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 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.

Decoupled degradation and translation enables noise-modulation by poly(A)-tails

“…The finding that the levels of mRNAs measured for all tails 3 hours post-transfection is comparable (Figure S2C) indicates that there are no discernible differences in mRNA degradation occurring within the first 3 hours post-transfection. While a slight positive correlation has been observed between tail length and mRNA degradation for endogenous mRNAs 37–40 , our results suggest that the strong influence of poly(A)-tail length on mRNA degradation was previously concealed by the presence of other regulatory elements, that likely dampen the observed effect of tail length on mRNA stability.…”

“…In order to discern the role of poly(A)-tail length and its variability in protein expression regulation, we synthesized identical mRNAs that only differ in tail length. We first studied the effect of single poly(A)-tail lengths on mRNA degradation and translation kinetics (Figure 1), and found that in the cell model used (HEK293T/17) there is a strong negative correlation between poly(A)-tail length and mRNA half-life, as previously reported 13,38–40,90 . Strikingly, we observed a very different relationship between poly(A)-tail length and protein levels, where intermediate tails (i.e., 100 nt) show the highest protein production.…”