We previously identified hDcp2 as the catalytic subunit of the human decapping enzyme and observed, surprisingly, that it was enriched together with hDcp1a in cytoplasmic foci (van Dijk et al., 2002
). In this paper, we asked if additional factors were present in these foci, if these foci were identical to some previously known cytoplasmic structures present in human cells, and if we could establish a link between these sites and the location of mRNA decay in human cells.
Our results demonstrate that all factors involved in 5′–3′ mRNA decay tested accumulate at least to some extent in a unique set of structures despite the fact that these proteins show a somewhat different overall distribution in cells. These proteins include factors involved in mRNA deadenylation (hCcr4), mRNA decapping (hDcp1a, hDcp1b, and hDcp2), as well as factors required for decapping activation (hLsm proteins and rck/p54). While this work was in progress, the colocalization of Lsm1–7 proteins with hDcp1a and/or hDcp2 in cytoplasmic foci was observed independently (Ingelfinger et al., 2002
; Eystathioy et al., 2003
). These foci also correspond to sites of accumulation of the 5′–3′ exonuclease Xrn1 (Bashkirov et al., 1997
; Ingelfinger et al., 2002
). This clustering of 5′–3′ mRNA decay factors contrasts with the nuclear location of DcpS (involved in cap nucleotide breakdown after mRNA decay in both the 3′–5′ and 5′–3′ mRNA decay) or with the location of the human exosome, which appears mostly concentrated in the nucleus and the nucleolus (Raijmakers et al., 2003
). The accumulation of all factors tested in a single set of structures suggests that they represent the actual site of mRNA decay rather than storage sites. Indeed, in the latter case, one may have anticipated that some factors would be present in excess, and thus stored, whereas others, limiting for the degradation process, would not accumulate in the storage compartment. The recent observation that another RNA binding protein of unknown function, GW182, also localizes in the same structure in Hep-2 (Eystathioy et al., 2003
) and HEK293 cells (unpublished data) also supports a role for these structure bodies in RNA metabolism. Further, we note that after the observation of the peculiar distribution of hDcp1a and hDcp2, a related observation was made in yeast cells (Sheth and Parker, 2003
), even though in another study a more uniform distribution of the same proteins was reported (Tharun et al., 2000
). The structures described in yeast contain the Dcp proteins, other mRNA decay factors, and mRNA decay intermediates (Sheth and Parker, 2003
). Given the similarity between the factors involved in mRNA decay in yeast and humans, this observation reinforces our conclusion that Dcp-containing bodies containing 5′–3′ mRNA decay factors are mRNA degradation centers. However, these structures are unlikely to be identical because hCcr4 is present in human bodies, whereas yeast Ccr4 is not detected in the yeast structures.
The use of translational inhibitors that are known to inhibit mRNA decay strongly suggests that cytoplasmic foci represent sites of mRNA decay. Indeed, translational inhibitors induced the disappearance rather than an enlargement of these structures. Our observation that two factors required for mRNA decapping interact in vivo in these foci also supports an active role for these structures. Further arguments for the functional nature of these bodies come from their absence from mitotic cells (unpublished data) or after transcriptional inhibition, consistent with the proposed absence of mRNA degradation under these conditions (Ross, 1997
). To confirm this possibility, we inactivated Xrn1 by RNAi treatment to induce accumulation of otherwise undetectable mRNA decay intermediates. This strategy resulted in the accumulation of poly(A)+
RNA in specific cytoplasmic foci that coincided with the location of the hDcp2 protein. These results indicate definitively that the cytoplasmic structures concentrating mRNA decay factors are active mRNA decay centers. Given that factors involved in decapping and 5′–3′ exonucleolytic trimming are also involved in NMD, it is tempting to speculate that this process occurs at the same location. Consistent with this possibility, hDcp1a and hDcp1b have been found to interact with hUpf1 that is involved in NMD (Lykke-Andersen, 2002
). Even though our data indicate that 5′–3′ mRNA decay and NMD occur in a dedicated subcellular compartment, there is currently no obvious reason to explain why it should be so. One can hypothesize that this may serve to enhance the process through substrate channeling. Alternatively, a dedicated structure may prevent unwanted mRNA degradation in the cytoplasm. The cellular machinery involved in the targeting of the various components of these structures to their correct location and in the dynamic maintenance of these bodies remains to be identified. The localization of some individual factors gives also some interesting feedback information on their functions. Thus, rck/p54, the homologue of the yeast Dhh1 helicase, has been proposed to function in initiation of translation and translational control (Minshall et al., 2001
; Smillie and Sommerville, 2002
; Akao et al., 2003
). In yeast, its homologue, Dhh1, interacts with Dcp1 and enhances decapping in vitro (Coller et al., 2001
; Fischer and Weis, 2002
). We observed that a fraction of rck/p54 localizes in cytoplasmic foci. Based on this result and on the homology of rck/p54 with yeast Dhh1, we propose that the protooncogene rck/p54 plays a role in activation of decapping as does the yeast homologue. The cytoplasmic location of hCcr4 is also informative, as this protein has been implicated in both transcription and mRNA degradation. The presence of an evolutionarily conserved nuclease domain (Chen et al., 2002
), together with its accumulation in the cytoplasm and a (near-)complete nuclear exclusion (see Results), supports its role in mRNA degradation, even though a function in transcription cannot be ruled out. The exclusive nuclear localization of DcpS is also of interest. On the one hand, this feature appears evolutionarily conserved because the DcpS homologue from Schizosaccharomyces pombe
also localizes in the nucleus (Salehi et al., 2002
). On the other hand, we recently demonstrated that DcpS is involved in 7-methyl guanine–containing cap nucleotide breakdown in both the 5′–3′ and 3′–5′ pathways and hypothesized that this function might be required to prevent the deleterious consequences of incorporation of methylated G residues in RNA and/or DNA (van Dijk et al., 2003
). The localization of DcpS in the nucleus, the main cellular site for transcription and DNA replication, is fully consistent with this possibility.
An interesting aspect of the cytoplasmic structures involved in mRNA decay is their dynamic nature. This feature will also probably be reflected in a heterogeneous composition and shape that will reflect the kinetic of association of individual factors with each structure. Thus, this may explain the slight differences in the number of foci and variation in staining intensities for individual factors. The observation of these new dynamic cytoplasmic structures raised the question of whether or not they corresponded to previously described bodies or to new cellular entities. Our data show clearly that they are not identical to stress granules even though both are highly dynamic and connected to mRNA metabolism. Other cytoplasmic structures related to RNA metabolism have been identified in specific cells, such as polar granules in Caenorhabditis elegans
and Drosophila melanogaster
oocytes and germ plasm in X. laevis
oocytes (Micklem, 1995
). However, given their restricted tissues distribution, such structures are unlikely to have a relation to the cytoplasmic mRNA degradation foci that we observe in human cells. Overall, these observations indicate that the cytoplasm may be more organized than previously anticipated. Interestingly, during the past years, results demonstrating that the eukaryotic nucleus is divided in highly dynamic but functionally distinct compartments have also accumulated (Lamond and Earnshaw, 1998
). Further work will be necessary to understand the composition of the various cytoplasmic structures involved in mRNA metabolism and their functions in the precise control of gene expression.