Translation of the chloroplast psbC mRNA is controlled by interactions between its 5' leader and the nuclear loci TBC1 and TBC3 in Chlamydomonas reinhardtii.

Translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii has been shown previously to require interactions between its 5' untranslated region (5' UTR) and the functions encoded by two nuclear loci, which we name here TBC1 and TBC2. We show that a 97-nucleotide (nt) region located in the middle of the psbC 5' UTR is required for translation initiation. Unlike most procaryotic cis-acting translational control elements, this region has a translational activation function and is located 236 nt upstream from the GUG translation initiation codon. In vivo pulse-labeling of chloroplast-encoded proteins and analyses of the expression of chimeric reporter genes in vivo reveal that a mutation of a newly described locus, TBC3, restores translation from the psbC 5' UTR in the absence of either this cis-acting element or the wild-type trans-acting TBC1 function. These data demonstrate that sequences located in the middle of the psbC 5' UTR, TBC1, and TBC3 functionally interact to control the translation of the psbC mRNA.     

AU-rich-element-dependent translation repression requires the cooperation of tristetraprolin and RCK/P54

AU-rich elements (AREs), residing in the 3' untranslated region (UTR) of many labile mRNAs, are important cis-acting elements that modulate the stability of these mRNAs by collaborating with trans-acting factors such as tristetraprolin (TTP). AREs also regulate translation, but the underlying mechanism is not fully understood. Here we examined the function and mechanism of TTP in ARE-mRNA translation. Through a luciferase-based reporter system, we used knockdown, overexpression, and tethering assays in 293T cells to demonstrate that TTP represses ARE reporter mRNA translation. Polyribosome fractionation experiments showed that TTP shifts target mRNAs to lighter fractions. In murine RAW264.7 macrophages, knocking down TTP produces significantly more tumor necrosis factor alpha (TNF-α) than the control, while the corresponding mRNA level has a marginal change. Furthermore, knockdown of TTP increases the rate of biosynthesis of TNF-α, suggesting that TTP can exert effects at translational levels. Finally, we demonstrate that the general translational repressor RCK may cooperate with TTP to regulate ARE-mRNA translation. Collectively, our studies reveal a novel function of TTP in repressing ARE-mRNA translation and that RCK is a functional partner of TTP in promoting TTP-mediated translational repression.     

Rapamycin conditionally inhibits Hsp90 but not Hsp70 mRNA translation in <Emphasis Type="Italic">Drosophila</Emphasis>: implications for the mechanisms of Hsp mRNA translation

Rapamycin inhibits the activity of the target of rapamycin (TOR)-dependent signaling pathway, which has been characterized as one dedicated to translational regulation through modulating cap-dependent translation, involving eIF4E binding protein (eIF4E-BP) or 4E-BP. Results show that rapamycin strongly inhibits global translation in Drosophila cells. However, Hsp70 mRNA translation is virtually unaffected by rapamycin treatment, whereas Hsp90 mRNA translation is strongly inhibited, at normal growth temperature. Intriguingly, during heat shock Hsp90 mRNA becomes significantly less sensitive to rapamycin-mediated inhibition, suggesting the pathway for Hsp90 mRNA translation is altered during heat shock. Reporter mRNAs containing the Hsp90 or Hsp70 mRNAs’ 5′ untranslated region recapitulate these rapamycin-dependent translational characteristics, indicating this region regulates rapamycin-dependent translational sensitivity as well as heat shock preferential translation. Surprisingly, rapamycin-mediated inhibition of Hsp90 mRNA translation at normal growth temperature is not caused by 4E-BP-mediated inhibition of cap-dependent translation. Indeed, no evidence for rapamycin-mediated impaired eIF4E function is observed. These results support the proposal that preferential translation of different Hsp mRNA utilizes distinct translation mechanisms, even within a single species.     

Chloroplast translation regulation

Chloroplast gene expression is primarily controlled during the translation of plastid mRNAs. Translation is regulated in response to a variety of biotic and abiotic factors, and requires a coordinate expression with the nuclear genome. The translational apparatus of chloroplasts is related to that of bacteria, but has adopted novel mechanisms in order to execute the specific roles that this organelle performs within a eukaryotic cell. Accordingly, plastid ribosomes contain a number of chloroplast-unique proteins and domains that may function in translational regulation. Chloroplast translation regulation involves cis-acting RNA elements (located in the mRNA 5′ UTR) as well as a set of corresponding trans-acting protein factors. While regulation of chloroplast translation is primarily controlled at the initiation steps through these RNA-protein interactions, elongation steps are also targets for modulating chloroplast gene expression. Translation of chloroplast mRNAs is regulated in response to light, and the molecular mechanisms underlying this response involve changes in the redox state of key elements related to the photosynthetic electron chain, fluctuations of the ADP/ATP ratio and the generation of a proton gradient. Photosynthetic complexes also experience assembly-related autoinhibition of translation to coordinate the expression of different subunits of the same complex. Finally, the localization of all these molecular events among the different chloroplast subcompartments appear to be a crucial component of the regulatory mechanisms of chloroplast gene expression.     

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.     

Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5'' noncoding region.

Poliovirus mRNA contains a long 5' noncoding region of about 750 nucleotides (the exact number varies among the three virus serotypes), which contains several AUG codons upstream of the major initiator AUG. Unlike most eucaryotic mRNAs, poliovirus does not contain a m7GpppX (where X is any nucleotide) cap structure at its 5' end and is translated by a cap-independent mechanism. To study the manner by which poliovirus mRNA is expressed, we examined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. In this paper we report striking translation system-specific differences in the ability of the altered mRNAs to be translated. The results suggest the existence of an inhibitory cis-acting element(s) within the 5' noncoding region of poliovirus (between nucleotides 70 and 381) which restricts mRNA translation in reticulocyte lysate, wheat germ extract, and Xenopus oocytes, but not in HeLa cell extracts. In addition, we show that HeLa cell extracts contain a trans-acting factor(s) that overcomes this restriction.     

Glorund, a Drosophila hnRNP F/H homolog, is an ovarian repressor of nanos translation

Patterning of the anterior-posterior body axis of the Drosophila embryo requires production of Nanos protein selectively in the posterior. Spatially restricted Nanos synthesis is accomplished by translational repression of unlocalized nanos mRNA together with translational activation of posteriorly localized nanos. Repression of unlocalized nanos mRNA is mediated by a bipartite translational control element (TCE) in its 3' untranslated region. TCE stem-loop II functions during embryogenesis, through its interaction with the Smaug repressor. Stem-loop III represses unlocalized nanos mRNA during oogenesis, but trans-acting factors that carry out this function have remained elusive. Here we identify a Drosophila hnRNP, Glorund, that interacts specifically with stem-loop III. We establish that the ability of the TCE to repress translation in vivo reflects its ability to bind Glorund in vitro. These data, together with the analysis of a glorund null mutant, reveal a specific role for an hnRNP in repression of nanos translation during oogenesis.     

Cytoplasmic proteins interact with a translational control element in the protein-coding region of proopiomelanocortin mRNA

Previous studies have indicated that proopiomelanocortin (POMC) is translationally regulated. We proposed that the regulatory mechanism involves an interaction between trans-acting protein factors and a cis-acting stem-loop structure in the coding region of POMC mRNA. Functional interactions were tested by examining the translation of mouse POMC mRNA in a rabbit reticulocyte system. Specific binding was demonstrated with ultraviolet-crosslinking and RNA gel mobility shift assays. The evidence presented supports our hypothesis that the translational regulation of POMC gene expression involves recognition of the stem-loop by RNA-binding proteins. Furthermore, POMC stem-loop RNA-binding proteins specifically recognized a predicted stem-loop found in the coding region of corticotropin-releasing hormone, suggesting a novel mechanism of gene regulation that may extend to other neuropeptides as well.     

The role of IRES trans-acting factors in regulating translation initiation

The majority of mRNAs in eukaryotic cells are translated via a method that is dependent upon the recognition of, and binding to, the methylguanosine cap at the 5' end of the mRNA, by a set of protein factors termed eIFs (eukaryotic initiation factors). However, many of the eIFs involved in this process are modified and become less active under a number of pathophysiological stress conditions, including amino acid starvation, heat shock, hypoxia and apoptosis. During these conditions, the continued synthesis of proteins essential to recovery from stress or maintenance of a cellular programme is mediated via an alternative form of translation initiation termed IRES (internal ribosome entry site)-mediated translation. This relies on the mRNA containing a complex cis-acting structural element in its 5'-UTR (untranslated region) that is able to recruit the ribosome independently of the cap, and is often dependent upon additional factors termed ITAFs (IRES trans-acting factors). A limited number of ITAFs have been identified to date, particularly for cellular IRESs, and it is not yet fully understood how they exert their control and which cellular pathways are involved in their regulation.     

In vitro mutational analysis of cis-acting RNA translational elements within the poliovirus type 2 5'' untranslated region.

Initiation of translation on poliovirus RNA occurs by internal binding of ribosomes to a region within the 5' untranslated region (UTR) of the mRNA. This region has been previously roughly mapped between nucleotides 140 and 631 of the 5' UTR and termed the ribosome landing pad. To identify cis-acting elements in the 5' UTR of poliovirus type 2 (Lansing strain) RNA that confer cap-independent internal initiation, we determined the in vitro translational efficiencies of a series of deletion and point mutations within the 5' UTR of the mRNA. The results demonstrate that the 3' border of the core poliovirus ribosome landing pad is located between nucleotides 556 and 585, whereas a region extending between nucleotides 585 and 612 confers enhanced translation. We studied two cis-acting elements within this region of the 5' UTR: a pyrimidine stretch which is critical for translation and an AUG (number 7 from the 5' end) that is located approximately 20 nucleotides downstream from the pyrimidine stretch and augments translation. We also show that the stem-loop structure which contains this AUG is not required for translation.     

mRNA stability: in trans-it.

The regulation of mRNA stability is an important step in the control of gene expression. Characterization of the mechanisms involved in the turnover of individual mRNAs has identified a requirement for specific cis-acting sequences and trans-acting factors, as well as an involvement of the translation apparatus. In the past year, significant progress has been made in the identification of trans-acting factors by both biochemical and genetic approaches. This review summarizes that progress and promotes the notion that the ribosome itself should also be considered as a trans-acting component of the mRNA decay machinery.     

Requirement for both EDEN and AUUUA motifs in translational arrest of Mos mRNA upon fertilization of Xenopus eggs

Translational arrest of maternal Mos mRNA upon fertilization of Xenopus eggs is a prerequisite for the initiation of embryonic divisions. Recent studies suggest that an embryo deadenylation element (EDEN) present in the 3' untranslated region (3'UTR) is sufficient for deadenylation (and, hence, probably for translational arrest) of Mos mRNA after fertilization. By directly monitoring translation of numerous Mos mRNA constructs in Xenopus eggs, however, we show here that the EDEN is necessary but not sufficient for translational arrest of Mos mRNA. We demonstrate that two AUUUA motifs, each located solitarily and distantly from the EDEN, are also required for the translational arrest of Mos mRNA after fertilization. Significantly, translational arrest of Eg2 mRNA, another EDEN-containing maternal mRNA, also requires a single AUUUA motif located far from the EDEN. Analysis of the poly(A) tails of various Mos mRNA constructs indicates that the EDEN alone confers only partial deadenylation on Mos mRNA, and that the AUUUA motifs act to enhance EDEN-directed deadenylation in a position-dependent manner. Finally, introduction of an excess of the EDEN, but not the AUUUA motifs, into eggs can restore translation of endogenous Mos mRNA. These results suggest that the EDEN, only together with appropriately positioned AUUUA motifs and a trans-acting factor(s), can efficiently deadenylate and hence translationally arrest Mos (as well as Eg2) mRNA after fertilization.