Figure 1. Accumulation of mRNA to steady-state concentrations: impact of alteration in mRNA half-life at a fixed rate of transcription. The concentration of mRNA is shown on the y-axis and the time in hours is shown on the x-axis. A long-lived mRNA, a short-lived mRNA and an mRNA with ‘average’ stability are shown. The kinetics of mRNA accumulation and the plateau at steady state are direct reflections of the mRNA half-life.

Figure 2. Tetracycline system for determining mRNA decay rates. This system uses the control elements of the TN10 prokaryotic tet repressor (tetR). The tetR is fused to the transcription activation domain of the adenoviral VP16 protein (tet transactivator, tTA). A tissue-culture cell line is established that expresses tTA. The target gene, which encodes the mRNA of interest, is placed under the transcriptional control of a minimal TATA element linked to seven repeats of the tetracycline-binding sequence. This gene is introduced into the tTA-expressing cell line. When tetracycline is present in the culture media, the tTA cannot bind to the promoter elements. When cells are transferred to media lacking tetracycline, tTA binds and transcription is initiated. After a suitable time period (4 h in this figure), tetracycline is added back to the media and transcription is again silenced. The decay rate of the mRNA synthesized during the 4-h pulse can then be followed over time to determine its physical half-life.

Figure 3. Phenotypes of mRNA stability vary markedly and can be grouped according to cell function. The spectrum of half-lives is represented by the double-headed arrow at the top, followed by examples of mRNAs in each category, the ‘role’ of the mRNAs as a group, and examples of the structural determinants in the 3¢-UTR that contribute to the stability profile.

Figure 4. General pathways of mRNA decay. (a) A typical eukaryotic mRNA. The 5¢ cap (small circle) and associated cap-binding proteins, and the 3¢ poly(A) tail and associated PABP are shown. The interaction between the 5¢ and 3¢ terminal complexes is indicated by the double-headed arrow; this interaction is best documented in yeast systems but may also occur in mammalian cells. The rate-limiting reaction in mRNA decay, poly(A) shortening, by PAN is shown below. (b, c) Two alternative decay pathways that are triggered by poly(A) shortening. The enzymes that mediate decapping (DCP1, DCP2), the 5¢3¢ exonuclease (XRN1) and the 3¢5¢ exonuclease (exosome), are shown.

Figure 5. Specific determinants of mRNA decay. (a) Intrinsically unstable and intrinsically stable mRNAs contain conserved 3¢-UTR determinants that either accelerate (ARE) or retard (PRE) the rate of poly(A) shortening by PAN. (b) Decay of a subset of mRNAs is controlled by rate-limiting endonuclease cleavage. Several such cases have been described. An example is the mRNA that encodes the transferrin receptor (TfR). The TfR is responsible for cellular uptake of serum iron. The TfR mRNA has a series of stem–loop segments in its 3¢-UTR. When the cell is iron-deficient, an endonuclease-sensitive cleavage site in this region is protected by binding of cytosolic aconitase (also known as IRP). In a high-iron environment, this protein binds iron and undergoes an allosteric shift with loss of RNA-binding activity and exposure of the endonuclease site to cleavage. Thus, the iron status in the cell directly controls TfR mRNA stability and the consequent rates of iron uptake. (c) Nonsense-mediated mRNA decay is triggered by direct decapping of the nonsense mRNA followed by 5¢3¢ exonuclease digestion (XRN1). This pathway is best established in yeast systems. (d) No-go decay pathway. This pathway is triggered by the stalling of the ribosome by a stem–loop structure or rare codon in the open reading frame of the mRNA. This ribosome stalling leads to cleavage of the mRNA at the site of stalling. The resultant RNA fragments are cleared by regular mRNA exonuclease enzymes, Xrn1 degrading the mRNA from the 5¢ end and exosome complex degrading from the 3¢ end. (e) Nonstop mRNA decay. This pathway is triggered by the stalling of an 80S ribosome at the 3¢ end of the mRNA lacking a termination codon. The stalled ribosome is recognized by Ski7p protein which recruits the exosome to the mRNA, and mRNA is degraded from the 3¢ end by exosome. (f) siRNA-mediated mRNA decay. siRNAs are small noncoding RNAs derived from long double-stranded RNA (dsRNA). The siRNAs assemble into an RNA-induced silencing complex (RISC) and form a perfectly complementary duplex with their target RNAs. The Argonaute 2 protein in RISC makes an endonucleolytic cleavage in the middle of the siRNA:mRNA complementarity region; the mRNA fragments are degraded by xrn1 and exosome. (g) 21-nt miRNAs assemble into an RNA–protein complex and the miRNA in this miRNP pair, usually imperfectly, to a 3¢-UTR of target (miRNA recognition element; MRE). This results in either translational inhibition or mRNA degradation.