Understanding gene expression control requires defining the molecular and cellular basis of mRNA turnover. We have previously shown that the human decapping factors hDcp2 and hDcp1a are concentrated in specific cytoplasmic structures. Here, we show that hCcr4, hDcp1b, hLsm, and rck/p54 proteins related to 5′–3′ mRNA decay also localize to these structures, whereas DcpS, which is involved in cap nucleotide catabolism, is nuclear. Functional analysis using fluorescence resonance energy transfer revealed that hDcp1a and hDcp2 interact in vivo in these structures that were shown to differ from the previously described stress granules. Our data indicate that these new structures are dynamic, as they disappear when mRNA breakdown is abolished by treatment with inhibitors. Accumulation of poly(A)+ RNA in these structures, after RNAi-mediated inactivation of the Xrn1 exonuclease, demonstrates that they represent active mRNA decay sites. The occurrence of 5′–3′ mRNA decay in specific subcellular locations in human cells suggests that the cytoplasm of eukaryotic cells may be more organized than previously anticipated.

We have cloned cDNAs for the human homologues of the yeast Dcp1 and Dcp2 factors involved in the major (5¢±3¢) and NMD mRNA decay pathways. While yeast Dcp1 has been reported to be the decapping enzyme, we show that recombinant human Dcp2 (hDcp2) is enzymatically active. Dcp2 activity appears evolutionarily conserved. Mutational and biochemical analyses indicate that the hDcp2 MutT/Nudix domain mediates this activity. hDcp2 generates m7GDP and 5¢-phosphorylated mRNAs that are 5¢±3¢ exonuclease substrates. Corresponding decay intermediates are present in human cells showing the relevance of this activity. hDcp1 and hDcp2 co-localize in cell cytoplasm, consistent with a role in mRNA decay. Interestingly, these two proteins show a non-uniform distribution, accumulating in speci®c foci. Keywords: mRNA cap/mRNA decay/MutT/Nudix/ nuclease/turnover

distinctive feature of eukaryotic mRNA and small nuclear RNA (snRNA) that are transcribed by RNA polymerase II (Pol II) is the presence of a cap structure at their 5' end. This essential modification serves as an inviting 'landing pad' for factors that are involved in various cellular processes such as pre-mRNA splicing, nucleocytoplasmic RNA export and localization, and translation initiation. Because of the important functions mediated by the mRNA cap, this structure needs to be modified and/or degraded in a tightly controlled manner. Several cellular and viral systems implicated in cap metabolism have been uncovered recently; their analyses provide interesting new information on cell structure and function.