Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.     

Poly(A)-specific ribonuclease (PARN): An allosterically regulated,processive and mRNA cap-interacting deadenylase

Abstract

Deadenylation of eukaryotic mRNA is a mechanism critical for mRNA function by influencing mRNA turnover and efficiency of protein synthesis. Here, we review poly(A)-specific ribonuclease (PARN), which is one of the biochemically best characterized deadenylases. PARN is unique among the currently known eukaryotic poly(A) degrading nucleases, being the only deadenylase that has the capacity to directly interact during poly(A) hydrolysis with both the m⁷G-cap structure and the poly(A) tail of the mRNA. In short, PARN is a divalent metal-ion dependent poly(A)-specific, processive and cap-interacting 3′–5′ exoribonuclease that efficiently degrades poly(A) tails of eukaryotic mRNAs. We discuss in detail the mechanisms of its substrate recognition, catalysis, allostery and processive mode of action. On the basis of biochemical and structural evidence, we present and discuss a working model for PARN action. Models of regulation of PARN activity by trans-acting factors are discussed as well as the physiological relevance of PARN.     

Interactions of CCCH zinc finger proteins with mRNA: tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur in the absence of a poly(A) tail

The CCCH family of tandem zinc finger proteins has recently been shown to promote the turnover of certain mRNAs containing class II AU-rich elements (AREs). In the case of one member of this family, tristetraprolin (TTP), absence of the protein in knockout mice leads to stabilization of two mRNAs containing AREs of this type, those encoding tumor necrosis factor alpha (TNFalpha) and granulocyte-macrophage colony-stimulating factor. To begin to decipher the mechanism by which these zinc finger proteins stimulate the breakdown of this class of mRNAs, we co-transfected TTP and its related CCCH proteins into 293 cells with vectors encoding full-length TNFalpha, granulocyte-macrophage colony-stimulating factor, and interleukin-3 mRNAs. Co-expression of the CCCH proteins caused the rapid turnover of these ARE-containing mRNAs and also promoted the accumulation of stable breakdown intermediates that were truncated at the 3'-end of the mRNA, even further 5' than the 5'-end of the poly(A) tail. To determine whether an intact poly(A) tail was necessary for TTP to promote this type of mRNA degradation, we inserted the TNFalpha ARE into a nonpolyadenylated histone mRNA and also attached a histone 3'-end-processing sequence to the 3'-end of nonpolyadenylated interleukin-3 and TNFalpha mRNAs. In all three cases, TTP stimulated the turnover of the ARE-containing mRNAs, despite the demonstrated absence of a poly(A) tail. These studies indicate that members of this class of CCCH proteins can promote class II ARE-containing mRNA turnover even in the absence of a poly(A) tail, suggesting that the processive removal of the poly(A) tail may not be required for this type of CCCH protein-stimulated mRNA turnover.     

Identification of a human cytoplasmic poly(A) nuclease complex stimulated by poly(A)-binding protein

The poly(A) tail shortening in mRNA, called deadenylation, is the first rate-limiting step in eukaryotic mRNA turnover, and the polyadenylate-binding protein (PABP) appears to be involved in the regulation of this step. However, the precise role of PABP remains largely unknown in higher eukaryotes. Here we identified and characterized a human PABP-dependent poly(A) nuclease (hPAN) complex consisting of catalytic hPan2 and regulatory hPan3 subunits. hPan2 has intrinsically a 3' to 5' exoribonuclease activity and requires Mg2+ for the enzyme activity. On the other hand, hPan3 interacts with PABP to simulate hPan2 nuclease activity. Interestingly, the hPAN nuclease complex has a higher substrate specificity to poly(A) RNA upon its association with PABP. Consistent with the roles of hPan2 and hPan3 in mRNA decay, the two subunits exhibit cytoplasmic co-localization. Thus, the human PAN complex is a poly(A)-specific exoribonuclease that is stimulated by PABP in the cytoplasm.     

High-resolution analysis of gro alpha mRNA poly(A) shortening: regulation by interleukin-1 beta.

We have previously shown that destabilization of gro alpha mRNA is associated with poly(A) shortening. In this study, we used high-resolution Northern blots to determine the rate and extent of gro alpha mRNA poly(A) shortening. gro alpha mRNA was found to undergo complete deadenylation within 2 h following withdrawal of IL-1. However, the process was not uniform: at 1 h following IL-1 withdrawal, gro alpha mRNA poly(A) lengths ranged from 0 to 180 nucleotides. There was an accumulation of deadenylated gro alpha mRNA which suggested that there may be another step before the mRNA is destroyed. Cycloheximide was found to block gro alpha mRNA degradation at the level of poly(A) shortening. Northern blots revealed a previously unrecognized periodic distribution of poly(A) lengths that was consistent with endonucleolytic cleavage between complexes of poly(A)-binding protein. The findings indicate that the degradation pathway of gro alpha mRNA is a slower version of the c-fos mRNA model, with the important additional feature that deadenylation and degradation are subject to physiologic regulation. This study provides a detailed picture of gro alpha mRNA poly(A) shortening and establishes a basis for further investigation of the mechanism by which IL-1 stabilizes specific mRNAs.     

Opposing polymerase-deadenylase activities regulate cytoplasmic polyadenylation

Cytoplasmic polyadenylation is one mechanism that regulates translation in early animal development. In Xenopus oocytes, polyadenylation of dormant mRNAs, including cyclin B1, is controlled by the cis-acting cytoplasmic polyadenylation element (CPE) and hexanucleotide AAUAAA through associations with CPEB and CPSF, respectively. Previously, we demonstrated that the scaffold protein symplekin contacts CPEB and CPSF and helps them interact with Gld2, a poly(A) polymerase. Here, we report the mechanism by which poly(A) tail length is regulated. Cyclin B1 pre-mRNA acquires a long poly(A) tail in the nucleus that is subsequently shortened in the cytoplasm. The shortening is controlled by CPEB and PARN, a poly(A)-specific ribonuclease. Gld2 and PARN both reside in the CPEB-containing complex. However, because PARN is more active than Gld2, the poly(A) tail is short. When oocytes mature, CPEB phosphorylation causes PARN to be expelled from the ribonucleoprotein complex, which allows Gld2 to elongate poly(A) by default.     

Messenger RNA stability in Saccharomyces cerevisiae: the influence of translation and poly(A) tail length.

A comparison between the half-lives of 10 specific yeast mRNAs and their distribution within polysomes (fractionated on sucrose density gradients) was used to test the relationship between mRNA translation and degradation in the eukaryote Saccharomyces cerevisiae. Although the mRNAs vary in their distribution across the same polysome gradients, there is no obvious correlation between the stability of an mRNA and the number of ribosomes it carries in vivo. This suggests that ribosomal protection against nucleolytic attack is not a major factor in determining the stability of an mRNA in yeast. The relative lengths of the poly(A) tails of 9 yeast mRNAs were analysed using thermal elution from poly(U)-Sepharose. No dramatic differences in poly(A) tail length were observed amongst the mRNAs which could account for their wide ranging half-lives. Minor differences were consistent with shortening of the poly(A) tail as an mRNA ages.     

Size heterogeneity of polyadenylate sequences in mouse globin messenger RNA

Heterogeneity in the length of the poly(A) region has been demonstrated in mouse α and β-globin messenger RNAs. This finding is based on the initial observation that only 30% of the globin mRNA purified by oligo(dT)-cellulose affinity chromatography binds to Millipore filters under conditions where other poly(A)-containing mRNAs have been shown to bind, and the subsequent finding that the bound and non-bound fractions contain different size classes of poly(A). The poly(A) size was determined by polyacrylamide gel electrophoresis of the T₁ and pancreatic RNAase-resistant fragments. The unbound mRNA fraction gives a fragment 35 to 45 adenine nucleotides long, while the bound mRNA contains two fragments with average lengths of 55 to 65 and 75 to 120 nucleotides.The heterogeneity of the poly(A) region is present in both α and β-globin mRNAs as both Millipore-bound and unbound RNA fractions directed the synthesis of comparable amounts of mouse α and β-globin chains.Change in the distribution of the various size classes of poly(A) was analyzed by Millipore binding assays after various times of labeling in vivo. The percentage of labeled mRNA bound to Millipore filters decreased with time, suggesting either a shortening of the poly(A) region or differential synthesis of mRNAs containing shorter poly(A) at earlier stages in erythropoeisis.