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.

Abstract

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 32P04 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.We have previously demonstrated that the mRNA population in vegetatively growing amoebae of the cellular slime mold Dictyostelium discoideum decays with complex kinetics, consisting of at least two major components: a rapidly decaying component with a half-life of approximately 50 min, and a long-lived component with a half-life of approximately 10 h (14, 58). Similar complex kinetics have been observed for the decay of poly(A)+ RNA in a variety of other eucaryotic cells (5,25,39,57,77,78,81). It is likely that these complex decay kinetics reflect the sum of the decay rates of individual mRNAs (e.g., in D. discoideum, the most stable mRNAs have half-lives of approximately 10 h and the least stable mRNAs have half-lives which are approximately 10-fold shorter). It has been s...

Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein.

“…In conclusion, our data with the rpbl-J mutant and the thiolutin-inhibited wild-type strain confirm previous results dissociating mRNA stability and the presence and length of the poly(A) tail (41,49,69,72). Nevertheless, in our mutants these two parameters seem correlated since despite continuous transcription, the mRNA levels, as well as the lengths of the poly(A) tracts, are strongly reduced.…”

“…One of the major roles for the poly(A) tail was suggested by its involvement in protein biosynthesis (47,63), and in yeast cells the poly(A)-binding protein (PABP [1,68]) was shown to be required for translation initiation (69). More recently, in vitro data have supported the idea that the poly(A) tail enhances translation through the interaction of an initiation factor or the ribosomal subunit with the PABP (59).Besides the implication of the poly(A) tail in translation (reviewed in reference 46), its role in mRNA half-life has been evoked as many times (30,44,82,83,86), sometimes in association with the PABP (11, 12, 17), as refuted (41,49,69,72). Nevertheless, it appears that the poly(A) tail is not solely responsible for determination of the stability of mRNA in vivo, but that it acts in association with other factors.…”

“…Besides the implication of the poly(A) tail in translation (reviewed in reference 46), its role in mRNA half-life has been evoked as many times (30,44,82,83,86), sometimes in association with the PABP (11, 12, 17), as refuted (41,49,69,72). Nevertheless, it appears that the poly(A) tail is not solely responsible for determination of the stability of mRNA in vivo, but that it acts in association with other factors.…”

RNA pseudoknot domain of tobacco mosaic virus can functionally substitute for a poly(A) tail in plant and animal cells.

“…A number of examples demonstrate the positive effect that the poly(A) tail has on translation initiation (Jacobson and Favreau 1983;Palatnik et al 1984;Shapiro et al 1988). The rapid degradation of human interferon, granulocyte-macrophage colony-stimulating factor, and c-los mRNAs is mediated by an AUUUA element in the 3'-UTR, and this element can also regulate their translational efficiencies (Kruys et al 1987(Kruys et al , 1989.…”

Genetic and molecular analysis of the temperature-sensitive mutant un-17 carrying a mutation in the gene encoding poly(A)-polymerase in Neurospora crassa

“…The poly(A) tail plays roles in both stability and translocation into the cytoplasm of mRNA. There are several reports about the connection of the length of poly(A) tails with mRNA stability (Shapiro et al, 1988;Herrick et al, 1990;Nie et al, 2004). Also, the poly(A) tail provides a binding site for poly(A)-binding proteins (PABPs: for review, Mangus et al, 2003).…”