A dual inhibitory mechanism restricts msl-2 mRNA translation for dosage compensation in Drosophila

Drosophila MSL-2 is the limiting component of the dosage compensation complex. Female flies must inhibit msl-2 mRNA translation for survival, and this inhibition is mediated by Sex-lethal (SXL) binding to sites in both the 5' and the 3' untranslated regions (UTRs). Here, we uncover the mechanism by which SXL achieves tight control of translation initiation. SXL binding to the 3'UTR regulatory region inhibits the recruitment of 43S ribosomal preinitiation complexes to the mRNA. Ribosomal complexes escaping this block and binding to the 5' end of the mRNA are challenged by SXL bound to the 5'UTR, which interferes with scanning to the downstream initiation codon of the mRNA. This failsafe mechanism thus forms the molecular basis of a critical step in dosage compensation. The results also elucidate a two step principle of translational control via multiple regulatory sites within an mRNA.     

Mechanism of translational regulation by miR-2 from sites in the 5' untranslated region or the open reading frame

MicroRNAs (miRs) commonly regulate translation from target mRNA 3' untranslated regions (UTRs). While effective miR-binding sites have also been identified in 5' untranslated regions (UTRs) or open reading frames (ORFs), the mechanism(s) of miR-mediated regulation from these sites has not been defined. Here, we systematically investigate how the position of miR-binding sites influences translational regulation and characterize their mechanistic basis. We show that specific translational regulation is elicited in vitro and in vivo not only from the 3'UTR, but equally effectively from six Drosophila miR-2-binding sites in the 5'UTR or the ORF. In all cases, miR-2 triggers mRNA deadenylation and inhibits translation initiation in a cap-dependent fashion. In contrast, single or dual miR-2-binding sites in the 5'UTR or the ORF yield rather inefficient or no regulation. This work represents the first demonstration that 5'UTR and ORF miR-binding sites can function mechanistically similarly to the intensively investigated 3'UTR sites. Using single or dual binding sites, it also reveals a biological rationale for the high prevalence of miR regulatory sites in the 3'UTR.     

The Drosophila splicing regulator sex-lethal directly inhibits translation of male-specific-lethal 2 mRNA.

Male-specific expression of the protein male-specific-lethal 2 (MSL-2) controls dosage compensation in Drosophila. msl-2 gene expression is inhibited in females by Sex-lethal (SXL), an RNA binding protein known to regulate pre-mRNA splicing. An intron present at the 5' untranslated region (UTR) of msl-2 mRNA contains putative SXL binding sites and is retained in female flies. Here we show that SXL plays a dual role in the inhibition of msl-2 expression. Cotransfection of Drosophila Schneider cells with an SXL expression vector and a reporter containing the 5' UTR of msl-2 mRNA resulted in retention of the 5' UTR intron and efficient accumulation of the unspliced mRNA in the cytoplasm, where its translation was blocked by SXL, but not by the intron per se. Both splicing and translation inhibition by SXL were recapitulated in vitro and found to be dependent upon SXL binding to high-affinity sites within the intron, showing that SXL directly regulates these events. Our data reveal a coordinated mechanism for the regulation of msl-2 expression by the same regulatory factor: SXL enforces intron retention in the nucleus and subsequent translation inhibition in the cytoplasm.     

Functional domains of Drosophila UNR in translational control

Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (SXL) is an essential regulatory step of X chromosome dosage compensation in Drosophila. Translation inhibition requires that SXL recruits the protein upstream of N-ras (UNR) to the 3' UTR of msl-2 mRNA. UNR is a conserved, ubiquitous protein that contains five cold-shock domains (CSDs). Here, we dissect the domains of UNR required for translational repression and complex formation with SXL and msl-2 mRNA. Using gel-mobility shift assays, the domain involved in interactions with SXL and msl-2 was mapped specifically to the first CSD (CSD1). Indeed, excess of a peptide containing this domain derepressed msl-2 translation in vitro. The CSD1 of human UNR can also form a complex with SXL and msl-2. Comparative analyses of the CSDs of the Drosophila and human proteins together with site-directed mutagenesis experiments revealed that three exposed residues within CSD1 are required for complex formation. Tethering assays showed that CSD1 is not sufficient for translational repression, indicating that UNR binding to SXL and msl-2 can be distinguished from translation inhibition. Repression by tethered UNR requires residues from both the amino-terminal Q-rich stretch and the two first CSDs, indicating that the translational effector domain of UNR resides within the first 397 amino acids of the protein. Our results identify domains and residues required for UNR function in translational control.     

Drosophila sex-lethal inhibits the stable association of the 40S ribosomal subunit with msl-2 mRNA

The inhibition of male-specific lethal-2 (msl-2) mRNA translation in female flies is essential for X chromosome dosage compensation in Drosophila melanogaster. Translational repression of msl-2 requires sex-lethal (SXL) binding to uridine-rich sequences in both the 5' and 3' untranslated regions (UTRs) of the message. We delineate the msl-2 mRNA sequence elements that are important for regulation by SXL and identify functionally critical sequences adjacent to regulatory SXL binding sites. We demonstrate that SXL inhibits translation initiation and prevents the stable association of the 40S ribosomal subunit with the mRNA in a manner that does not require the presence of a cap structure at the 5' end of the mRNA. These results elucidate a critical regulatory step for dosage compensation in Drosophila melanogaster.     

Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5' and 3' UTRs of msl-2 mRNA is independent of the poly(A) tail.

Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (SXL) controls dosage compensation in Drosophila. In vivo regulation involves cooperativity between SXL-binding sites in the 5' and 3' untranslated regions (UTRs). To investigate the mechanism of msl-2 translational control, we have developed a novel cell-free translation system from Drosophila embryos that recapitulates the critical features of mRNA translation in eukaryotes: cap and poly(A) tail dependence. Importantly, tight regulation of msl-2 translation in this system requires cooperation between the SXL-binding sites in both the 5' and 3' UTRs, as seen in vivo. However, in contrast to numerous other developmentally regulated mRNAs, the regulation of msl-2 mRNA occurs by a poly(A) tail-independent mechanism. The approach described here allows mechanistic analysis of translational control in early Drosophila development and has revealed insights into the regulation of dosage compensation by SXL.     

A GG nucleotide sequence of the 3' untranslated region of amyloid precursor protein mRNA plays a key role in the regulation of translation and the binding of proteins

The alternative polyadenylation of the mRNA encoding the amyloid precursor protein (APP) involved in Alzheimer's disease generates two molecules, with the first of these containing 258 additional nucleotides in the 3' untranslated region (3'UTR). We have previously shown that these 258 nucleotides increase the translation of APP mRNA injected in Xenopus oocytes (5). Here, we demonstrate that this mechanism occurs in CHO cells as well. We also present evidence that the 3'UTR containing 8 nucleotides more than the short 3'UTR allows the recovery of an efficiency of translation similar to that of the long 3'UTR. Moreover, the two guanine residues located at the 3' ends of these 8 nucleotides play a key role in the translational control. Using gel retardation mobility shift assay, we show that proteins from Xenopus oocytes, CHO cells, and human brain specifically bind to the short 3'UTR but not to the long one. The two guanine residues involved in the translational control inhibit this specific binding by 65%. These results indicate that there is a correlation between the binding of proteins to the 3'UTR of APP mRNA and the efficiency of mRNA translation, and that a GG motif controls both binding of proteins and translation.     

Proteins binding to 5'' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.     

Translational control via protein-regulated upstream open reading frames

Analysis of the regulation of msl-2 mRNA by Sex lethal (SXL), which is critical for dosage compensation in Drosophila, has uncovered a mode of translational control based on common 5' untranslated region elements, upstream open reading frames (uORFs), and interaction sites for RNA-binding proteins. We show that SXL binding downstream of a short uORF imposes a strong negative effect on major reading frame translation. The underlying mechanism involves increasing initiation of scanning ribosomes at the uORF and augmenting its impediment to downstream translation. Our analyses reveal that SXL exerts its effect controlling initiation, not elongation or termination, at the uORF. Probing the generality of the underlying mechanism, we show that the regulatory module that we define experimentally functions in a heterologous context, and we identify natural Drosophila mRNAs that are regulated via this module. We propose that protein-regulated uORFs constitute a systematic principle for the regulation of protein synthesis.     

Rapid deadenylation and Poly(A)-dependent translational repression mediated by the Caenorhabditis elegans tra-2 3' untranslated region in Xenopus embryos

The 3' untranslated region (3'UTR) of many eukaryotic mRNAs is essential for their control during early development. Negative translational control elements in 3'UTRs regulate pattern formation, cell fate, and sex determination in a variety of organisms. tra-2 mRNA in Caenorhabditis elegans is required for female development but must be repressed to permit spermatogenesis in hermaphrodites. Translational repression of tra-2 mRNA in C. elegans is mediated by tandemly repeated elements in its 3'UTR; these elements are called TGEs (for tra-2 and GLI element). To examine the mechanism of TGE-mediated repression, we first demonstrate that TGE-mediated translational repression occurs in Xenopus embryos and that Xenopus egg extracts contain a TGE-specific binding factor. Translational repression by the TGEs requires that the mRNA possess a poly(A) tail. We show that in C. elegans, the poly(A) tail of wild-type tra-2 mRNA is shorter than that of a mutant mRNA lacking the TGEs. To determine whether TGEs regulate poly(A) length directly, synthetic tra-2 3'UTRs with and without the TGEs were injected into Xenopus embryos. We find that TGEs accelerate the rate of deadenylation and permit the last 15 adenosines to be removed from the RNA, resulting in the accumulation of fully deadenylated molecules. We conclude that TGE-mediated translational repression involves either interference with poly(A)'s function in translation and/or regulated deadenylation.     

YB-1 autoregulates translation of its own mRNA at or prior to the step of 40S ribosomal subunit joining

YB-1 is a member of the numerous families of proteins with an evolutionary ancient cold-shock domain. It is involved in many DNA- and RNA-dependent events and regulates gene expression at different levels. Previously, we found a regulatory element within the 3' untranslated region (UTR) of YB-1 mRNA that specifically interacted with YB-1 and poly(A)-binding protein (PABP); we also showed that PABP positively affected YB-1 mRNA translation in a poly(A) tail-independent manner (O. V. Skabkina, M. A. Skabkin, N. V. Popova, D. N. Lyabin, L. O. Penalva, and L. P. Ovchinnikov, J. Biol. Chem. 278:18191-18198, 2003). Here, YB-1 is shown to strongly and specifically inhibit its own synthesis at the stage of initiation, with accumulation of its mRNA in the form of free mRNPs. YB-1 and PABP binding sites have been mapped on the YB-1 mRNA regulatory element. These were UCCAG/ACAA for YB-1 and a approximately 50-nucleotide A-rich sequence for PABP that overlapped each other. PABP competes with YB-1 for binding to the YB-1 mRNA regulatory element and restores translational activity of YB-1 mRNA that has been inhibited by YB-1. Thus, YB-1 negatively regulates its own synthesis, presumably by specific interaction with the 3'UTR regulatory element, whereas PABP restores translational activity of YB-1 mRNA by displacing YB-1 from this element.     

The Escherichia coli threonyl-tRNA synthetase gene contains a split ribosomal binding site interrupted by a hairpin structure that is essential for autoregulation

The expression of the gene encoding Escherichia coli threonyl-tRNA synthetase (ThrRS) is negatively autoregulated at the translational level. ThrRS binds to its own mRNA leader, which consists of four structural and functional domains: the Shine–Dalgarno (SD) sequence and the initiation codon region (domain 1); two upstream hairpins (domains 2 and 4) connected by a single-stranded region (domain 3). Using a combination of in vivo and in vitro approaches, we show here that the ribosome binds to thrS mRNA at two non-contiguous sites: region −12 to +16 comprising the SD sequence and the AUG codon and, unexpectedly, an upstream single-stranded sequence in domain 3. These two regions are brought into close proximity by a 38-nucleotide-long hairpin structure (domain 2). This domain, although adjacent to the 5' edge of the SD sequence, does not inhibit ribosome binding as long as the single-stranded region of domain 3 is present. A stretch of unpaired nucleotides in domain 3, but not a specific sequence, is required for efficient translation. As the repressor and the ribosome bind to interspersed domains, the competition between ThrRS and ribosome for thrS mRNA binding can be explained by steric hindrance.     

A U-rich element in the 5' untranslated region is necessary for the translation of p27 mRNA

Increased translation of p27 mRNA correlates with withdrawal of cells from the cell cycle. This raised the possibility that antimitogenic signals might mediate their effects on p27 expression by altering complexes that formed on p27 mRNA, regulating its translation. In this report, we identify a U-rich sequence in the 5' untranslated region (5'UTR) of p27 mRNA that is necessary for efficient translation in proliferating and nonproliferating cells. We show that a number of factors bind to the 5'UTR in vitro in a manner dependent on the U-rich element, and their availability in the cytosol is controlled in a growth- and cell cycle-dependent fashion. One of these factors is HuR, a protein previously implicated in mRNA stability, transport, and translation. Another is hnRNP C1 and C2, proteins implicated in mRNA processing and the translation of a specific subset of mRNAs expressed in differentiated cells. In lovastatin-treated MDA468 cells, the mobility of the associated hnRNP C1 and C2 proteins changed, and this correlated with increased p27 expression. Together, these data suggest that the U-rich dependent RNP complex on the 5'UTR may regulate the translation of p27 mRNA and may be a target of antimitogenic signals.     

Iron regulatory protein prevents binding of the 43S translation pre-initiation complex to ferritin and eALAS mRNAs.

Translation of ferritin and erythroid 5-aminolevulinate synthase (eALAS) mRNAs is regulated by iron via mRNA-protein interactions between iron-responsive elements (IREs) and iron regulatory protein (IRP). In iron-depleted cells, IRP binds to single IREs located in the 5' untranslated regions of ferritin and eALAS mRNAs and represses translation initiation. The molecular mechanism underlying this translational repression was investigated using reconstituted, IRE-IRP-regulated, cell-free translation systems. The IRE-IRP interaction is shown to prevent the association of the 43S translation pre-initiation complex (including the small ribosomal subunit) with the mRNA. Studies with the spliceosomal protein U1A and mRNAs which harbour specific binding sites for this protein in place of an IRE furthermore reveal that the 5' termini of mRNAs are generally sensitive to repressor protein-mediated inhibition of 43S pre-initiation complex binding.     

The DICE-binding activity of KH domain 3 of hnRNP K is affected by c-Src-mediated tyrosine phosphorylation

hnRNP K and hnRNP E1/E2 are RNA-binding proteins comprised of three hnRNP K-homology (KH) domains. These proteins are involved in the translational control and stabilization of mRNAs in erythroid cells. hnRNP E1 and hnRNP K regulate the translation of reticulocyte 15-lipoxygenase (r15-LOX) mRNA. Both proteins bind specifically to the differentiation control element (DICE) in the 3' untranslated region (3'UTR) of the r15-LOX mRNA. It has been shown that hnRNP K is a substrate of the tyrosine kinase c-Src and that tyrosine phosphorylation by c-Src inhibits the binding of hnRNP K to the DICE. Here, we investigate which of the three KH domains of hnRNP E1 and hnRNP K mediate the DICE interaction. Using RNA-binding assays, we demonstrate DICE-binding of the KH domains 1 and 3 of hnRNP E1, and KH domain 3 of hnRNP K. Furthermore, with RNA-binding assays, NMR experiments and in vitro translation studies, we show that tyrosine 458 in KH domain 3 of hnRNP K is important for the DICE interaction and we provide evidence that it is a target of c-Src.