Interplay of two functionally and structurally distinct domains of the c-fos AU-rich element specifies its mRNA-destabilizing function

Abstract

AU-rich elements (ARE) in the 3' untranslated region of many highly labile mRNAs for proto-oncogenes, lymphokines, and cytokines can act as an RNA-destabilizing element. The absence of a clear understanding of the key sequence and structural features of the ARE that are required for its destabilizing function has precluded the further elucidation of its mode of action and the basis of its specificity. Combining extensive mutagenesis of the c-fos ARE with in vivo analysis of mRNA stability, we were able to identify mutations that exhibited kinetic phenotypes consistent with the biphasic decay characteristic of a two-step mechanism: accelerated poly(A) shortening and subsequent decay of the transcribed portion of the mRNA. These mutations, which affected either an individual step or both steps, all changed the mRNA stability. Our experiments further revealed the existence of two structurally distinct and functionally interdependent domains that constitute the c-fos ARE. Domain I, which is located within the 5' 49-nucleotide segment of the ARE and contains the three AUUUA motifs, can function as an RNA destabilizer by itself. It forms the essential core unit necessary for the ARE-destabilizing function. Domain II is a 20-nucleotide U-rich sequence which is located within the 3' part of the c-fos ARE. Although it alone can not act as an RNA destabilizer, this domain serves two critical roles: (i) its presence enhances the destabilizing ability of domain I by accelerating the deadenylation step, and (ii) it has a novel capacity of buffering decay-impeding effects exerted by mutations introduced within domain I. A model is proposed to explain how these critical structural features may be involved in the c-fos ARE-directed mRNA decay pathway. These findings have important implications for furthering our understanding of the molecular basis of differential mRNA decay mediated by different AREs.

AUUUA Is Not Sufficient To Promote Poly(A) Shortening and Degradation of an mRNA: the Functional Sequence within AU-Rich Elements May Be UUAUUUA(U/A)(U/A)

“…A further analysis of the c-fos ARE was published while our manuscript was in the final stages of preparation (17) and has shown, consistent with our findings, that the AUUUA-containing region is sufficient to promote deadenylation and degradation of a heterologous mRNA, while the downstream U-tract alone is not. In contrast to our findings, however, this U tract was found to have an enhancing effect on deadenylation when placed either 5' or 3' of the AUUUA motifs.…”

“…1) containing the promoter and 5'UTR from c-fos, a translated region encoding human growth hormone, and a 3'UTR and downstream polyadenylation signals from bovine growth hormone. The fos promoter responds transiently to a shift from low to high serum in the culture medium, resulting in a pulse of transcription lasting less than 1 h, and has been used by others in studies of mRNA degradation (3,17,47,48). This inducible promoter was used in preference to a constitutive promoter to allow mRNA degradation rates to be mea- 2.…”

“…DISCUSSION A model system with which to study the effects of synthetic AREs on mRNA stability. Chimeric genes have been used in a number of studies to assess the destabilizing effect of various elements within the 3'UTRs of natural mRNAs (3,17,21,45,47,48). Such systems have several advantages: by using a coding region from a stable mRNA (which presumably does not contain a destabilizing element such as those identified in the coding regions of the c-fos [30,48] and c-myc [54] mRNAs) and by inserting all sequences at a fixed location in the 3'UTR, other influences on mRNA stability can be minimized, allowing the observed effects to be attributed to the insertion of these sequences.…”

RNA stabilization by the AU-rich element binding protein, HuR, an ELAV protein

“…Previously, we have shown that in the ARE‐mediated mRNA decay deadenylation precedes decay of the body of the mRNA (Chen and Shyu, 1994; Chen et al ., 1994, 1995). To address further which step in the c‐ fos ARE‐mediated decay pathway is affected by HuR, we have performed additional time‐course experiments with time points up to 480 min.…”

Analysis of the AU-Rich Elements in the 3‘-Untranslated Region of β2-Adrenergic Receptor mRNA by Mutagenesis and Identification of the Homologous AU-Rich Region from Different Species

“…β 2 -AR mRNA appears to be similar to other highly regulated mRNAs [e.g., mRNAs of granulocyte/macrophage colonystimulating factor (GM-CSF), tumor necrosis factor-R (TNF-R), and the oncogenes c-myc and c-fos] that are markedly influenced by alterations of their rates of degradation (15)(16)(17)(18)(19). In addition to the ARE of β 2 -AR mRNA, cognate sequences of 3′-untranslated regions (UTR) of mRNA, such as the tandem repeats of AUUUA pentamers (10)(11)(12)(13)(20)(21)(22) and nonamers, such as UUAUUUA(U/A) (U/A) (23) and UUAUUUAUU (24), are important elements in determining RNA stability.…”