Specific interaction of HeLa cell proteins with coxsackievirus B3 3'UTR: La autoantigen binds the 3' and 5'UTR independently of the poly(A) tail

Coxsackievirus B3 (CVB3) is a positive, single-stranded RNA virus. The secondary structure of the 3' untranslated region (3'UTR) of CVB3 RNA consists of three stem-loops and is followed by a poly(A) tail sequence. These stem-loop structures have been suggested to participate in the regulation of viral replication through interaction with cellular proteins that are yet to be identified. In this study, by competitive UV cross-linking using mutated 3'UTR probes we have demonstrated that the poly(A) tail is essential for promoting HeLa cell protein interactions with the 3'UTR because deletion of this sequence abolished most of the protein interactions. Unexpectedly, mutations that disrupted the tertiary loop-loop interactions without affecting the stem-loops did not apparently affect these protein interactions, indicating that secondary structure rather than the high-order structure may play a major role in recruiting these RNA binding proteins. Among the observed 3'UTR RNA binding proteins, we have confirmed a 52 kDa protein as the human La autoantigen by using purified recombinant protein and a polyclonal La antibody. This protein can interact with both the 3' and 5'UTRs independently of the poly(A) tail. Further analysis by two-stage UV cross-linking, we found that the 3' and 5'UTR sequences may share the same binding site on the La protein.     

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.     

Translational regulation of the expression of ribosomal protein genes in Xenopus laevis

The mRNAs coding for ribosomal proteins (rp-mRNA) are subjected to translational control during Xenopus oogenesis and embryogenesis, and also during nutritional changes in Xenopus cultured cells. This regulation, which appears to respond to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA engaged on polysomes, each translated rp-mRNA molecule always remaining fully loaded with ribosomes. All rp-mRNAs analyzed up to now show this translational behavior, and also share some structural features in their untranslated portions. In particular they all have rather short 5' untranslated regions, similar to each other, and always start at the very 5' end with a stretch of several pyrimidines. Fusion to a reporter-coding sequence of the 5' untranslated region of r-protein S19 has shown that this is involved in the translational regulation.     

Translational repression restricts expression of the C. elegans Nanos homolog NOS-2 to the embryonic germline

Members of the nanos gene family are evolutionarily conserved regulators of germ cell development. In several organisms, Nanos protein expression is restricted to the primordial germ cells (PGCs) during early embryogenesis. Here, we investigate the regulation of the Caenorhabditis elegans nanos homolog nos-2. We find that the nos-2 RNA is translationally repressed. In the adult germline, translation of the nos-2 RNA is inhibited in growing oocytes, and this inhibition depends on a short stem loop in the nos-2 3'UTR. In embryos, nos-2 translation is repressed in early blastomeres, and this inhibition depends on a second region in the nos-2 3'UTR. nos-2 RNA is also degraded in somatic blastomeres by a process that is independent of translational repression and requires the CCCH finger proteins MEX-5 and MEX-6. Finally, the germ plasm component POS-1 activates nos-2 translation in the PGCs. A combination of translational repression, RNA degradation, and activation by germ plasm has also been implicated in the regulation of nanos homologs in Drosophila and zebrafish, suggesting the existence of conserved mechanisms to restrict Nanos expression to the germline.     

A competition mechanism regulates the translation of the Escherichia coli operon encoding ribosomal proteins L35 and L20

Escherichia coli ribosomal protein (r-protein) L20 is essential for the assembly of the 50S ribosomal subunit and is also a translational regulator of its own rpmI-rplT operon, encoding r-proteins L35 and L20 in that order. L20 directly represses the translation of the first cistron and, through translational coupling, that of its own gene. The translational operator of the operon is 450 nt in length and includes a long-range pseudoknot interaction between two RNA sequences separated by 280 nt. L20 has the potential to bind both to this pseudoknot and to an irregular hairpin, although only one site is occupied at a time during regulation. This work shows that the rpmI-rplT operon is regulated by competition between L20 and the ribosome for binding to mRNA in vitro and in vivo. Detailed studies on the regulatory mechanisms of r-protein synthesis have only been performed on the rpsO gene, regulated by r-protein S15, and on the alpha operon, regulated by S4. Both are thought to be controlled by a trapping mechanism, whereby the 30S ribosomal subunit, the mRNA, and the initiator tRNA are blocked as a nonfunctional preternary complex. This alternative mode of regulation of the rpmI-rplT operon raises the possibility that control is kinetically and not thermodynamically limited in this case. We show that the pseudoknot, which is known to be essential for L20 binding and regulation, also enhances 30S binding to mRNA as if this structure is specifically recognised by the ribosome.     

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.     

Multiple maternal proteins coordinate to restrict the translation of C. elegans nanos-2 to primordial germ cells

Although germ cell formation has been relatively well understood in worms and insects, how germ cell-specific developmental programs are initiated is not clear. In Caenorhabditis elegans, translational activation of maternal nos-2 mRNA is the earliest known molecular event specific to the germline founder cell P(4). Cis-elements in nos-2 3'UTR have been shown to mediate translational control; however, the trans-acting proteins are not known. Here, we provide evidence that four maternal RNA-binding proteins, OMA-1, OMA-2, MEX-3 and SPN-4, bind nos-2 3'UTR to suppress its translation, and POS-1, another maternal RNA-binding protein, relieves this suppression in P(4). The POS-1: SPN-4 ratio in P(4) increases significantly over its precursor, P(3); and POS-1 competes with SPN-4 for binding to nos-2 RNA in vitro. We propose temporal changes in the relative concentrations of POS-1 and SPN-4, through their effect on the translational status of maternal mRNAs such as nos-2, initiate germ cell-specific developmental programs in C. elegans.     

Binding of the La autoantigen to the 5' untranslated region of a chimeric human translation elongation factor 1A reporter mRNA inhibits translation in vitro.

Human translation elongation factor 1A (EF1A) is a member of a large class of mRNAs, including ribosomal proteins and other translation elongation factors, which are coordinately translationally regulated under various conditions. Each of these mRNAs contains a terminal oligopyrimidine tract (TOP) that is required for translational control. A human growth hormone (hGH) expression construct containing the promoter region and 5' untranslated region (UTR) of EF1A linked to the hGH coding region (EF1A/hGH) was translationally repressed following rapamycin treatment in similar fashion to endogenous EF1A in human B lymphocytes. Mutation of two nucleotides in the TOP motif abolished the translational regulation. Gel mobility shift assays showed that both La protein from human B lymphocyte cytoplasmic extracts as well as purified recombinant La protein specifically bind to an in vitro-synthesized RNA containing the 5' UTR of EF1A mRNA. Moreover, extracts prepared from rapamycin-treated cells showed increased binding activity to the EF1A 5' UTR RNA, which correlates with TOP mRNA translational repression. In an in vitro translation system, recombinant La dramatically decreased the expression of EF1A/hGH construct mRNA, but not mRNAs lacking an intact TOP element. These results indicate that TOP mRNA translation may be modulated through La binding to the TOP element.