The Rpb4/7 module of RNA polymerase II is required for carbon catabolite repressor protein 4-negative on TATA (Ccr4-not) complex to promote elongation

Abstract

Gene expression relies on the balance between mRNA synthesis in the nucleus and decay in the cytoplasm, processes once thought to be separate. We now know that transcription and decay rates are coordinated, but the factors or molecular mechanisms are unclear. The Ccr4-Not complex regulates multiple stages of gene expression, from mRNA synthesis to protein destruction. One of its functions is to promote RNA polymerase II elongation by reactivating arrested elongation complexes. Here we explored the features of polymerase required for Ccr4-Not to promote elongation and found that the Rpb4/7 module is important for Ccr4-Not to associate with elongation complexes and stimulate elongation. Rpb4/7 has also been implicated in coordinating mRNA synthesis and decay, but its role in this process is controversial. The interplay between Ccr4-Not and Rpb4/7 described here suggests a mechanism for how the cell coordinates mRNA synthesis and decay.

Keywords: Ccr4-Not; RNA Polymerase II; RNA Synthesis; Transcription Elongation Factor; Transcription Regulation; mRNA Decay.

FIGURE 1.

Rpb4/7 is required for Ccr4-Not to rescue arrested RNAPII. A, purification of intact (WT) and Rpb4/7-less RNA polymerase (Rpb4/7Δ). Gels were stained with Coomassie Blue. Rpb8 migrates differently in the last lane because of the calmodulin binding peptide (CBP) tag. M, molecular size markers. B, schematic diagram of elongation complex formation and transcription run-off assays. C, representative transcription run-off gels using intact (12-subunit) and Rpb4/7Δ RNAPII. Reactions were stopped at 1, 2, 4, and 8 min after the addition of nucleotides. Where indicated, 1.5 μg of Ccr4-Not was added to the reactions. D, averages of the quantification of three separate experiments. Error bars represent S.D. Intact RNAPII (black lines) or Rpb4/7Δ RNAPII (gray lines) reactions were incubated with Ccr4-Not (C/N), (solid lines), or an equivalent amount of bovine serum albumin carrier protein (dashed lines). The percentage of run off of ECs was plotted over time.

FIGURE 2.

Rpb9 is not required for Ccr4-Not to stimulate elongation. A, RNAPII was isolated from wild type cells (WT) and from an rpb9Δ mutant (Rpb9Δ). Gels were stained by Coomassie Blue. Rpb9 and Rpb11 could not be resolved in the gels and co-migrate as a doublet. A reduction in the lower band is observed in preparations of Rpb9Δ polymerase, indicating an absence of Rpb9. M, molecular size markers. B, a representative run-off gel using Rpb9-less polymerase. Assays were performed as described for Fig. 1C. C, as described for Fig. 1D, except that Rpb9Δ polymerase was used. Transcription reactions with carrier protein (gray dashed lines) or Ccr4-Not (C/N), (black solid lines) is shown. Error bars represent S.D.

FIGURE 3.

Rpb4/7 is required for the stable association of Ccr4-Not with RNAPII elongation complexes. A, schematic of EC formation and binding assay. Approximately 250 fmol of intact or mutant RNAPII (Rpb4/7Δ and Rpb9Δ) was used to form ECs on 100 ng of template (EC70). ECs were incubated for 10 min with 0.5 and 1.5 μg of Ccr4-Not complex or carrier protein (RNAPII-only lanes). nt, nucleotides. B, products were separated on native polyacrylamide gels and visualized by the incorporation of radiolabeled UTP into the nascent transcript (see “Experimental Procedures”). C, schematic of the pulldown assay to measure the binding between recombinant Rpb4/7 and Ccr4-Not. Rpb4/7 were co-expressed using a bicistronic expression system, and Rpb4 was purified separately. Proteins (∼5 μg) were immobilized on Ni-NTA-agarose by a His₆ tag incorporated into the Rpb4 subunit. Beads were incubated for 1 h with TAP-purified Ccr4-Not containing Myc-tagged Caf1. D, the fractions were analyzed by Western blotting using the 9E10 antibody. Naked beads were used as a control. 200 ng of purified Ccr4-Not is shown in the input lane. Bands were detected using a Cy-5-conjugated secondary antibody. Half of the total of each fraction was loaded onto the gel.