Isolation and characterization of human orthologs of yeast CCR4-NOT complex subunits - PubMed (original) (raw)

Isolation and characterization of human orthologs of yeast CCR4-NOT complex subunits

T K Albert et al. Nucleic Acids Res. 2000.

Abstract

The yeast CCR4-NOT protein complex is a global regulator of RNA polymerase II transcription. It is comprised of yeast NOT1 to NOT5, yeast CCR4 and additional proteins like yeast CAF1. Here we report the isolation of cDNAs encoding human NOT2, NOT3, NOT4 and a CAF1-like factor, CALIF. Analysis of their mRNA levels in different human tissues reveals a common ubiquitous expression pattern. A multitude of two-hybrid interactions among the human cDNAs suggest that their encoded proteins also form a complex in mammalian cells. Functional conservation of these proteins throughout evolution is supported by the observation that the isolated human NOT3 and NOT4 cDNAs can partially com-plement corresponding not mutations in yeast. Interestingly, human CALIF is highly homologous to, although clearly different from, a recently described human CAF1 protein. Conserved interactions of this factor with both NOT and CCR4 proteins and co-immunoprecipitation experiments suggest that CALIF is a bona fide component of the human CCR4-NOT complex.

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Figures

Figure 1

Figure 1

Schematic presentation of conserved regions shared between S.cerevisiae yeast (y) and human (h) CCR4–NOT proteins. The percentage of identity between two proteins was calculated using the ALIGN algorithm (global alignment with no short-cuts). Different shadings denote different percentages of identity as indicated in the bottom panel; the percentage of the overall identity between two proteins is shown to the right. Special motifs and hallmarks are indicated by black boxe. For details see text (supplementary information can be obtained at http://ruummc.med.uu.nl/publications/publtxp.htm ).

Figure 2

Figure 2

Expression of CCR4–NOT mRNAs in various human tissues. A multiple human tissue northern blot was hybridized with probes specific for h_NOT1_, h_NOT2_, h_NOT3_, h_CALIF_ and, as a control, with a β-ACTIN specific probe. Molecular sizes (in kb) are indicated to the left.

Figure 3

Figure 3

Complementation of mutant not alleles in yeast by human cDNAs. (A) Complementation of not4::LEU2 by the h_NOT4-N_ cDNA. Strain YOU637 was transformed with pGEN-h_NOT4-N_, pRS314-y_NOT4_ or pRS314 alone. Transformants were streaked on FOA plates to determine for loss of the pRS316-y_NOT5_ plasmid essential in this genetic background. Both pRS314-yNOT4 and pGEN-hNOT4-N transformants could grow on FOA (left). Growth of the FOA-resistant transformants was then compared on YPD rich medium (right). (B) Strains YOU578 (not3_Δ/not5_Δ_N_) and YOU555 (_not5_Δ) were transformed with multicopy plasmids expressing the indicated chimeric proteins. Equivalent expression levels for the proteins examined were confirmed by western blot (data not shown). Human and yeast chimeric proteins recombined the natural sequences found within the native NOT3 and NOT5 proteins, but incorporated a few residues in frame that were necessary for creation of the protein junctions (indicated by white border lines in illustrations). Transformants were tested for growth at 37°C. Wild-type growth, (+++); slower than wild-type growth, (+); barely detectable growth, (+/–); no growth, (–).

Figure 3

Figure 3

Complementation of mutant not alleles in yeast by human cDNAs. (A) Complementation of not4::LEU2 by the h_NOT4-N_ cDNA. Strain YOU637 was transformed with pGEN-h_NOT4-N_, pRS314-y_NOT4_ or pRS314 alone. Transformants were streaked on FOA plates to determine for loss of the pRS316-y_NOT5_ plasmid essential in this genetic background. Both pRS314-yNOT4 and pGEN-hNOT4-N transformants could grow on FOA (left). Growth of the FOA-resistant transformants was then compared on YPD rich medium (right). (B) Strains YOU578 (not3_Δ/not5_Δ_N_) and YOU555 (_not5_Δ) were transformed with multicopy plasmids expressing the indicated chimeric proteins. Equivalent expression levels for the proteins examined were confirmed by western blot (data not shown). Human and yeast chimeric proteins recombined the natural sequences found within the native NOT3 and NOT5 proteins, but incorporated a few residues in frame that were necessary for creation of the protein junctions (indicated by white border lines in illustrations). Transformants were tested for growth at 37°C. Wild-type growth, (+++); slower than wild-type growth, (+); barely detectable growth, (+/–); no growth, (–).

Figure 4

Figure 4

Human CCR4–NOT proteins interact in the yeast two-hybrid assay. (A) Yeast cells of strain EGY48 were transformed with the indicated LexA- and B42-fusion expression plasmids together with a lacZ reporter gene containing one LexA operator. Interactions were assayed by blue staining of transformants that were streaked on Xgal-containing selective plates, and incubated for 16–20 h at 30°C. (B) Relative β-galactosidase activities of EGY48 derivatives expressing the indicated proteins. Yeast extracts from EGY48 transformants selected by trp+his+ura+ prototrophy were assayed for β-galactosidase activity. Three to four independent experiments were performed for each combination and the results of one representative set of experiments are shown, with similar results obtained for the other independent sets of experiments. Measured β-galactosidase activities (in RLUs) were normalized for the amount of total protein in extracts and expressed as fold inductions relative to the β-galactosidase activity of cells expressing the B42 activation domain only. A table summarizing the observed two-hybrid interactions is presented in (C).

Figure 5

Figure 5

hCALIF and hNOT3 interact in vivo. C33A cell lysates were subjected to immunoprecipitation with protein A–agarose beads alone (lane 2), beads coupled to anti-CALIF antibody (lane 3) or to a control antibody (anti-ERK2, lane 4). The input (lane 1) contains one-tenth the quantity of extract used for each immunoprecipitation. Protein extraction and binding conditions are as described in Materials and Methods. The upper panel shows an immunoblot (IB) with anti-CALIF, the middle panel with anti-NOT3 and the lower panel with anti-ERK2. The arrows indicate the proteins found in the immunoprecipitates. Molecular weight markers are indicated on the left.

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