Dimerization of the 3'UTR of bicoid mRNA involves a two-step mechanism.

The proper localization of bicoid (bcd) mRNA requires cis-acting signals within its 3' untranslated region (UTR) and trans-acting factors such as Staufen. Dimerization of bcd mRNA through intermolecular base-pairing between two complementary loops of domain III of the 3'UTR was proposed to be important for particle formation in the embryo. The participation in the dimerization process of each domain building the 3'UTR was evaluated by thermodynamic and kinetic analysis of various mutated and truncated RNAs. Although sequence complementarity between the two loops of domain III is required for initiating mRNA dimerization, the initial reversible loop-loop complex is converted rapidly into an almost irreversible complex. This conversion involves parts of RNA outside of domain III that promote initial recognition, and dimerization can be inhibited by sense or antisense oligonucleotides only before conversion has proceeded. Injection of the different bcd RNA variants into living Drosophila embryos shows that all elements that inhibit RNA dimerization in vitro prevent formation of localized particles containing Staufen. Particle formation appeared to be dependent on both mRNA dimerization and other element(s) in domains IV and V. Domain III of bcd mRNA could be substituted by heterologous dimerization motifs of different geometry. The resulting dimers were converted into stable forms, independently of the dimerization module used. Moreover, these chimeric RNAs were competent in forming localized particles and recruiting Staufen. The finding that the dimerization domain of bcd mRNA is interchangeable suggests that dimerization by itself, and not the precise geometry of the intermolecular interactions, is essential for the localization process. This suggests that the stabilizing interactions that are formed during the second step of the dimerization process might represent crucial elements for Staufen recognition and localization.     

RNA-RNA interaction is required for the formation of specific bicoid mRNA 3'' UTR-STAUFEN ribonucleoprotein particles.

The formation of the anterior pattern of the Drosophila embryo is dependent on the localization of the mRNA of the morphogen Bicoid (bcd) to the anterior pole of the egg cell. Staufen protein (STAU) is required in a late step of the localization to anchor the bcd mRNA in the anterior cytoplasm. We have shown previously that endogenous STAU associates specifically with injected bcd mRNA 3'-untranslated region (UTR), resulting in the formation of characteristic RNA-protein particles that are transported along microtubules of the mitotic spindles in a directed manner. The regions recognized by STAU in this in vivo assay are predicted to form three stem-loop structures involving large double-stranded stretches. Here, we show that the STAU interaction requires a double-stranded conformation of the stems within the RNA localization signal. In addition, base pairing between two single-stranded loops plays a major role in particle formation. This loop-loop interaction is intermolecular, not intramolecular; thus dimers or multimers of the RNA localization signal must be associated with STAU in these particles. The bcd mRNA 3' UTR can also dimerize in vitro in the absence of STAU. Thus, in addition to RNA-protein interactions, RNA-RNA interaction might be involved in the formation of ribonucleoprotein particles for transport and localization.     

The molecular motor dynein is involved in targeting swallow and bicoid RNA to the anterior pole of Drosophila oocytes

Localization of bicoid (bcd) messenger RNA to the anterior pole of the Drosophila oocyte requires the exuperantia ( exu), swallow (swa) and staufen (stau) genes. We show here that Swa protein transiently co-localizes with bcd RNA in mid-oogenesis. Swa also localizes to the anterior pole of the oocyte in the absence of bcd RNA. This localization does not require Exu, but depends on intact microtubules. In mutant ovaries with duplicated polarity of microtubules, Swa and bcd RNA are ectopically localized at the posterior pole, as well as being present at the anterior pole. We identify dynein light chain-1 (Ddlc-1), a component of the minus-end-directed microtubule motor cytoplasmic dynein, as a Swa-binding protein. We propose that Swa acts as an adaptor for the dynein complex and thereby enables dynein to transport bcd RNA along microtubules to their minus ends at the anterior pole of the oocyte.     

Comparative sequence analysis and patterns of covariation in RNA secondary structures

A novel method of RNA secondary structure prediction based on a comparison of nucleotide sequences is described. This method correctly predicts nearly all evolutionarily conserved secondary structures of five different RNAs: tRNA, 5S rRNA, bacterial ribonuclease P (RNase P) RNA, eukaryotic small subunit rRNA, and the 3' untranslated region (UTR) of the Drosophila bicoid (bcd) mRNA. Furthermore, covariations occurring in the helices of these conserved RNA structures are analyzed. Two physical parameters are found to be important determinants of the evolution of compensatory mutations: the length of a helix and the distance between base-pairing nucleotides. For the helices of bcd 3' UTR mRNA and RNase P RNA, a positive correlation between the rate of compensatory evolution and helix length is found. The analysis of Drosophila bcd 3' UTR mRNA further revealed that the rate of compensatory evolution decreases with the physical distance between base-pairing residues. This result is in qualitative agreement with Kimura's model of compensatory fitness interactions, which assumes that mutations occurring in RNA helices are individually deleterious but become neutral in appropriate combinations.     

Insulin-mediated suppression of apolipoprotein B mRNA translation requires the 5' UTR and is characterized by decreased binding of an insulin-sensitive 110-kDa 5' UTR RNA-binding protein

Insulin has been shown to acutely regulate hepatic apolipoprotein B (apoB) secretion at both translational and post-translational levels; however, mechanisms of apoB mRNA translational control are largely unknown. Recent studies of apoB untranslated regions (UTRs) revealed a potentially important role for cis-trans interactions at the 5' and 3' UTRs. In the present paper, deletion constructs of the UTR regions of apoB revealed that the 5' UTR was necessary and sufficient for insulin to inhibit synthesis of apoB15. Metabolic radiolabeling and in vitro translation experiments in the presence of protease inhibitors confirmed that the effect of insulin on the apoB 5' UTR was translational in nature. Using the nondenaturing electrophoretic mobility shift assay (EMSA), protein-RNA complexes were detected binding to the apoB 5' and 3' UTRs. Denaturing EMSA identified a 110-kDa protein interacting at the 5' UTR. Nondenaturing EMSA determined that insulin altered binding of large protein complexes to the 5' UTR. Binding specificity was determined by competition with both specific and nonspecific competitors. Insulin treatment decreased binding of the 110-kDa protein to the 5' UTR as visualized by EMSA. Absence of insulin increased binding of this trans-acting factor to the 5' UTR by 2-fold. Analysis of the 3' UTR showed no significant insulin-mediated changes in binding of trans-acting factors. We thus propose the existence of a novel RNA-binding insulin-sensitive factor that binds to the 5' UTR and may regulate apoB mRNA translation. Perturbations in hepatic insulin signaling as observed in insulin-resistant states may alter cis-trans interactions at the 5' UTR, leading to alterations in the rate of apoB mRNA translation, thus contributing to apoB-lipoprotein overproduction.     

A software tool-box for analysis of regulatory RNA elements

We describe an integrated tool-box to identify regulatory RNA elements. The RNA analyzer collects general and specific information on any submitted RNA sequence or batch of sequences in FASTA format. It determines and rapidly scans the different regions of an RNA (including 5' UTR, CDS, 3' UTR in mRNA) and screens for specific RNA signals (in each of these regions, e.g. polyA-site, AU rich region etc. in 3' UTR). It runs a fast folding RNA routine to provide an overview of the RNA fold. Furthermore it analyzes structure content, fold energy and stem loops. In addition, consensus templates are used to determine whether there are any functional structures present for translational control (template: IRE), structured RNA (template: tRNA consensus) or catalytic RNA (template: trans-splicing RNA), giving indications as to how well the structures found match to these templates. The tool box has been implemented as a WWW server at http://wb2x01.biozentrum.uni-wuerzburg.de/.     

Defining the orientation of the human U1A RBD1 on its UTR by tethered-EDTA(Fe) cleavage.

The N-terminal RNA binding domain of the human U1A protein (RBD1) specifically binds an RNA hairpin of U1 snRNA as well as two internal loops in the 3' UTR of its own mRNA. Here, a single cysteine has been introduced into Loop 1 of RBD1, which is subsequently used to attach (EDTA-2-aminoethyl) 2-pyridyl disulfide-Fe3+ (EPD-Fe). This EDTA-Fe derivative is used to generate hydroxyl radicals to cleave the proximal RNA sugar-phosphate backbone in the RNA-RBD complexes. RBD1(K20C)-EPD-Fe cleaves the 5' strand of the RNA hairpin stem, centered four base pairs away from the base of the loop, and cleaves the UTR in two places, again centered on the 5' side of the fourth base pair from each internal loop. These data, extrapolated to the position of Lys 20 in RBD1, orient the two proteins bound to the UTR, and provide direct biochemical evidence for the proposed model of the RBD1:UTR complex.     

Pyrimidine tract binding protein and La autoantigen interact differently with the 5' untranslated regions of lentiviruses and oncoretrovirus mRNAs

Retrovirus genomic mRNA exhibits a several hundred nucleotides-long untranslated region (5' UTR) which encloses many control elements required for retrovirus replication. In addition, this 5' UTR contains translation regulatory elements, such as internal ribosome entry sites (IRESes) that have been described in oncoretroviruses, as well as in lentiviruses. UV cross-linking experiments suggested that the pyrimidine tract binding protein (PTB), a cellular protein known to regulate the activity of several picornaviral IRESes, binds to human T-cell leukemia virus (HTLV)-I RNA but not to lentiviral human immunodeficiency virus (HIV)-1, HIV-2 or simian immunodeficiency virus RNAs. To calculate the affinity of such RNA-protein interactions, we developed a new method based on the BIAcore technology. The absence of affinity of PTB for lentiviral RNAs was confirmed, whereas its affinity for HTLV-I RNAs was 1000-fold lower than for picornaviral RNAs. The BIAcore technology also revealed a significant affinity of the La autoantigen, previously described for its involvement in translational control of viral mRNAs, for HIV-1 and HTLV-I RNAs. Addition of recombinant PTB to in vitro translation experiments weakly enhanced translation initiation in the presence of HTLV-I IRES, suggesting that such an IRES requires additional trans-acting factors.     

The 5' untranslated region of encephalomyocarditis virus contains a sequence for very efficient binding of eukaryotic initiation factor eIF-2/2B.

The mechanism by which internal ribosomal binding on the picornaviral RNA takes place is still not known. An important role has been suggested for eukaryotic initiation factors eIF-4A, eIF-4B, as well as for some not yet defined trans-acting factors like p52 for poliovirus and p58 for encephalomyocarditis virus (EMCV). In this paper we describe the competition between the 5' untranslated region (UTR) of EMCV and globin mRNA for the translational apparatus in rabbit reticulocyte lysates and show that the factor that is competed for is eIF-2/2B. The EMC 5' UTR is a very strong inhibitor of globin synthesis in the rabbit reticulocyte lysate because of a 30-fold higher eIF-2/2B binding capacity. Mutations 100 to 140 nucleotides upstream of the initiation codon led to a decreased efficiency to initiate translation and to a decreased ability to inhibit globin mRNA translation. The results suggest an important role for eIF-2/2B binding in EMC RNA translation and therefore in internal initiation.     

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.     

Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1

In C. elegans, the Notch receptor GLP-1 is localized within the germline and early embryo by translational control of glp-1 mRNA. RNA elements in the glp-1 3'untranslated region (3' UTR) are necessary for repression of glp-1 translation in germ cells, and for localization of translation to anterior cells of the early embryo. The direct regulators of glp-1 mRNA are not known. Here, we show that a 34 nucleotide region of the glp-1 3' UTR contains two regulatory elements, an element that represses translation in germ cells and posterior cells of the early embryo, and an element that inhibits repressor activity to promote translation in the embryo. Furthermore, we show that the STAR/KH domain protein GLD-1 binds directly and specifically to the repressor element. Depletion of GLD-1 activity by RNA interference causes loss of endogenous glp-1 mRNA repression in early meiotic germ cells, and in posterior cells of the early embryo. Therefore, GLD-1 is a direct repressor of glp-1 translation at two developmental stages. These results suggest a new function for GLD-1 in regulating early embryonic asymmetry. Furthermore, these observations indicate that precise control of GLD-1 activity by other regulatory factors is important to localize this Notch receptor, and contributes to the spatial organization of Notch signaling.     

Changes in bicoid mRNA anchoring highlight conserved mechanisms during the oocyte-to-embryo transition

Intracellular mRNA localization directs protein synthesis to particular subcellular domains to establish embryonic polarity in a variety of organisms. In Drosophila, bicoid (bcd) mRNA is prelocalized at the oocyte anterior. After fertilization, translation of this RNA produces a Bcd protein gradient that determines anterior cell fates [1] and [2]. Analysis of bcd mRNA during late stages of oogenesis suggested a model for steady-state bcd localization by continual active transport [3]. However, this mechanism cannot explain maintenance of bcd localization throughout the end of oogenesis, when microtubules disassemble in preparation for embryogenesis [4] and [5], or retention of bcd at the anterior in mature oocytes, which can remain dormant for weeks before fertilization [6]. Here, we elucidate the path and mechanism of sustained bcd mRNA transport by direct observation of bcd RNA particle translocation in living oocytes. We show that bcd mRNA shifts from continuous active transport to stable actin-dependent anchoring at the end of oogenesis. Egg activation triggers bcd release from the anterior cortex for proper deployment in the embryo, probably through reorganization of the actin cytoskeleton. These findings uncover a surprising parallel between flies and frogs, as cortically tethered Xenopus Vg1 mRNA undergoes a similar redistribution during oocyte maturation [7]. Our results thus highlight a conserved mechanism for regulating mRNA anchoring and redeployment during the oocyte-to-embryo transition.     

Regulatory Role of the Conserved Stem-Loop Structure at the 5′ End of Collagen α1(I) mRNA

Three fibrillar collagen mRNAs, alpha1(I), alpha2(I), and alpha1(III), are coordinately upregulated in the activated hepatic stellate cell (hsc) in liver fibrosis. These three mRNAs contain sequences surrounding the start codon that can be folded into a stem-loop structure. We investigated the role of this stem-loop structure in expression of collagen alpha1(I) reporter mRNAs in hsc's and fibroblasts. The stem-loop dramatically decreases accumulation of mRNAs in quiescent hsc's and to a lesser extent in activated hsc's and fibroblasts. The stem-loop decreases mRNA stability in fibroblasts. In activated hsc's and fibroblasts, a protein complex binds to the stem-loop, and this binding requires the presence of a 7mG cap on the RNA. Placing the 3' untranslated region (UTR) of collagen alpha1(I) mRNA in a reporter mRNA containing this stem-loop further increases the steady-state level in activated hsc's. This 3' UTR binds alphaCP, a protein implicated in increasing stability of collagen alpha1(I) mRNA in activated hsc's (B. Stefanovic, C. Hellerbrand, M. Holcik, M. Briendl, S. A. Liebhaber, and D. A. Brenner, Mol. Cell. Biol. 17:5201-5209, 1997). A set of protein complexes assembles on the 7mG capped stem-loop RNA, and a 120-kDa protein is specifically cross-linked to this structure. Thus, collagen alpha1(I) mRNA is regulated by a complex interaction between the 5' stem-loop and the 3' UTR, which may optimize collagen production in activated hsc's.