An unconventional human Ccr4-Caf1 deadenylase complex in nuclear cajal bodies

Abstract

mRNA deadenylation is a key process in the regulation of translation and mRNA turnover. In Saccharomyces cerevisiae, deadenylation is primarily carried out by the Ccr4p and Caf1p/Pop2p subunits of the Ccr4-Not complex, which is conserved in eukaryotes including humans. Here we have identified an unconventional human Ccr4-Caf1 complex containing hCcr4d and hCaf1z, distant human homologs of yeast Ccr4p and Caf1p/Pop2p, respectively. The hCcr4d-hCaf1z complex differs from conventional Ccr4-Not deadenylase complexes, because (i) hCaf1z and hCcr4d concentrate in nuclear Cajal bodies and shuttle between the nucleus and cytoplasm and (ii) the hCaf1z subunit, in addition to rapid deadenylation, subjects substrate RNAs to slow exonucleolytic degradation from the 3' end in vitro. Exogenously expressed hCaf1z shows both of those activities on reporter mRNAs in human HeLa cells and stimulates general mRNA decay when restricted to the cytoplasm by deletion of its nuclear localization signal. These observations suggest that the hCcr4d-hCaf1z complex may function either in the nucleus or in the cytoplasm after its nuclear export, to degrade polyadenylated RNAs, such as mRNAs, pre-mRNAs, or those RNAs that are polyadenylated prior to their degradation in the nucleus.

FIG. 1.

hCaf1z forms a specific complex with hCcr4d. A. Schematic representation of the human Ccr4 homologs designated hCcr4a to hCcr4e. Each has a conserved nuclease domain (shown in gray) related to the Mg²⁺-dependent apurinic/apyrimidinic endonucleases (AP Endo). Numbers refer to amino acid (aa) positions. hCcr4a and hCcr4b possess N-terminal LRRs, which are conserved in yeast Ccr4p. hCcr4c has a leucine zipper motif (LZ) and is the ortholog of the Xenopus protein nocturnin, while hCcr4e is related to the Drosophila melanogaster protein angel. GenBank accession numbers are as given: hCcr4a, AB033020; hCcr4b, XM_939929; hCcr4c, AF183961; hCcr4d, XM_034232; hCcr4e, AL137268. B. Schematic representation of human Caf1 proteins, hCaf1a, hCaf1b, and hCaf1z. The nuclease domains (shown in gray) of the Caf1 proteins are members of the DEDD superfamily of exonucleases. hCaf1z contains a C₃H-type zinc finger (Zn) and a basic NLS. GenBank accession numbers are as given: hCaf1a, NM_013354; hCaf1b, NM_004779; hCaf1z, NP_079353. C. Coimmunoprecipitation assays performed in RNase A-treated HEK293T extracts derived from cells coexpressing FLAG-tagged hCaf1a (lanes 3 and 4), hCaf1b (lanes 7 and 8), hCaf1z (lanes 11 and 12), or hNot2 (lanes 15 and 16) with Myc-tagged hCcr4a to hCcr4e and Myc-hnRNP A1 as indicated. FLAG fusion proteins were immunoprecipitated using anti-FLAG M2 agarose, and immunoprecipitates were subjected to immunoblotting with anti-Myc. Lanes 1, 2, 5, 6, 9, 10, 13, and 14 are negative immunoprecipitation controls from cells expressing no FLAG-tagged proteins. Myc-hnRNP A1 served as a negative coimmunoprecipitation control. Pellet (P) and 5% of total (T) fractions were loaded as indicated. D. Schematic of human Ccr4-Caf1 complexes based on the observations in panel C. The proteins tested in panel C are shown in light gray. E. Endogenous hCcr4d was immunoprecipitated from HeLa cell extracts and probed for the presence of endogenous hCaf1z and hCaf1a. Lane 1, 5% total HeLa extract; lane 2, preimmune serum immunoprecipitate; lane 3, anti-hCcr4d immunoprecipitate. IP, immunoprecipitation; IgG, immunoglobulin G.

FIG. 2.

hCaf1z copurifies with fast deadenylase and slow non-poly(A) nuclease activity. A. FLAG-hCaf1z deadenylation time course. The deadenylation substrate (A₆₀, lane 2) and the nonadenylated control (C, lane 1) were incubated with ∼10 ng FLAG-hCaf1z for 0 to 60 min as indicated (lanes 4 to 13). Deadenylated substrate A₀, generated by RNase H/DNA oligonucleotide cleavage, is shown in lane 3. B. deadenylation time course with FLAG-hCaf1b, as described for panel A. BC, buffer control; Mo, “mock”-purified protein. C. Same as panel A but using a mutant hCaf1z DEAA (D64A, E66A) protein. D. hCaf1z zinc finger mutants C300A (lanes 9 and 10) and C309A (lanes 11 and 12) are active in deadenylation.

FIG. 3.

hCaf1z is a poly(A)-specific nuclease. A. deadenylation time course as in Fig. 2 but using recombinant His₆-tagged hCaf1z purified from E. coli. B. deadenylation time course with mutant His₆-hCaf1z DEAA protein purified from E. coli. C. Incubation of poly(U) substrate with FLAG-hCaf1z or His₆-hCaf1z does not result in removal of the poly(U) tail. A polyuridylated substrate (U₄₀) and a nonuridylated/nonadenylated control RNA (C) were incubated with ∼10 ng FLAG-hCaf1z (lanes 4 to 7) or His₆-hCaf1z (lanes 8 to 11) for 0 to 60 min as indicated. The migration of the U₀ product, generated by RNase H/oligo(dA) cleavage, is shown in lane 3.

FIG. 4.

Localization of human Ccr4d and Caf1z proteins in Cajal bodies. A. Myc-tagged hCaf1z, hCcr4d, and hCaf1z lacking the putative nuclear localization signal, hCaf1z ΔNLS, were transiently expressed in HeLa cells and visualized with anti-Myc antibody. Nuclei are stained with DAPI (4′,6′-diamidino-2-phenylindole). B. Endogenous hCaf1z and hCcr4d were stained with affinity-purified polyclonal antibodies and costained with the Cajal body marker protein coilin/p80. Merging of these panels reveals that both hCaf1z and hCcr4d (red) completely colocalize with coilin/p80 (green).

FIG. 5.

hCaf1z and hCcr4d are nucleocytoplasmic shuttling proteins. A. HeLa cells coexpressing Myc-hCaf1z with GFP-hnRNP A1 or GFP-hnRNP C were fused with mouse NIH 3T3 cells. Nuclei are stained with Hoechst stain. Mouse nuclei are indicated by white arrows. B. Myc-hCcr4d was coexpressed with GFP-hnRNP A1 or GFP-hnRNP C in HeLa cells and fused to form heterokaryons with NIH 3T3 cells. DIC, differential interference contrast.

FIG. 6.

Exogenously expressed hCaf1z can stimulate deadenylation and decay of reporter mRNAs in human HeLa cells. A. Transcriptional pulse-chase mRNA decay assays of a stable β-globin (β-wt) reporter transcript in the presence of no protein (none) or exogenously expressed wild-type or mutant hCaf1z ΔNLS proteins as indicated. Assays were performed in cells coexpressing a constitutive β-GAP control mRNA. After a 6-hour transcriptional pulse, tetracycline was used to stop reporter β-globin mRNA transcription, regulated by a tetracycline-repressible promoter, and total RNA was isolated from cells at intervals thereafter indicated below the panels and subjected to Northern blotting. Calculated mRNA t_1/2s are given on the right in minutes. B. Decay of an unstable β-globin transcript (β-ARE), which contains the granulocyte-macrophage colony-stimulating factor ARE in the 3′ untranslated region, in the presence of wild-type hCaf1z or mutant hCaf1z ΔNLS, hCaf1z DEAA, or hCaf1z DEAA ΔNLS proteins as indicated. C. Decay of an intronless β-globin mRNA reporter (β-wtΔ12) in the presence of hCaf1z proteins. Asterisks in panels A to D indicate the shortened β-globin transcripts observed in the presence of exogenously expressed hCaf1z. E. Northern blotting of mRNAs isolated from cells transfected with the control (β-GAP) and intronless (β-wtΔ12) reporter constructs together with either an empty expression vector (lanes 1 to 3) or an expression vector encoding FLAG-hCaf1z (lanes 4 to 6) or FLAG-hCaf1z ΔNLS (lanes 7 to 9). Isolated RNA was treated with RNase H in the presence of oligo(dT) (lanes 3, 6, and 9) or a 3′-H oligonucleotide which hybridizes ∼30 nucleotides upstream of the poly(A) site in both the β-GAP control and β-wtΔ12 mRNAs (lanes 2, 5, and 8; hybridization site illustrated below the panel). Lanes 1, 4, and 7 show RNase H treatment in the absence of DNA oligonucleotide.