Faculty Bibliography

Labile mRNAs that encode cytokine and immediate-early gene products often contain AU-rich sequences within their 3' untranslated region (UTR). These AU-rich sequences appear to be key determinants of the short half-lives of these mRNAs, although the sequence features of these elements and the mechanism by which they target mRNAs for rapid decay have not been fully defined. We have examined the features of AU-rich elements (AREs) that are crucial for their function as determinants of mRNA instability in mammalian cells by testing the ability of various mutant c-fos AREs and synthetic AREs to direct rapid mRNA deadenylation and decay when inserted within the 3' UTR of the normally stable beta-globin mRNA. Evidence is presented that the pentamer AUUUA, which previously was suggested to be the minimal determinant of instability present in mammalian AREs, cannot direct rapid mRNA deadenylation and decay. Instead, the nonomer UUAUUUAUU is the elemental AU-rich sequence motif that destabilizes mRNA. Removal of one uridine residue from either end of the nonamer (UUAUUUAU or UAUUUAUU) results in a decrease of potency of the element, while removal of a uridine residue from both ends of the nonamer (UAUUUAU) eliminates detectable destabilizing activity. The inclusion of an additional uridine residue at both ends of the nonamer (UUUAUUUAUUU) does not further increase the efficacy of the element. Taken together, these findings suggest that the nonamer UUAUUUAUU is the minimal AU-rich motif that effectively destabilizes mRNA. Additional ARE potency is achieved by combining multiple copies of this nonamer in a single mRNA 3' UTR. Furthermore, analysis of poly(A) shortening rates for ARE-containing mRNAs reveals that the UUAUUUAUU sequence also accelerates mRNA deadenylation and suggests that the UUAUUUAUU motif targets mRNA for rapid deadenylation as an early step in the mRNA decay process

RNase E is a key regulatory enzyme that appears to control the principal pathway for mRNA degradation in Escherichia coli. Here, we show that RNase E represses its own synthesis by reducing the cellular concentration of the rne (RNase E) gene transcript. Autoregulation is achieved by modulating the longevity of this 3.6-kb mRNA, whose half-life ranges from < 40 sec to > 8 min depending on the level of RNase E activity in the cell. Feedback regulation is mediated in cis by the 5'-terminal 0.44-kb segment of rne mRNA, which is sufficient to confer this property onto a heterologous transcript to which it is fused. Like the intact protein, an amino-terminal fragment of RNase E lacking 563 amino acid residues can act in trans to repress rne gene expression. Paradoxically, raising the rne gene copy number 21-fold in E. coli causes an unexpected reduction in the concentration of the full-length rne transcript, yet results in a small increase in RNase E protein production. These surprising phenomena are explained in terms of a model in which the degradation of this long and highly labile mRNA commences before elongation of the nascent transcript has been completed. In such circumstances, gene expression can be unusually sensitive to changes in mRNA stability

RNase E plays a central role in controlling mRNA degradation in E. coli. We have investigated the mechanism of RNase E autoregulation. Our data indicate that RNase E autoregulates its synthesis by controlling the decay rate of its own transcript (rne mRNA), which is unusually sensitive to the level of cellular RNase E activity. Feedback regulation is mediated in cis by the rne 5' untranslated region (5' UTR), which can confer this property onto heterologous mRNAs to which it is fused. The marked sensitivity of rne mRNA to regulation by RNase E is also due in part to the susceptibility of nascent rne transcripts to RNase E-mediated degradation

The unusual longevity of the Escherichia coli ompA transcript is determined by its 5' untranslated region (UTR), which functions in vivo as an mRNA stabilizer. Here we show that this 5' UTR can prolong the lifetime in E. coli of a variety of heterologous mRNAs to which it is joined, either as a gene fusion or as an operon fusion. Statistical extrapolation suggests that it is quite likely that most E. coli mRNAs could be stabilized in this manner. We conclude that the ompA 5' UTR impedes a major pathway for mRNA degradation in E. coli and that stabilization by fusion to this UTR does not require translational readthrough of the heterologous mRNA segment by ribosomes that initiate translation at the ompA ribosome-binding site. Additional experiments indicate that the E. coli ribonuclease whose action is slowed by the ompA 5' UTR is not RNase III

The c-fos proto-oncogene transcript is one of the most labile mammalian mRNAs known. Rapid degradation of c-fos mRNA is mediated by both the c-fos protein-coding region and an AU-rich element in the 3'-untranslated region. Here we present evidence that the c-fos coding region contains multiple destabilizing elements that can function independently to facilitate both deadenylation and decay of mRNA. The ability of these coding region destabilizing elements to direct deadenylation and decay requires the assembly of ribosomes at the 5' end of this domain and, most likely, translation of the message

The protein-coding region of the c-fos proto-oncogene transcript contains elements that direct the rapid deadenylation and decay of this mRNA in mammalian cells. The function of these coding region instability determinants requires movement of ribosomes across mRNAs containing them. Three types of mechanisms could account for this translational requirement. Two of these possibilities, (i) that rapid mRNA decay might be mediated by the nascent polypeptide chain and (ii) that it might result from an unusual codon usage, have experimental precedent. Here, we present evidence that the destabilizing elements in the c-fos coding region are not recognized in either of these two ways. Instead, the ability of the c-fos coding region to function as a potent mRNA destabilizer when translated in the +1 reading frame indicates that the signals for rapid deadenylation and decay reside in the sequence or structure of the RNA comprising this c-fos domain

Despite the variety of messenger RNA half-lives in bacteria (0.5-30 min in Escherichia coli) and their importance in controlling gene expression, their molecular basis remains obscure. The lifetime of an entire mRNA molecule can be determined by features near its 5' end, but no 5' exoribonuclease has been identified in any prokaryotic organism. A mutation that inactivates E. coli RNase E also increases the average lifetime of bulk E. coli mRNA and of many individual messages, suggesting that cleavage by this endonuclease may be the rate-determining step in the degradation of most mRNAs in E. coli. We have investigated the substrate preference of RNase E in E. coli by using variants of RNA I, a small untranslated RNA whose swift degradation in vivo is initiated by RNase E cleavage at an internal site. We report here that RNase E has an unprecedented substrate specificity for an endoribonuclease, as it preferentially cleaves RNAs that have several unpaired nucleotides at the 5' end. The sensitivity of RNase E to 5'-terminal base pairing may explain how determinants near the 5' end can control rates of mRNA decay in bacteria