Visualization of primordial germ cells in vivo using GFP-nos1 3'UTR mRNA

In some teleost fish, primordial germ cells (PGCs) inherit specific maternal cytoplasmic factors such as vasa and nanos 1 (nos1) mRNA. It has been shown that the 3'untranslated regions (UTRs) of vasa and nos1 have critical roles for stabilization of these RNAs in zebrafish PGCs. In this study, to determine whether this role of the nos 1 3'UTR is conserved between teleost species, we injected artificially synthesized mRNA, combining green fluorescent protein (GFP) and the zebrafish nos 1 3'UTR (GFP-nos 1 3'UTR mRNA), into the fertilized eggs of various fish species. The 3'UTR of the Oryzias latipes vasa homologue (olvas ) mRNA was assayed in the same manner. We demonstrate that the PGCs of seven teleost species could be visualized using GFP-nos 1 3'UTR mRNA. GFP-olvas 3'UTR mRNA did not identify PGCs in herring or loach embryos, but did enable visualization of the PGCs in medaka embryos. Our results indicate that the 3'UTR of the zebrafish nos1 mRNA can promote maintenance of RNAs in the PGCs of different fish species. Finally, we describe and compare the migration routes of PGCs in seven teleost species.     

RNA-protein interactions within the 3 ' untranslated region of vimentin mRNA.

Several functions have been attributed to protein binding within the 3'untranslated region (3'UTR) of mRNA, including mRNA localization, stability, and translational repression. Vimentin is an intermediate filament protein whose 3'untranslated sequence is highly conserved between species. In order to identify sequences that might play a role in vimentin mRNA function, we synthesized32P-labeled RNA from different regions of vimentin's 3'UTR and assayed for protein binding with HeLa extracts using band shift assays. Sequences required for binding are contained within a region 61-114 nucleotides downstream of the stop codon, a region which is highly conserved from Xenopus to man. As judged by competition assays, binding is specific. Solution probing studies of 32P-labeled RNA with various nucleases and lead support a complex stem and loop structure for this region. Finally, UV cross-linking of the RNA-protein complex identifies an RNA binding protein of 46 kDa. Fractionation of a HeLa extract on a sizing column suggests that in addition to the 46 kDa protein, larger complexes containing additional protein(s) can be identified. Vimentin mRNA has been shown to be localized to the perinuclear region of the cytoplasm, possibly at sites of intermediate filament assembly. To date, all sequences required for localization of various mRNAs have been confined to the 3'UTR. Therefore, we hypothesize that this region and associated protein(s) might be important for vimentin mRNA function such as in localization.     

The AU-rich 3' untranslated region of human MDR1 mRNA is an inefficient mRNA destabilizer.

The human multidrug resistance gene MDR1 encodes a membrane-bound protein, referred to as P-glycoprotein, that acts as a pump to extrude toxins from cells. The 3' untranslated region (3'UTR) of the human MDR1 mRNA is very AU-rich (70%) and contains AU-rich sequences similar to those shown to confer rapid decay on c-myc, c-fos, and lymphokine mRNAs. We tested the ability of the MDR1 3'UTR to act as an mRNA destabilizing element in the human hepatoma cell line HepG2. The MDR1 mRNA has an intermediate half-life of 8 h in HepG2 cells compared to a half-life of 30 min for c-myc mRNA. The MDR1 mRNA half-life was prolonged to >20 h upon treatment with the protein synthesis inhibitor cycloheximide. We constructed expression vectors containing the human beta-globin coding region with the 3'UTR from either MDR1 or c-myc. The c-myc 3'UTR increased the decay of the chimeric mRNA, but the MDR1 3'UTR had no effect. We tested the ability of MDR1 3'UTR sequences to compete for interaction with AU-binding proteins in cell extracts; MDR1 RNA probes had a fivefold lower affinity for AU-binding proteins that interact with the c-myc AU-rich 3'UTR. Overall, our data suggest that the MDR1 3'UTR does not behave as an active destabilizing element in HepG2 cells.     

The mammalian exosome mediates the efficient degradation of mRNAs that contain AU-rich elements.

HeLa cytoplasmic extracts contain both 3'-5' and 5'-3' exonuclease activities that may play important roles in mRNA decay. Using an in vitro RNA deadenylation/decay assay, mRNA decay intermediates were trapped using phosphothioate-modified RNAs. These data indicate that 3'-5' exonucleolytic decay is the major pathway of RNA degradation following deadenylation in HeLa cytoplasmic extracts. Immunodepletion using antibodies specific for the exosomal protein PM-Scl75 demonstrated that the human exosome complex is required for efficient 3'-5' exonucleolytic decay. Furthermore, 3'-5' exonucleolytic decay was stimulated dramatically by AU-rich instability elements (AREs), implicating a role for the exosome in the regulation of mRNA turnover. Finally, PM-Scl75 protein was found to interact specifically with AREs. These data suggest that the interaction between the exosome and AREs plays a key role in regulating the efficiency of ARE-containing mRNA turnover.     

Regulated expression of nuclear protein(s) in myogenic cells that binds to a conserved 3'' untranslated region in pro alpha 1 (I) collagen cDNA.

We describe the identification and DNA-binding properties of nuclear proteins from rat L6 myoblasts which recognize an interspecies conserved 3' untranslated segment of pro alpha 1 (I) collagen cDNA. Levels of the two pro alpha 1 (I) collagen RNAs, present in L6 myoblasts, decreased drastically between 54 and 75 h after induction of myotube formation in serum-free medium. Both mRNAs contained a conserved sequence segment of 135 nucleotides (termed tame sequence) in the 3' untranslated region that had 96% homology to the human and murine pro alpha 1 (I) collagen genes. The cDNA of this tame sequence was specifically recognized by nuclear protein(s) from L6 myoblasts, as judged by gel retardation assays and DNase I footprints. The tame-binding protein(s) was able to recognize its target sequence on double-stranded DNA but bound also to the appropriate single-stranded oligonucleotide. Protein that bound to the tame sequence was undetectable in nuclear extracts of L6 myotubes that did not accumulate the two collagen mRNAs. Therefore, the activity of this nuclear protein seems to be linked to accumulation of the sequences that it recognizes in vitro. The collagen RNAs and the nuclear tame-binding proteins reappeared after a change of medium, which further suggests that the RNAs and the protein(s) are coordinately regulated.     

miR-148 targets human DNMT3b protein coding region

MicroRNAs (miRNAs) are small noncoding RNA molecules of 20-24 nucleotides that regulate gene expression. In animals, miRNAs form imperfect interactions with sequences in the 3' Untranslated region (3'UTR) of mRNAs, causing translational inhibition and mRNA decay. In contrast, plant miRNAs mostly associate with protein coding regions. Here we show that human miR-148 represses DNA methyltransferase 3b (Dnmt3b) gene expression through a region in its coding sequence. This region is evolutionary conserved and present in the Dnmt3b splice variants Dnmt3b1, Dnmt3b2, and Dnmt3b4, but not in the abundantly expressed Dnmt3b3. Whereas overexpression of miR-148 results in decreased DNMT3b1 expression, short-hairpin RNA-mediated miR-148 repression leads to an increase in DNMT3b1 expression. Interestingly, mutating the putative miR-148 target site in Dnmt3b1 abolishes regulation by miR-148. Moreover, endogenous Dnmt3b3 mRNA, which lacks the putative miR-148 target site, is resistant to miR-148-mediated regulation. Thus, our results demonstrate that the coding sequence of Dnmt3b mediates regulation by the miR-148 family. More generally, we provide evidence that coding regions of human genes can be targeted by miRNAs, and that such a mechanism might play a role in determining the relative abundance of different splice variants.     

Conservation and divergence of Bmp2a, Bmp2b, and Bmp4 expression patterns within and between dentitions of teleost fishes

The diversity of tooth location in teleost fishes provides an excellent system for comparing genetic divergence between teeth in different species (phylogenetic homologs) with divergence between teeth within one species (iterative homologs). We have chosen to examine the expression of three members of the bone morphogenetic protein (Bmp) family because they are known to play multiple roles in tooth development and evolution in tetrapod vertebrates. We characterized expression of Bmp2a, Bmp2b, and Bmp4 during the development of oral and pharyngeal dentitions in three species of teleost fishes, the zebrafish (Danio rerio), Mexican tetra (Astyanax mexicanus), and Japanese medaka (Oryzias latipes). We found that expression in teleosts is generally highly conserved, with minor differences found among both iteratively homologous and phylogenetically homologous teeth. Expression of orthologous genes differs in several ways between the teeth of teleost fishes and those of the mouse, but between these vertebrate groups the summed expression pattern of Bmp genes is highly conserved. Significantly, the toothless oral region of the zebrafish lacks Bmp expression domains found in teleosts with oral teeth, implicating these genes in evolutionary tooth loss. We conclude that Bmp expression has been largely conserved in vertebrate tooth development over evolutionary time, and that loss of Bmp expression is correlated with region-specific loss of the dentition in a major group of fishes.