Binding of U1A protein to the 3' untranslated region of its pre-mRNA.

We have studied the global structure of the U1A 3' untranslated region (UTR) element using fluorescence resonance energy transfer. Comparison of a single UTR-box with a series of oligoadenine bulges indicates that the UTR-box introduces a significant kink into the axis of the RNA, and quantification of the results suggests an included bend angle of approximately 100 degrees (i.e. 80 degrees from linear). The complete 3'-UTR element is also severely kinked by the two UTR-boxes. We can observe binding of U1A protein to the 3'-UTR element by a change in the fluorescence anisotropy of Cy3 attached to one of the helical ends. In parallel with the binding, we observe a marked increase in fluoresence resonance energy transfer efficiency between fluorophores attached at the two 5' termini, indicating a significant change in global conformation induced by the binding of the protein.     

Polymorphisms in the transcribed 3' untranslated region of eukaryotic genes.

In this review we present preliminary evidence for a new class of polymorphism that may be used in a systematic way to map cDNAs efficiently and to expedite the construction of a high-resolution genetic map of the human genome. Ultimately, transcribed 3' untranslated polymorphisms will warrant further study because they should be widely distributed throughout the genome within transcribed sequences, and they can be readily identified as a result of cDNA cloning and sequencing. Furthermore, these markers should be universally available on the basis of the sequence data and highly useful in linkage analyses.     

Skeletal muscle actin mRNA. Characterization of the 3' untranslated region.

Plasmids p749, p106, and p150 contain cDNA inserts complementary to rat skeletal muscle actin mRNA. Nucleotide sequence analysis indicates the following sequence relationships: p749 specifies codons 171 to 360; p150 specifies codons 357 to 374 together with 120 nucleotides of the 3'-non-translated region; p106 specifies the last actin amino acid codon, the termination codon and the entire 3' non-translated region. Plasmid p749 hybridized with RNA extracted from rat skeletal muscle, cardiac muscle, smooth (stomach) muscle, and from brain. It also hybridizes well with RNA extracted from skeletal muscle and brain of dog and chick. Plasmid p106 hybridized specifically with rat striated muscles (skeletal and cardiac muscle) mRNA but not with mRNA from rat stomach and from rat brain. It also hybridized to RNA extracted from skeletal muscle of rabbit and dog but not from chick. Thermal stability of the hybrids and sensitivity to S1 digestion also indicated substantial divergence between the 3' untranslated end of rat and dog skeletal muscle actins. The investigation shows that the coding regions of actin genes are highly conserved, whereas the 3' non-coding regions diverged considerably during evolution. Probes constructed from the 3' non-coding regions of actin mRNAs can be used to identify the various actin mRNA and actin genes.     

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.     

Prosaposin: a new player in cell death prevention of U937 monocytic cells

We report that prosaposin binds to U937 and is active as a protective factor on tumor necrosis factor alpha (TNFalpha)-induced cell death. The prosaposin-derived saposin C binds to U937 cells in a concentration-dependent manner, suggesting that prosaposin behaves similarly. Prosaposin binding induces U937 cell death prevention, reducing both necrosis and apoptosis. This effect was inhibited by mitogen-activated protein ERK kinase (MEK) and sphingosine kinase (SK) inhibitors, indicating that prosaposin prevents cell apoptosis by activation of extracellular signal-regulated kinases (ERKs) and sphingosine kinase. Prosaposin led to rapid ERK phosphorylation in U937 cells as detected by anti-phospho-p44/42 mitogen-activated protein (MAP) kinase and anti-phosphotyrosine reactivity on ERK immunoprecipitates. It was partially prevented by apo B-100 and pertussis toxin (PT), suggesting that both lipoprotein receptor-related protein (LRP) receptor and Go-coupled receptor may play a role in the prosaposin-triggered pathway. Moreover, sphingosine kinase activity was increased by prosaposin treatment as demonstrated by the enhanced intracellular formation of sphingosine-1-phosphate (S-1-P). The observation that the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented the prosaposin effect on cell apoptosis suggests that sphingosine kinase exerts its anti-apoptotic activity by the PI3K-Akt pathway. Thus, cell apoptosis prevention by prosaposin occurs through ERK phosphorylation and sphingosine kinase. The biological effect triggered by prosaposin might be extended to primary cells because it triggers Erk phosphorylation in peripheral blood mononuclear cells (PBMCs). This is the first evidence of a biological effect consequent to a signal transduction pathway triggered by prosaposin in cells of non-neurological origin.     

Programmed cell death in 2',3'-dideoxycytidine-resistant human monoblastoid U937 cells

2,3-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O₂ and nitric oxide.Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.     

Ribosome binding to a 5' translational enhancer is altered in the presence of the 3' untranslated region in cap-independent translation of turnip crinkle virus

Plus-strand RNA viruses without 5' caps require noncanonical mechanisms for ribosome recruitment. A translational enhancer in the 3' untranslated region (UTR) of Turnip crinkle virus (TCV) contains an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. We now report that the 63-nucleotide (nt) 5' UTR of TCV contains a 19-nt pyrimidine-rich element near the initiation codon that supports translation of an internal open reading frame (ORF) independent of upstream 5' UTR sequences. Addition of 80S ribosomes to the 5' UTR reduced the flexibility of the polypyrimidine residues and generated a toeprint consistent with binding to this region. Binding of salt-washed 40S ribosomal subunits was reduced 6-fold when the pyrimidine-rich sequence was mutated. 40S subunit binding generated the same toeprint as 80S ribosomes but also additional ones near the 5' end. Generation of out-of-frame AUGs upstream of the polypyrimidine region reduced translation, which suggests that 5'-terminal entry of 40S subunits is followed by scanning and that the polypyrimidine region is needed for an alternative function that requires ribosome binding. No evidence for RNA-RNA interactions between 5' and 3' sequences was found, suggesting that TCV utilizes an alternative means for circularizing its genome. Combining 5' and 3' UTR fragments in vitro had no discernible effect on the structures of the RNAs. In contrast, when 80S ribosomes were added to both fragments, structural changes were found in the 5' UTR polypyrimidine tract that were not evident when ribosomes interacted with the individual fragments. This suggests that ribosomes can promote an interaction between the 5' and 3' UTRs of TCV.