How does the cell stop an mRNA?

To prevent the continued expression of its protein product, an mRNA must, at some point, stop being translated and be destroyed.

The key to understanding how mRNA degrade, therefore, is to understand how they are translated.

Work in the Coller lab focuses on the destruction of messenger RNA (mRNA). mRNA must be destroyed. mRNA decay ensures that previously transcribed messages do not translate indefinitely. Importantly, mRNAs degrade at different rates; some fast, some slow. The spectrum of decay rates is achieved via the interplay between three fundamental principles. First mRNA decay is the default state; all messages will succumb. Second, RNA degradation is intimately connected to protein synthesis; a message that translates better is more stable and vice versa. Third, stabilization requires the mRNA be maintained in an ideal ribonucleoprotein context (mRNP); deviants are destroyed. The long term focus of my lab is to understand how these three principles interconnect and are regulated by the cell to forge the cellular mRNA landscape.

A major pathway for mRNA decay initiates by shortening the 3’ polyadenosine tail (deadenylation), followed by 5’ cap cleavage (decapping), and 5’-3’ exonucleolytic digestion. A popular model posits that deadenylation occurs while the mRNA is actively translated, while, decapping and exonucleolytic decay occur only after ribosomes are cleared from the mRNA and in the cytoplasmic structures termed P-bodies (Fig. 1 right panel). Several pieces of data support this hypothesis including the observation that decapping rate is reciprocally proportional to translational initiation rate, and decapping regulators can directly repress mRNA translation. Moreover, several decay factors, their regulators and mRNA decay intermediates co-localize in ribosome-free P-bodies. Finally, P-body size and abundance are inversely proportional with mRNA translation and decay status in the cell. Our current work set out to validate this model by determining if mRNA decapping occurs before or after ribosome dissociation.

In sharp contrast to the prevailing model of mRNA decay, we have shown that mRNA decapping occurs while mRNA is ribosome bound (Fig. 1 left panel & Fig. 2). First, ribosome-free mRNA does not accumulate when decapping is inhibited. Second, decapped mRNAs are associated with polyribosomes, implying decapping has occurred on ribosome-bound mRNAs.. Third, kinetic analysis demonstrates that mRNA decapping is initiated on polyribosomes. Fourth, blocking exoribonucleolytic decay in cis leads to accumulation of polyribosome-bound mRNA decay intermediates that are dependent on decapping. In total, these findings strongly argue that under normal conditions, mRNA decapping occurs while mRNA is associated with polyribosomes.

Our current data indicate that polyribosomes represent a major site of mRNA decapping within the cell under normal growth conditions. Importantly, however, we cannot determine if mRNA decapping also occurs, at some level, in P-bodies, or if mRNA decay in P-bodies is restricted to certain circumstances, such as under stress (when global translation is altered) or for mRNA populations in which ribosomal run-off is kinetically faster than mRNA decapping. Indeed mechanistic insight into the evolution of mRNA decay may come from understanding that decapping and 5’-3’ degradation occurs while mRNA is polyribosome associated. Specifically, the three steps of decay each serve to systematically limit translational events without interfering with them. Deadenylation reduces translational efficiency perhaps through loss of the poly(A) binding protein, Pab1p or association of decapping regulators. mRNA decapping precludes continued translation and will occur on polyribosomes if kinetically faster than ribosomal run-off (Fig. 1). Under conditions in which mRNA translational initiation or decapping are rate-limiting (such as during stress), ribosomal run-off would predominate even in the absence of mRNA deadenylation and decay would be predicted to occur on ribosome-free mRNAs which may assemble into cytoplasmic P-bodies (Fig. 1). Under normal conditions, however, we propose that decapping and degradation from the 5’ end while the mRNA is ribosome associated ensures decay does not impede any residual ribosomes undergoing translocation. In this way, the final polypeptides expressed prior to the mRNA being destroyed are full length and functional.

Figure 1. Our Current Model of Eukaryotic mRNA decapping. We propose that under normal circumstances mRNA decapping and exonucleolytic decay occur predominately while the mRNA is bound to polyribosomes (left panel). Deadenylation presumably reduces the rate of translational initiation. If under these conditions the decapping rate is faster than the rate of ribosomal run-off, then decay would be observed on polyribosomes. Under conditions of stress (which accelerates translational repression ) or when mRNA decapping rates are slow, ribosomal run-off following deadenylation may predominate (right panel). In these biological context, translational quiescent mRNA may accumulate into ribosome-free P-bodies where they are either stored or ultimately destroyed.