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

The control of mRNA degradation is an important component of the regulation of gene expression since the steady-state concentration of mRNA is determined both by the rates of synthesis and of decay. Two general pathways of mRNA decay have been described in eukaryotes. Both pathways share the exonucleolytic removal of the poly(A) tail (deadenylation) as the first step. In one pathway, deadenylation is followed by the hydrolysis of the cap and processive degradation of the mRNA body by a 5' exonuclease. In the second pathway, the mRNA body is degraded by a complex of 3' exonucleases before the remaining cap structure is hydrolyzed. This review discusses the proteins involved in the catalysis and control of both decay pathways.

The CCR4-NOT complex is the major enzyme catalyzing mRNA deadenylation in Saccharomyces cerevisiae. We have identified homologs for almost all subunits of this complex in the Drosophila genome. Biochemical fractionation showed that the two likely catalytic subunits, CCR4 and CAF1, were associated with each other and with a poly(A)-specific 3 0 exonuclease activity. In Drosophila, the CCR4 and CAF1 proteins were ubiquitously expressed and present in cytoplasmic foci. Individual knock-down of several potential subunits of the Drosophila CCR4-NOT complex by RNAi in tissue culture cells led to a lengthening of bulk mRNA poly(A) tails. Knock-down of two individual subunits also interfered with the rapid deadenylation of Hsp70 mRNA during recovery from heat shock. Similarly, ccr4 mutant flies had elongated bulk poly(A) and a defect in Hsp70 mRNA deadenylation. A minor increase in bulk poly(A) tail length was also observed in Rga mutant flies, which are affected in the NOT2 subunit. The data show that the CCR4-NOT complex is conserved in Drosophila melanogaster and plays a role in general and regulated mRNA deadenylation.

An essential constituent of the integrated stress response (ISR) is a reversible translational suppression. This mRNA silencing occurs in distinct cytoplasmic foci called stress granules (SGs), which transiently associate with processing bodies (PBs), typically serving as mRNA decay centers. How mRNAs are protected from degradation in these structures remains elusive. We identify that Zipcode-binding protein 1 (ZBP1) regulates the cytoplasmic fate of specific mRNAs in nonstressed cells and is a key regulator of mRNA turnover during the ISR. ZBP1 association with target mRNAs in SGs was not essential for mRNA targeting to SGs. However, ZBP1 knockdown induced a selective destabilization of target mRNAs during the ISR, whereas forced expression increased mRNA stability. Our results indicate that although targeting of mRNAs to SGs is nonspecific, the stabilization of mRNAs during cellular stress requires specific protein–mRNA interactions. These retain mRNAs in SGs and prevent premature decay in PBs. Hence, mRNA-binding proteins are essential for translational adaptation during cellular stress by modulating mRNA turnover.

The nuclear poly(A)-binding protein (PABPN1) is involved in the synthesis of the mRNA poly(A) tails in most eukaryotes. We report that the protein contains two RNA binding domains, a ribonucleoprotein-type RNA binding domain (RNP domain) located approximately in the middle of the protein sequence and an arginine-rich C-terminal domain. The C-terminal domain also promotes self-association of PABPN1 and moderately cooperative binding to RNA. Whereas the isolated RNP domain binds specifically to poly(A), the isolated C-terminal domain binds non-specifically to RNA and other polyanions. Despite this nonspecific RNA binding by the C-terminal domain, selection experiments show that adenosine residues throughout the entire minimal binding site of ϳ11 nucleotides are recognized specifically. UV-induced cross-links with oligo(A) carrying photoactivatable nucleotides at different positions all map to the RNP domain, suggesting that most or all of the basespecific contacts are made by the RNP domain, whereas the C-terminal domain may contribute nonspecific contacts, conceivably to the same nucleotides. Asymmetric dimethylation of 13 arginine residues in the C-terminal domain has no detectable influence on the interaction of the protein with RNA. The N-terminal domain of PABPN1 is not required for RNA binding but is essential for the stimulation of poly(A) polymerase.In the cell, mRNA molecules and their precursors are always bound by proteins. These proteins not only protect the RNA from nucleases and undesirable interactions of its highly charged surface but influence enzymes and other proteins that act upon the RNA at all stages of its maturation, function, and decay (1). Characteristically, a single RNA-binding protein very often contains more than one RNA binding domain. Different kinds of RNA binding domains have been described (2). Among them, the RNA recognition motif or RNP 1 -type RNA binding domain is probably the best understood (1, 3, 4). The RNP domain consists of ϳ90 amino acids forming a ␤␣␤␤␣␤ fold, in which a four-stranded ␤-sheet is backed by two ␣-helices. The two central antiparallel ␤-strands carry the highly conserved amino acids of the RNP1 and RNP2 motifs. Different members of the RNP protein family can bind structured or extended RNA molecules in a sequence-specific manner. As seen in several co-crystals, the RNA is bound on the surface of the ␤-sheet by hydrogen bonds and stacking interactions between bases and amino acid side chains (5-8).Another common RNA binding domain is the so-called RGG domain, characterized by multiple copies of the amino acid sequence arginine-glycine-glycine, interspersed with phenylalanine and tyrosine residues (9). The structure of the domain is not known, although a spiral of ␤-turns has been proposed based on spectroscopic data (10). A possibly related argininerich domain found at the C termini of several of the spliceosomal Sm core proteins was not ordered in a crystal structure in the absence of RNA (11). The RGG domain usually occurs in proteins in conjunction wit...