Rapid degradation of AU-rich element (ARE) mRNAs is activated by ribosome transit and blocked by secondary structure at any position 5' to the ARE.

The 3' noncoding region (NCR) AU-rich element (ARE) selectively confers rapid degradation on many mRNAs via a process requiring translation of the message. The role of cotranslation in destabilization of ARE mRNAs was examined by insertion of translation-blocking stable secondary structure at different sites in test mRNAs containing either the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE or a control sequence. A strong (-80 kcal/mol [1 kcal = 4.184 kJ]) but not a moderate (-30 kcal/mol) secondary structure prevented destabilization of mRNAs when inserted at any position upstream of the ARE, including in the 3' NCR. Surprisingly, a strong secondary structure did not block rapid mRNA decay when placed immediately downstream of the ARE. Studies are also presented showing that the turnover of mRNAs containing control or ARE sequences is not altered by insertion of long (1,000-nucleotide) intervening segments between the stop codon and the ARE or between the ARE and poly(A) tail. Characterization of ARE-containing mRNAs in polyadenylated and whole cytoplasmic RNA fractions failed to find evidence for decay intermediates degraded to the site of strong secondary structure from either the 5' or 3' end. From these and other data presented, this study demonstrates that complete translation of the coding region is essential for activation of rapid mRNA decay controlled by the GM-CSF ARE and that the structure of the 3' NCR can strongly influence activation. The results are consistent with activation of ARE-mediated decay by possible entry of translation-linked decay factors into the 3' NCR or translation-coupled changes in 3' NCR ribonucleoprotein structure or composition.     

Inferred functions of "novel" genes identified in fibroblasts chondroinduced by demineralized bone

Little is known about the cellular mechanisms that control postnatal chondrocyte differentiation. As a first step towards identifying those mechanisms, gene expression shifts were characterized in an in vitro model of chondroinduction. In previous studies, several functional classes of genes (cytoskeletal and matrix elements, cell adhesion proteins, peptide growth factors, and signal transduction proteins) were found to be altered in human dermal fibroblasts (hDFs) cultured in porous collagen sponges with chondroinductive demineralized bone powder (DBP) for 3 days. In addition, a number of "novel" sequences were identified. In this study, molecular techniques were combined with computational methods to characterize those sequences. Gene expression of all 10 novel sequences tested was found in hDFs by RT-PCR. The sequences were compared to the human genome, and their cellular functions were inferred from genes that mapped to the same chromosomal coordinates. Only one of the novel sequences contained a protein-coding region (kinesin superfamily protein 26B). The others contained 3' untranslated (osteonectin, alpha-V integrin, RAP2B) or other untranslated regions (PTPN21, GAS6) of mRNAs. The cellular functions of the DBP-regulated genes described in this study fall into similar categories as those previously identified. These results provide new details on the cellular response of hDFs exposed to DBP.     

Facilitation of mRNA deadenylation and decay by the exosome-bound, DExH protein RHAU

The AU-rich element (ARE) in the 3' untranslated region of unstable mRNAs mediate their rapid degradation. ARE binding proteins (AUBPs) have been described that either stabilize or otherwise degrade ARE-mRNAs by recruiting the exosome, a complex of 3'-to-5' exoribonucleases. We have identified RHAU, a putative DExH RNA helicase that was isolated in association with the ARE of urokinase plasminogen activator mRNA (ARE(uPA)). RHAU physically interacts with the deadenylase PARN and the human exosome and enhances the deadenylation and decay of ARE(uPA)-mRNAs. An alternatively spliced isoform of RHAU that localized to the cytoplasm had a more pronounced effect on ARE(uPA)-mRNA destabilization than full-length RHAU. Furthermore, the ATPase activity of RHAU is essential for its mRNA-destabilizing function. ARE(uPA)-mRNA recognition by RHAU may be mediated through its RNA-dependent interaction with the AUBPs HuR and NFAR1. A model is presented to describe the action of RHAU in ARE(uPA)-directed mRNA turnover.     

Genome-wide computational identification of bicistronic mRNA in humans

Mammalian bicistronic mRNA is a recently discovered mammalian gene structure. Several reported cases of mammalian bicistronic mRNA indicated that genes of this structure play roles in some important biological processes. However, a genome-wide computational identification of bicistronic mRNA in mammalian genome, such as human genome, is still lacking. Here we used a comparative genomics approach to identify the frequency of human bicistronic mRNA. We then validated the result by using a new support vector machine (SVM) model. We identified 43 human bicistronic mRNAs in 30 distinct genes. Our literature analysis shows that our method recovered 100 % (6/6) of the previously known bicistronic mRNAs which had been experimentally confirmed by other groups. Our graph theory-based analysis and GO analysis indicated that human bicistronic mRNAs are prone to produce different yet closely functionally related proteins. In addition, we also described and analyzed three different mechanisms of ORF fusion. Our method of identifying bicistronic mRNAs in human genome provides a model for the computational identification of characteristic gene structures in mammalian genomes. We anticipate that our data will facilitate further molecular characterization and functional study of human bicistronic mRNA.     

AU binding proteins recruit the exosome to degrade ARE-containing mRNAs.

Inherently unstable mammalian mRNAs contain AU-rich elements (AREs) within their 3' untranslated regions. Although found 15 years ago, the mechanism by which AREs dictate rapid mRNA decay is not clear. In yeast, 3'-to-5' mRNA degradation is mediated by the exosome, a multisubunit particle. We have purified and characterized the human exosome by mass spectrometry and found its composition to be similar to its yeast counterpart. Using a cell-free RNA decay system, we demonstrate that the mammalian exosome is required for rapid degradation of ARE-containing RNAs but not for poly(A) shortening. The mammalian exosome does not recognize ARE-containing RNAs on its own. ARE recognition requires certain ARE binding proteins that can interact with the exosome and recruit it to unstable RNAs, thereby promoting their rapid degradation.     

At least 104 nucleotides are transposed from the 5' terminus of the avian sarcoma virus genome to the 5' termini of smaller viral mRNAs.

Cells producing avian sarcoma virus (ASV) contain at least three virus-specific mRNAs, two of which are encoded within the 3' half of the viral genome. Each of these viral RNAs can hybridize with single-stranded DNA(cDNA5') that is complementary to a sequence of 101 nucleotides found at the 5' terminus of the ASV genome, but not within the 3' half of the genome. We proposed previously (Weiss, Varmus and Bishop, 1977) that this nucleotide sequence may be transposed to the 5' termini of viral mRNAs during the genesis of these RNAs. We now substantiate this proposal by reporting the isolation and chemical characterization of the nucleotide sequences complementary to cDNA5' in the genome and mRNAs of the Prague B strain of ASV. We isolated the three identified classes of ASVmRNA (38, 28 and 21S) by molecular hybridization; each class of RNA contained a "capped" oligonucleotide identical to that found at the 5' terminus of the ASV genome. When hybridized with cDNA5', each class of RNA gave rise to RNAase-resistant duplex hybrids that probably encompassed the full extent of cDNA5'. The molar yields of duplex conformed approximately to the number of virus-specific RNA molecules in the initial samples; hence most if not all of the molecules of virus-specific RNA could give rise to the duplexes. The duplexes prepared from the various RNAs all contained the capped oligonucleotide found at the 5' terminus of the viral genome and had identical "fingerprints" when analyzed by two-dimensional fractionation following hydrolysis with RNAase T1. In contrast, RNA representing the 3' half of the ASV genome did not form hybrids with cDNA5'. We conclude that a sequence of more than 100 nucleotides is transposed from the 5' end of the ASV genome to the 5' termini of smaller viral RNAs during the genesis of these RNAs. Transposition of nucleotide sequences during the production of mRNA has now been described for three families of animal viruses and may be a common feature of mRNA biogenesis in eucaryotic cells. The mechanism of transposition, however, and the function of the transposed sequences are not known.     

The 5' ends of Hantaan virus (Bunyaviridae) RNAs suggest a prime-and-realign mechanism for the initiation of RNA synthesis.

We examined the 5' ends of Hantaan virus (HTN) genomes and mRNAs to gain insight into the manner in which these chains were initiated. Like those of all members of the family Bunyaviridae described so far, the HTN mRNAs contained 5' terminal extensions that were heterogeneous in both length and sequence, presumably because HTN also "cap snatches" host mRNAs to initiate the viral mRNAs. Unexpectedly, however, almost all of the mRNAs contained a G residue at position -1, and a large fraction also lacked precisely one of the three UAG repeats at the termini. The genomes, on the other hand, commenced with a U residue at position +1, but only 5' monophosphates were found here, indicating that these chains may not have initiated with UTP at this position. Taken together, these unusual findings suggest a prime-and-realign mechanism of chain initiation in which mRNAs are initiated with a G-terminated host cell primer and genomes with GTP, not at the 3' end of the genome template but internally (opposite the template C at position +3), and after extension by one or a few nucleotides, the nascent chain realigns backwards by virtue of the terminal sequence repeats, before processive elongation takes place. For genome initiation, an endonuclease, perhaps that involved in cap snatching, is postulated to remove the 5' terminal extension of the genome, leaving the 5' pU at position +1.     

Inhibition of translation by UAUUUAU and UAUUUUUAU motifs of the AU-rich RNA instability element in the HPV-1 late 3' untranslated region

The human papillomavirus type 1 (HPV-1) late mRNAs contain a 57-nucleotide adenosine- and uridine-rich RNA instability element termed h1ARE in their late 3' untranslated regions. Here we show that five sequence motifs in the h1ARE (named I-V) affect the mRNA half-life in an additive manner. The minimal inhibitory sequence in motifs I and II was mapped to UAUUUAU, and the minimal inhibitory sequence in motifs III-V was mapped to UAUUUUUAU. We also provide evidence that the same motifs in the AU-RNA instability element inhibit mRNA translation, an effect that was entirely dependent on the presence of a poly(A) tail on the mRNA. Additional experiments demonstrated that the h1ARE interacted directly with the poly(A)-binding protein, suggesting that the h1ARE inhibits translation by interfering with the function of the poly(A)-binding protein.     

The association of Alu repeats with the generation of potential AU-rich elements (ARE) at 3' untranslated regions.

Background  

A significant portion (about 8% in the human genome) of mammalian mRNA sequences contains AU (Adenine and Uracil) rich elements or AREs at their 3' untranslated regions (UTR). These mRNA sequences are usually stable. However, an increasing number of observations have been made of unstable species, possibly depending on certain elements such as Alu repeats. ARE motifs are repeats of the tetramer AUUU and a monomer A at the end of the repeats ((AUUU)_nA). The importance of AREs in biology is that they make certain mRNA unstable. Proto-oncogene, such as c-fos, c-myc, and c-jun in humans, are associated with AREs. Although it has been known that the increased number of ARE motifs caused the decrease of the half-life of mRNA containing ARE repeats, the exact mechanism is as of yet unknown. We analyzed the occurrences of AREs and Alu and propose a possible mechanism for how human mRNA could acquire and keep AREs at its 3' UTR originating from Alu repeats.