Mutations Altering the Predicted Secondary Structure of a Chloroplast 5′ Untranslated Region Affect Its Physical and Biochemical Properties as Well as Its Ability To Promote Translation of Reporter mRNAs Both in the Chlamydomonas reinhardtii Chloroplast and in Escherichia coli

Random mutations were generated in the sequence for the 5' untranslated region (5'UTR) of the Chlamydomonas reinhardtii chloroplast rps7 mRNA by PCR, the coding sequence for the mutant leaders fused upstream of the lacZ' reporter in pUC18, and transformed into Escherichia coli, and white colonies were selected. Twelve single base pair changes were found at different positions in the rps7 5'UTR in 207 white colonies examined. Seven of the 12 mutant leaders allowed accumulation of abundant lacZ' message. These mutant rps7 leaders were ligated into an aadA expression cassette and transformed into the chloroplast of C. reinhardtii and into E. coli. In vivo spectinomycin-resistant growth rates and in vitro aminoglycoside adenyltransferase enzyme activity varied considerably between different mutants but were remarkably similar for a given mutant expressed in the Chlamydomonas chloroplast and in E. coli. The variable effect of the mutants on aadA reporter expression and their complete abolition of lacZ' reporter expression in E. coli suggests differences in the interaction between the 5'UTR of rps7 and aadA or lacZ' coding regions. Several rps7 5'UTR mutations affected the predicted folding pattern of the 5'UTR by weakening the stability of stem structures. Site-directed secondary mutations generated to restore these structures in the second stem suppressed the loss of reporter activity caused by the original mutations. Additional site-directed mutations that were predicted to further strengthen (A-U-->G-C) or weaken (G-C-->A-U) the second stem of the rps7 leader both resulted in reduced reporter expression. This genetic evidence combined with differences between mutant and wild-type UV melting profiles and RNase T1 protection gel shifts further indicate that the predicted wild-type folding pattern in the 5'UTR is likely to play an essential role in translation initiation.     

Efficient translation in chloroplasts requires element(s) upstream of the putative ribosome binding site from atpI

Thousands of proteins make up a chloroplast, but fewer than 100 are encoded by the chloroplast genome. Despite this low number, expression of chloroplast-encoded genes is essential for plant survival. Every chloroplast has its own gene expression system with a major regulatory point at the initiation of protein synthesis (translation). In chloroplasts, most protein-encoding genes contain elements resembling the ribosome binding sites (RBS) found in prokaryotes. In vitro, these putative chloroplast ribosome binding sequences vary in their ability to support translation. Here we report results from an investigation into effects of the predicted RBS for the tobacco chloroplast atpI gene on translation in vivo. Two reporter constructs, differing only in their 5'-untranslated regions (5'UTRs) were stably incorporated into tobacco chloroplast genomes and their expression analyzed. One 5'UTR was derived from the wild-type (WT) atpI gene. The second, Holo-substitution (Holo-sub), had nonchloroplast sequence replacing all wild-type nucleotides, except for the putative RBS. The abundance of reporter RNA was the same for both 5'UTRs. However, translation controlled by Holo-sub was less than 4% that controlled by WT. These in vivo experiments support the idea that translation initiation in land plant chloroplasts depends on 5'UTR elements outside the putative RBS.     

Three-hybrid strategy reveals a peptide segment that specifically binds to the 3'-untranslated region of NF-IL6 mRNA

The 3'UTR of eukaryotic mRNA is an important regulation region, on which many trans factors act. In recent years, a series of 3'UTRs were shown to have tumor suppressor function, including the 3'UTR of the human nuclear factor for interleukin-6 (NF-IL6 3'UTR). To understand molecular basis for this function, we have tried to isolate genes encoding protein factors acting on the RNA of NF-IL6 3'UTR. Here we show that, by using a yeast three-hybrid system, a cDNA fragment was successfully isolated. This cDNA was allowed to express in E. coli, and its expression product, a polypeptide of ca. 70 amino acids long, was shown to specifically bind to the NF-IL6 3'UTR RNA. A search in GenBank did not reveal homologous sequences. Therefore, this cDNA fragment may be a part of the gene of a novel NF-IL6 3'UTR specific binding protein.     

Interactions of intergenic microRNAs with mRNAs of genes involved in carcinogenesis

miRNAs regulate gene expression by binding with mRNAs of many genes. Studying their effects on genes involved in oncogenesis is important in cancer diagnostics and therapeutics. The RNAHybrid 2.1 program was used to predict the strong miRNA binding sites (p < 0.0005) in target mRNAs. The program Finder 2.2 was created to verify 784 intergenic miRNAs (ig-miRNA) origin. Among 54 considered oncogenes and tumor suppressor genes, 47 genes are the best targets for ig-miRNAs. Accordingly, these genes are strongly regulated by 111 ig-miRNAs. Some miRNAs bind several mRNAs, and some mRNAs have several binding sites for miRNAs. Of the 54 mRNAs, 21.8%, 43.0%, and 35.2% of the miRNA binding sites are present in the 5'UTRs, CDSes, and 3'UTRs, respectively. The average density of the binding sites for miRNAs in the 5'UTR was 4.4 times and 4.1 times greater than in the CDS and the 3'UTR, respectively. Three types of interactions between miRNAs and mRNAs were identified, which differ according to the region of the miRNA bound to the mRNA: 1) binding occurs predominantly via the 3'-region of the miRNA; 2) binding occurs predominantly through the central region of the miRNA; and 3) binding occurs predominantly via the 5'-region of the miRNA. Several miRNAs effectively regulate only one gene, and this information could be useful in molecular medicine to modulate translation of the target mRNA. We recommend described new sites for validation by experimental investigation.     

RAS is regulated by the let-7 microRNA family

MicroRNAs (miRNAs) are regulatory RNAs found in multicellular eukaryotes, including humans, where they are implicated in cancer. The let-7 miRNA times seam cell terminal differentiation in C. elegans. Here we show that the let-7 family negatively regulates let-60/RAS. Loss of let-60/RAS suppresses let-7, and the let-60/RAS 3'UTR contains multiple let-7 complementary sites (LCSs), restricting reporter gene expression in a let-7-dependent manner. mir-84, a let-7 family member, is largely absent in vulval precursor cell P6.p at the time that let-60/RAS specifies the 1 degrees vulval fate in that cell, and mir-84 overexpression suppresses the multivulva phenotype of activating let-60/RAS mutations. The 3'UTRs of the human RAS genes contain multiple LCSs, allowing let-7 to regulate RAS expression. let-7 expression is lower in lung tumors than in normal lung tissue, while RAS protein is significantly higher in lung tumors, providing a possible mechanism for let-7 in cancer.     

Post-transcriptional regulation of human cathepsin L expression

The expression of cathepsin L, a lysosomal protease, is known to be elevated in cancer and other pathologies. Multiple splice variants of human cathepsin L with variable 5'UTRs exist, which encode for the same protein. Previously we have observed that variant hCATL A (bearing the longest 5'UTR) was translated in vitro with significantly lower efficiency than variant hCATL AIII (bearing the shortest 5'UTR). Contrary to these findings, results of the present study reveal that in cancer cells, hCATL A mRNA exhibits higher translatability in spite of having lower stability than AIII. This is the first report demonstrating a highly contrasting trend in translation efficiencies of hCATL variants in rabbit reticulocytes and live cells. Expression from chimeric mRNAs containing 5'UTRs of A or AIII upstream to luciferase reporter cDNA established the A UTR to be the sole determinant for this effect. Transient transfections of bicistronic plasmids and mRNAs confirmed the presence of a functional Internal Ribosome Entry Site in this UTR. Our data suggest that differential stability and translation initiation modes mediated by the 5'UTRs of human cathepsin L variants are involved in regulating its expression.     

Sequence and translation initiation properties of the xenopus TGFbeta5, PDGF-A, and PDGF-alpha receptor 5' untranslated regions

The properties of the architecturally complex Xenopus laevis TGFbeta5, PDGF-A and PDGF-alpha receptor 5'UTRs were investigated. 5' extended cDNAs were obtained by 5'RACE, resulting in long 5'UTRs (478-710 nt) with multiple upstream AUGs (3-13), andthe potential to fold into stable structures. Injection studies suggested that the cloned PDGF-alphaR 5'UTR contains an intron. Splicing at potential 5' and 3' splice sites would result in a non-complex 5'UTR of 142 nt. The above mentioned 5'UTR characteristics are inhibitory for ribosomal scanning. Indeed, relative to the beta-globin 5'UTR, the complex 5'UTRs strongly repressed initiation of protein synthesis in pre-MBT Xenopus embryos. However, later in embryogenesis, the inhibition was partly relieved. The results show temporal translational control by these 5'UTRs. Transgenic embryos showed that the 5'UTRs allowed translation throughout the embryo; spatial control could not be observed. Interestingly, a fragment in the PDGF-A 5'UTR highly similar to an element in the human PDGF-A 5'UTR is complementary to Xenopus 18S ribosomal RNA. None of these Xenopus 5'UTRs contains an IRES, as determined by injecting bicistronic constructs.     

Phosphorylation of the spinach chloroplast 24 kDa RNA-binding protein (24RNP) increases its binding to petD and psbA 3' untranslated regions

The chloroplast 24 kDa RNA binding protein (24RNP) from Spinacea oleracea is a nuclear encoded protein that binds the 3' untranslated region (3'UTR) of some chloroplast mRNAs and seems to be involved in some processes of mRNA metabolism, such as 3'UTR processing, maturation and stabilization. The 24RNP is similar to the 28RNP which is involved in the correct maturation of petD and psbA 3'UTRs, and when phosphorylated, decreases its binding affinity for RNA. In the present work, we determined that the recombinant 24RNP was phosphorylated in vitro either by an animal protein kinase C, a plant Ca(2+)-dependent protein kinase, or a chloroplastic kinase activity present in a protein extract with 3'-end processing activity in which the 24RNP is also present. Phosphorylation of 24RNP increased the binding capacity (B(max)) 0.25 time for petD 3'UTR, and three times for psbA 3'UTR; the affinity for P-24RNP only increased when the interaction with petD was tested. Competition experiments suggested that B(max), not K(d), might be a more important factor in the P-24RNP-3'UTR interaction. The data suggested that the 24RNP role in chloroplast mRNA metabolism may be regulated in vivo by changes in its phosphorylation status carried out by a chloroplastic kinase.