Computational modeling of eukaryotic mRNA turnover

Abstract

The process of eukaryotic gene expression involves a diverse number of steps including transcription, RNA processing, transport, translation, and mRNA turnover. A critical step in understanding this process will be the development of mathematical models that quantitatively describe and predict the behavior of this complex system. We have simulated eukaryotic mRNA turnover in a linear multicomponent model based on the known mRNA decay pathways in yeast. Using rate constants based on experimental data for the yeast unstable MFA2 and stable PGK1 transcripts, the computational modeling reproduces experimental observations after minor adjustments. Subsequent analysis and a series of in silico experiments led to several conclusions. First, we demonstrate that mRNA half-life as commonly measured underestimates the average life span of an mRNA. Second, due to the properties of the pathways, the measurement of a half-life can predominantly measure different steps in the decay network. A corollary of this fact is that different mRNAs will be affected differentially by changes in specific rate constants. Third, the way to obtain the largest change of levels of mRNA for the smallest changes in rate is by changing the rate of deadenylation, where a large amount of regulation of mRNA decay occurs. Fourth, the 3'-to-5' degradation of mRNA shows mRNA-specific rates of degradation that are dependent on the 5' structure of the mRNA. These programs can be run over the Web, are adaptable to other eukaryotes, and provide outputs as graphs and virtual northern gels, which can be directly compared to experimental data. Therefore, this model constitutes a useful tool for the quantitative analysis of the process and control of mRNA degradation in eukaryotic cells.

Different nuclease requirements for exosome-mediated degradation of normal and nonstop mRNAs

“…The initiating step for decay of normal mRNAs is shortening of the poly(A) tail (14,15). Removal of the poly(A) tail predominantly triggers cytoplasmic 5′-to-3′ decay in yeast (16)(17)(18)(19). Deadenylation can also trigger the degradation of an mRNA from its 3′ end, in a process catalyzed by the cytoplasmic exosome (20).…”

The DEAD box helicase, Dhh1p, functions in mRNA decapping and interacts with both the decapping and deadenylase complexes

“…To determine if Dhh1p is involved in mRNA turnover, the half-life of both reporters and endogenous mRNAs were assayed in dhh1⌬ mutant strains+ The reporters used were the MFA2pG and PGK1pG (Decker & Parker, 1993)+ These two constructs are derivatives of the endogenous genes for MFA2 and PGK1 that contain a poly-guanosine tract (poly(G)) in their 39 UTR and are under the transcriptional control of the GAL1 UAS+ These modifications allow mRNA turnover to be assessed by two means+ First, the decay rate of full-length mRNA can be quantitated following repression of transcription by the addition of glucose to the growth medium+ Second, the poly(G) tract inhibits exonucleolytic digestion thereby trapping decay intermediates (Vreken & Raue, 1992;Decker & Parker, 1993)+ The ratio of full-length mRNA to the mRNA decay intermediate is indicative of the efficiency of turnover Cao & Parker, 2001)+ Analysis of the MFA2pG and PGK1pG transcripts in dhh1⌬ strains provided two observations that mRNA decay was deficient in the dhh1⌬ cells+ First, both reporters were stabilized in dhh1⌬ mutants as compared to wild-type cells+ The half-life of the unstable MFA2pG reporter was 8 min in the dhh1⌬ mutant background compared to 3 min in wild-type (Fig+ 1A)+ For the stable PGK1pG transcript the half-life was 33 min in the mutant as compared to 25 min in wild type, which is consistent with the stable PGK1 mRNA being less responsive to subtle changes in decapping rate (see Cao & Parker, 2001)+ A change in mRNA decay rate in dhh1⌬ cells was also supported by the observation that the level of mRNA decay intermediate was approximately fourfold less in dhh1⌬ cells versus+ wild type (Fig+ 1A,B)+ We also observed that the endogenous GAL1 mRNA (7 min wild type vs+ 18 min dhh1⌬ ), GAL7 mRNA (12 min wild type vs+ 26 min dhh1⌬ ), and GAL10 mRNA (7 min wild type vs+ 18 min dhh1⌬ ) were stabilized in dhh1⌬ cells+ These results suggest that Dhh1p is required for efficient mRNA turnover in yeast+…”

Poly(A) Tail Length-dependent Stabilization of GAP-43 mRNA by the RNA-binding Protein HuD

“…In fact, in all cases examined, there is a strict correlation between GAP-43 mRNA and protein levels in the cell (1, 2, 8, 18, 19). It is becoming apparent that deadenylation is the first and rate-limiting step in mRNA degradation (49). Deadenylation rates depend on sequences at the 5Ј and 3Ј ends of the mRNAs and on the interactions of many RNA-binding proteins, including the poly(A)-binding protein, the cap-binding protein (eIF4E), the initiation factor eIF4G, and the poly(A)-specific RNA nuclease PARN (43,50,51).…”