gene expression in all domains of life. A number of cases of mRNA cleavage have been documented in Archaea, but available data are fragmentary. We have examined RNAs present in Methanocaldococcus (Methanococcus) jannaschii for evidence of RNA processing upstream of protein-coding genes. Of 123 regions covered by the data, 31 were found to be processed, with 30 including a cleavage site 12–16 nucleotides upstream of the corresponding translation start site. Analyses with 3′-RACE (rapid amplification of cDNA ends) and 5′-RACE indicate that the processing is endonucleolytic. Analyses of the sequences surrounding the processing sites for functional sites, sequence motifs, or potential RNA secondary structure elements did not reveal any recurring features except for an AUG translation start codon and (in most cases) a ribosome binding site. These properties differ from those of all previously described mRNA processing systems. Our data suggest that the processing alters the representation of various genes in the RNA pool and therefore, may play a significant role in defining the balance of proteins in the cell.     

Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs

Escherichia coli mRNA translation is facilitated by sequences upstream and downstream of the initiation codon, called Shine–Dalgarno (SD) and downstream box (DB) sequences, respectively. In E.coli enhancing the complementarity between the DB sequences and the 16S rRNA penultimate stem resulted in increased protein accumulation without a significant affect on mRNA stability. The objective of this study was to test whether enhancing the complementarity of plastid mRNAs downstream of the AUG (downstream sequence or DS) with the 16S rRNA penultimate stem (anti-DS or ADS region) enhances protein accumulation. The test system was the tobacco plastid rRNA operon promoter fused with the E.coli phage T7 gene 10 (T7g10) 5′-untranslated region (5′-UTR) and DB region. Translation efficiency was tested by measuring neomycin phosphotransferase (NPTII) accumulation in tobacco chloroplasts. We report here that the phage T7g10 5′-UTR and DB region promotes accumulation of NPTII up to ~16% of total soluble leaf protein (TSP). Enhanced mRNA stability and an improved NPTII yield (~23% of TSP) was obtained from a construct in which the T7g10 5′-UTR was linked with the NPTII coding region via a NheI site. However, replacing the T7g10 DB region with the plastid DS sequence reduced NPTII and mRNA levels to 0.16 and 28%, respectively. Reduced NPTII accumulation is in part due to accelerated mRNA turnover.     

Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start codon

Translation initiation represents a key step during regulation of gene expression in chloroplasts. Here, we report on the identification and characterization of three suppressor point mutations which overcome a translational defect caused by the deletion of a U-rich element in the 5′-untranslated region (5′-UTR) of the psbD mRNA in the green alga Chlamydomonas reinhardtii. All three suppressors affect a secondary RNA structure encompassing the psbD AUG initiation codon within a double-stranded region as judged by the analysis of site-directed chloroplast mutants as well as in vitro RNA mapping experiments using RNase H. In conclusion, the data suggest that these new element serves as a negative regulator which mediates a rapid shut-down of D2 synthesis.