Post-transcriptional regulation of RNase-L expression is mediated by the 3'-untranslated region of its mRNA

RNase-L mediates critical cellular functions including antiviral, pro-apoptotic, and tumor suppressive activities; accordingly, its expression must be tightly regulated. Little is known about the control of RNASEL expression; therefore, we examined the potential regulatory role of a conserved 3'-untranslated region (3'-UTR) in its mRNA. The 3'-UTR mediated a potent decrease in the stability of RNase-L mRNA, and of a chimeric beta-globin-3'-UTR reporter mRNA. AU-rich elements (AREs) are cis-acting regulatory regions that modulate mRNA stability. Eight AREs were identified in the RNase-L 3'-UTR, and deletion analysis identified positive and negative regulatory regions associated with distinct AREs. In particular, AREs 7 and 8 served a strong positive regulatory function. HuR is an ARE-binding protein that stabilizes ARE-containing mRNAs, and a predicted HuR binding site was identified in the region comprising AREs 7 and 8. Co-transfection of HuR and RNase-L enhanced RNase-L expression and mRNA stability in a manner that was dependent on this 3'-UTR region. Immunoprecipitation demonstrated that RNase-L mRNA associates with a HuR containing complex in intact cells. Activation of endogenous HuR by cell stress, or during myoblast differentiation, increased RNase-L expression, suggesting that RNase-L mRNA is a physiologic target for HuR. HuR-dependent regulation of RNase-L enhanced its antiviral activity demonstrating the functional significance of this regulation. These findings identify a novel mechanism of RNase-L regulation mediated by its 3'-UTR.     

Sequences of a hairpin structure in the 3'-untranslated region mediate regulation of human pulmonary surfactant protein B mRNA stability

The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate expression of surfactant protein B (SP-B). Dexamethasone (DEX, 10(-7) M) increases human SP-B mRNA stability by a mechanism that requires a 126-nt-long segment (the 7.6S region) of the 3'-untranslated region (3'-UTR). The objective of this study was to identify sequences in the 7.6S region that mediate regulation of SP-B mRNA stability. The 7.6S region was found to be sufficient for DEX-mediated stabilization of mRNA. Sequential substitution mutagenesis of the 7.6S region indicates that a 90-nt region is required for DEX-mediated stabilization and maintenance of intrinsic stability. In this region, one 30-nt-long element (002), predicted to form a stem-loop structure, is sufficient for DEX-mediated stabilization of mRNA and intrinsic mRNA stability. Cytosolic proteins specifically bind element 002, and binding activity is unaffected whether proteins are isolated from cells incubated in the absence or presence of DEX. While loop sequences of element 002 have no role in regulation of SP-B mRNA stability, the proximal stem sequences are required for DEX-mediated stabilization and specific binding of proteins. Mutation of the sequences that comprise the proximal or distal arm of the stem negates the destabilizing activity of element 002 on intrinsic SP-B mRNA stability. These results indicate that cytosolic proteins bind a single hairpin structure that mediates intrinsic and hormonal regulation of SP-B mRNA stability via mechanisms that involve sequences of the stems of the hairpin structure.     

A human genomic sequence highly homologous to the 3'-untranslated region of rat uricase mRNA

A human genomic sequence was isolated from a library using a rat uricase cDNA probe. Sequence analysis has shown that it is highly homologous to the 3'-untranslated region of rat uricase mRNA. Total loss of uricase activity in human is, therefore, not due to total loss of the gene. Discovery of high degree of conservation of the non-coding region of the gene would be of great interest as we attempt to learn the process of gene evolution.     

Structure and function of the selenium translation element in the 3'-untranslated region of human cellular glutathione peroxidase mRNA.

In eukaryotes, incorporation of selenocysteine into the polypeptide chain at a UGA codon requires a unique sequence motif, or "selenium translation element" (STE), located in the 3'-untranslated region of the mRNA. The present study examines structure-function relationships of conserved sequence elements and of the putative stem-loop secondary structure in the STE of human GPX1 mRNA, which encodes the important antioxidant enzyme cellular glutathione peroxidase (EC 1.11.1.9). Deletion of the basal stem, upper stem, or apical loop of the stem-loop structure eliminated the ability of the STE to direct selenocysteine incorporation at the UGA codon of an epitope-tagged GPX1 reporter construct transfected into COS1 cells. However, mutations that change the primary nucleotide sequence of nonconserved portions of the stem-loop, but preserve its overall secondary structure, by inversion of apical loop sequences or exchange of 5' and 3' sides of stem segments, had little or no effect on selenocysteine incorporation. Effects of single- and double-nucleotide substitutions in three short, highly conserved elements in the GPX1 STE depended in large part on their computer-predicted perturbation of the stem-loop and its midstem bulge. Only in the conserved "AAA" apical loop sequence did mutations show major effects on function without predicted changes in secondary structure. Our results demonstrate the critical role of the three short, highly conserved sequences. However, outside of these elements, the function of the human GPX1 STE appears to depend strongly on the stem-loop secondary structure.     

Multiple,conserved iron-responsive elements in the 3'-untranslated region of transferrin receptor mRNA enhance binding of iron regulatory protein 2

Synthesis of proteins for iron homeostasis is regulated by specific, combinatorial mRNA/protein interactions between RNA stem-loop structures (iron-responsive elements, IREs) and iron-regulatory proteins (IRP1 and IRP2), controlling either mRNA translation or stability. The transferrin receptor 3'-untranslated region (TfR-3'-UTR) mRNA is unique in having five IREs, linked by AU-rich elements. A C-bulge in the stem of each TfR-IRE folds into an IRE that has low IRP2 binding, whereas a loop/bulge in the stem of the ferritin-IRE allows equivalent IRP1 and IRP2 binding. Effects of multiple IRE interactions with IRP1 and IRP2 were compared between the native TfR-3'-UTR sequence (5xIRE) and RNA with only 3 or 2 IREs. We show 1) equivalent IRP1 and IRP2 binding to multiple TfR-IRE RNAs; 2) increased IRP-dependent nuclease resistance of 5xIRE compared with lower IRE copy-number RNAs; 3) distorted TfR-IRE helix structure within the context of 5xIRE, detected by Cu-(phen)(2) binding/cleavage, that coincides with ferritin-IRE conformation and enhanced IRP2 binding; and 4) variable IRP1 and IRP2 expression in human cells and during development (IRP2-mRNA predominated). Changes in TfR-IRE structure conferred by the full length TfR-3'-UTR mRNA explain in part evolutionary conservation of multiple IRE-RNA, which allows TfR mRNA stabilization and receptor synthesis when IRP activity varies, and ensures iron uptake for cell growth.     

Three-hybrid strategy reveals a peptide segment that specifically binds to the 3'-untranslated region of NF-IL6 mRNA

The 3'UTR of eukaryotic mRNA is an important regulation region, on which many trans factors act. In recent years, a series of 3'UTRs were shown to have tumor suppressor function, including the 3'UTR of the human nuclear factor for interleukin-6 (NF-IL6 3'UTR). To understand molecular basis for this function, we have tried to isolate genes encoding protein factors acting on the RNA of NF-IL6 3'UTR. Here we show that, by using a yeast three-hybrid system, a cDNA fragment was successfully isolated. This cDNA was allowed to express in E. coli, and its expression product, a polypeptide of ca. 70 amino acids long, was shown to specifically bind to the NF-IL6 3'UTR RNA. A search in GenBank did not reveal homologous sequences. Therefore, this cDNA fragment may be a part of the gene of a novel NF-IL6 3'UTR specific binding protein.     

A repressor element in the 5'-untranslated region of human Pax5 exon 1A

Five members of the RecQ helicase family, RECQL, WRN, BLM, RTS and RECQL5, have been found in human and three of them (WRN, BLM and RTS) were disclosed to be the genes responsible for Werner, Bloom and Rothmund–Thomson syndromes, respectively. RECQL5 (RecQ helicase protein-like 5) was isolated as the fifth member of the family in humans through a search of homologous expressed sequence tags. The gene is expressed with at least three alternative splicing products, , β and γ. Here, we isolated mouse RECQL5β and determined the DNA sequence of full-length cDNA as well as the genome organization and chromosome locus. The mouse RECQL5β gene consists of 2949 bp coding 982 amino acid residues. Comparison of amino acid sequence among human (Homo sapiens), mouse (Mus musculus), Drosophila melanogaster and Caenorhabditis elegans RECQL5β homologs revealed three portions of highly conserved regions in addition to the helicase domain. Nineteen exons are dispersed over 40 kbp in the genome and all of the acceptor and donor sites for the splicing of each exon conform to the GT/AG rule. The gene is localized to the mouse chromosome 11E2, which has a syntenic relation to human 17q25.2-q25.3 where human RECQL5β exists. Our genetic characterizations of the mouse RECQL5β gene will contribute to functional studies on the RECQL5β products.