Regulation of cytoplasmic mRNA decay

Discoveries made over the past 20 years highlight the importance of mRNA decay as a means of modulating gene expression and thereby protein production. Up until recently, studies largely focused on identifying cis-acting sequences that serve as mRNA stability or instability elements, the proteins that bind these elements, how the process of translation influences mRNA decay and the ribonucleases that catalyse decay. Now, current studies have begun to elucidate how the decay process is regulated. This Review examines our current understanding of how mammalian cell mRNA decay is controlled by different signalling pathways and lays out a framework for future research.     

Regulation of nonsense-mediated mRNA decay: Implications for physiology and disease

Nonsense-mediated mRNA decay (NMD) is an mRNA quality control mechanism that destabilizes aberrant mRNAs harboring premature termination (nonsense) codons (PTCs). Recent studies have shown that NMD also targets mRNAs transcribed from a large subset of wild-type genes. This raises the possibility that NMD itself is under regulatory control. Indeed, several recent studies have shown that NMD activity is modulated in specific cell types and that key components of the NMD pathway are regulated by several pathways, including microRNA circuits and NMD itself. Cellular stress also modulates the magnitude of NMD by mechanisms that are beginning to be understood. Here, we review the evidence that NMD is regulated and discuss the physiological role for this regulation. We propose that the efficiency of NMD is altered in some cellular contexts to regulate normal biological events. In disease states—such as in cancer—NMD is disturbed by intrinsic and extrinsic factors, resulting in altered levels of crucial NMD-targeted mRNAs that lead to downstream pathological consequences. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

Regulation of c-myc mRNA decay by translational pausing in a coding region instability determinant

A 249-nucleotide coding region instability determinant (CRD) destabilizes c-myc mRNA. Previous experiments identified a CRD-binding protein (CRD-BP) that appears to protect the CRD from endonuclease cleavage. However, it was unclear why a CRD-BP is required to protect a well-translated mRNA whose coding region is covered with ribosomes. We hypothesized that translational pausing in the CRD generates a ribosome-deficient region downstream of the pause site, and this region is exposed to endonuclease attack unless it is shielded by the CRD-BP. Transfection and cell-free translation experiments reported here support this hypothesis. Ribosome pausing occurs within the c-myc CRD in tRNA-depleted reticulocyte translation reactions. The pause sites map to a rare arginine (CGA) codon and to an adjacent threonine (ACA) codon. Changing these codons to more common codons increases translational efficiency in vitro and increases mRNA abundance in transfected cells. These data suggest that c-myc mRNA is rapidly degraded unless it is (i) translated without pausing or (ii) protected by the CRD-BP when pausing occurs. Additional mapping experiments suggest that the CRD is bipartite, with several upstream translation pause sites and a downstream endonuclease cleavage site.     

Shutdown decay of mRNA

Although plasmid-borne and chromosomal toxin-antitoxin (TA) operons have been known for some time, the recent identification of mRNA as the target of at least two different classes of toxins has led to a dramatic renewal of interest in these systems as mediators of stress responses. Members of the MazF/PemK family, the so-called mRNA interferases, are ribonucleases that inhibit translation by destroying cellular mRNAs under stress conditions, while the founder member of the RelE family promotes cleavage of mRNAs through the ribosome. Detailed structures of these enzymes, often in complex with their inhibitors, have provided vital clues to their mechanisms of action. The primary role and regulation of these systems has been the subject of some controversy. One model suggests they play a beneficial role by wiping the slate clean and preventing wasteful energy consumption by the translational apparatus during adaptation to stress conditions, while another favours the idea that their main function is programmed cell death. The two models might not be mutually exclusive if a side-effect of prolonged exposure to toxic RNase activity without de novo synthesis of the inhibitor were a state of dormancy for which we do not yet understand the key to recovery. In this review, I discuss the recent developments in the rapidly expanding field of what I refer to as bacterial shutdown decay.     

Translation and mRNA decay

Summary Degradation of messenger RNA from the lactose operon (lac mRNA) was measured during the inhibition of protein synthesis by chloramphenicol (CM) or of translation-initiation by kasugamycin (KAS). With increasing CM concentration mRNA decay becomes slower, but there is no direct proportionality between rates of chemical decay and polypeptide synthesis. During exponential growth lac mRNA is cleaved endonucleolytically (Blundell and Kennell, 1974). At a CM concentration which completely inhibits all polypeptide synthesis this cleavage is blocked. In contrast, if only the initiation of translation is blocked by addition of KAS, the cleavage rate as well as the rate of chemical decay are increased significantly without delay. These faster rates do not result from immediate degradation of the lengthening stretch of ribosome-free proximal message, since the full-length size is present and the same discrete message sizes are generated during inhibition.These results suggest that neither ribosomes nor translation play an active role in the degradative process. Rather, targets can be protected by the proximity of a ribosome, and without nearby ribosomes the probability of cleavage becomes very high. During normal growth there is a certain probability that any message is in such a vulnerable state, and the fraction of vulnerable molecules determines the inactivation rate of that species.     

Regulation of c-myc mRNA decay in vitro by a phorbol ester-inducible, ribosome-associated component in differentiating megakaryoblasts

The K562 leukemia cell line is bipotential for erythroid and megakaryoblastic differentiation. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) activates a genetic program of gene expression in these cells leading to their differentiation into megakaryoblasts, a platelet precursor. Thus, K562 cells offer a means to examine early changes in gene expression necessary for megakaryoblastic commitment and differentiation. An essential requirement for differentiation of many hematopoietic cell types is the down-regulation of c-myc expression, because its constitutive expression blocks differentiation. TPA-induced differentiation of K562 cells causes rapid down-regulation of c-myc expression, due in part to an mRNA decay rate that is 4-fold faster compared with dividing cells. A cell-free mRNA decay system reconstitutes TPA-induced destabilization of c-myc mRNA, but it requires at least two components for reconstitution. One component fractionates to the post-ribosomal supernatant from either untreated or treated cells. This component is sensitive to cycloheximide and micrococcal nuclease. The other component is polysome-associated and is induced or activated by TPA. Although in dividing cells c-myc mRNA decays via a sequential pathway involving removal of the poly(A) tract followed by degradation of the mRNA body, TPA activates a deadenylation-independent pathway. The cell-free mRNA decay system reconstitutes this alternate decay pathway as well.     

Emerging features of mRNA decay in bacteria

The problem of mRNA decay in E. coli has recently seen exciting progress, with the discoveries that key degradation enzymes are associated together in a high molecular weight degradosome and that polyadenylation promotes decay. Recent advances make it clear that mRNA decay in bacteria is far more interesting enzymatically than might have been predicted. In-depth study of specific mRNAs has revealed multiple pathways for degradation. Which pathway a given mRNA follows appears to depend in large part on the location of the initiating endonucleolytic cleavage within the mRNA. During the steps of mRNA decay, stable RNA structures pose formidable barriers to the 3' --> 5' exonucleases. However, polyadenylation is now emerging as a process that plays an important role in maintaining the momentum of exonucleolytic degradation by adding single-stranded extensions to the 3' ends of mRNAs and their decay intermediates, thereby facilitating further exonuclease digestion.     

c-Src activates endonuclease-mediated mRNA decay

The mRNA endonuclease PMR1 initiates mRNA decay by forming a selective complex with its translating substrate mRNA. Previous work showed that the ability of PMR1 to target to polysomes and activate decay depends on the phosphorylation of a tyrosine residue at position 650. The current study shows that c-Src is responsible for activating this mRNA decay pathway. c-Src was recovered with immunoprecipitated PMR1, and it phosphorylates PMR1 in vitro and in vivo. The interaction with c-Src involves two domains of PMR1: Y650 and a series of proline-rich SH3 peptides in the N terminus. In cells with little c-Src, PMR1 targeting to polysomes is induced by constitutively active c-Src but not by inactive forms of the kinase. Similarly, only active c-Src induces PMR1-mediated mRNA decay. Finally, we show that EGF rapidly induces c-Src phosphorylation of PMR1, providing a direct link between tyrosine kinase-mediated signal transduction and mRNA decay.