Recent years have seen recognition that a diversity of post-transcriptional control mechanisms influences the rate and regulation of eukaryotic gene expression. Yet our understanding of the interplay between the component processes of post-transcriptional gene expression is very limited. A prime example is the relationship between translation and mRNA degradation, which is not only fundamental to the correct functioning of gene expression but also a potential cause of disease if defective. It has been proposed that translational repression, as for example observed under stress conditions, is a key step in promoting mRNA decapping, thus leading to the formation of P bodies (1
). P bodies, like stress granules, are RNA/protein foci that form under certain (mostly stress-related) conditions in eukaryotic cells. P bodies generally contain non-translating mRNAs as well as the mRNA decapping machinery, Lsm1-7, the 5′-3′ exonuclease Xrn1 and other RNA-binding proteins (3
), although the physical nature and degree of heterogeneity of P body populations is unclear.
Two proteins, Dhh1 and Pat1, are thought to lie at the heart of the relationship between translation and mRNA degradation (4
). Dhh1 and Pat1 act as activators of decapping and, at least under conditions of overexpression, they are capable of repressing translation in vivo
). However, other results suggest that Pat1 (at normal cellular levels) acts to promote
translation initiation at a step before or during 40S ribosomal recruitment onto mRNA (5
). In other eukaryotes, such as Xenopus
, Dhh1 orthologues have been shown to be involved in translational repression of specific mRNAs during early development (6
). It has previously been postulated that translational repression generically drives mRNAs into P bodies (and thus accelerated decay), and that translational repression is in constant competition with active translation (4
). Other reports have suggested that the decay rate may be modulated differentially in response to distinct types of translational control event and that translation–decay relationships can be mRNA species specific (8
). While mRNA decapping, which plays a key role in mRNA degradation (4
), can be inhibited by the cap-binding protein eIF4E in vitro
and in vivo
), it is neither clear how this apparently competitive relationship is controlled nor at what stage it features in modulating the balance between translation and decay. Very recent work has also shifted the emphasis of current thinking by revealing that, as in bacteria (9
), mRNA decay in Saccharomyces cerevisiae
can be co-translational (14
) although this does not rule out the possibility that translation and decay mutually influence or regulate each other. Against this complex background of previous findings, it is important to know how Dhh1 and Pat1 participate in controlling the relationship between the translation apparatus and the decay machinery.
Dhh1 belongs to a family of closely related DEAD-box RNA helicases that associate with components of mRNA decapping, deadenylation and transcription complexes (1
). Dhh1 stimulates mRNA decapping by the decapping enzyme complex Dcp1/Dcp2, and has been shown to localize partly to P-bodies (15
). Orthologues of Dhh1 in other eukaryotes, such as Xenopus
, play roles in repressing translation of specific mRNAs during early development (6
). Dhh1 has been suggested to play a role in partitioning mRNAs between translatable and non-translatable pools, which has been implicated in the recovery from G1/S cell-cycle arrest following DNA damage (16
is orthologous to the human putative proto-oncogene p54/RCK, indicating that the mechanisms of action suggested by studies of yeast are relevant to human health/disease. Moreover, a fascinating parallel exists to the involvement of Lsm1-7/Pat1/Dhh1 in the transition from an actively translating state to a non-translating state (replication or decay competent) observed in Brome Mosaic Virus (BMV). In addition, a comparable transition is promoted in Hepatitis C Virus (HCV) by the virus-encoded NS3 helicase (e.g. 17
), suggesting that there may be common molecular principles (for example, responsible for remodelling ribonucleoprotein complex structures) operating in diverse subcellular systems.
In this study, we examine the undefined relationship between Dhh1/Pat1 and the translation machinery. We focus on their respective cellular distributions, since these are directly relevant to the functions of these proteins. For example, if the spatial distributions of a regulatory molecule and its target do not overlap, this exercises a limiting effect on the regulatory competence of the regulator. Imaging of fluorescently tagged cellular components, combined with analyses of the composition of polysomal complexes, reveals a remarkable degree of separation of these proteins from ribosomal populations during exponential cell growth, i.e. in cells lacking P bodies. This is found to correlate with spatial segregation of these proteins from actively translating polysomal complexes. In contrast, Dhh1 and Pat1 gain greatly increased access to actively translating polysomes in the phase of growth that is associated with the shift from glucose fermentation to ethanol oxidation (the diauxic growth shift). This has prompted us to investigate whether there is a control relationship between translation rate and these relocation events, and to characterize protein interactions involving Dhh1 and the translation machinery that are likely to be relevant to the functional roles of these decapping modulators in the cell. The results suggest that segregation of Dhh1 and Pat1 from actively translating polysomes, like the formation of P bodies, reflects modulation of the access of these translational repressors to the translational machinery. Thus modulation of the subcellular localization of translational effectors may play a role in post-transcriptional control.