The CCR4-NOT complex physically and functionally interacts with TRAMP and the nuclear exosome

Abstract

Background: Ccr4-Not is a highly conserved multi-protein complex consisting in yeast of 9 subunits, including Not5 and the major yeast deadenylase Ccr4. It has been connected functionally in the nucleus to transcription by RNA polymerase II and in the cytoplasm to mRNA degradation. However, there has been no evidence so far that this complex is important for RNA degradation in the nucleus.

Methodology/principal findings: In this work we point to a new role for the Ccr4-Not complex in nuclear RNA metabolism. We determine the importance of the Ccr4-Not complex for the levels of non-coding nuclear RNAs, such as mis-processed and polyadenylated snoRNAs, whose turnover depends upon the nuclear exosome and TRAMP. Consistently, mutation of both the Ccr4-Not complex and the nuclear exosome results in synthetic slow growth phenotypes. We demonstrate physical interactions between the Ccr4-Not complex and the exosome. First, Not5 co-purifies with the exosome. Second, several exosome subunits co-purify with the Ccr4-Not complex. Third, the Ccr4-Not complex is important for the integrity of large exosome-containing complexes. Finally, we reveal a connection between the Ccr4-Not complex and TRAMP through the association of the Mtr4 helicase with the Ccr4-Not complex and the importance of specific subunits of Ccr4-Not for the association of Mtr4 with the nuclear exosome subunit Rrp6.

Conclusions/significance: We propose a model in which the Ccr4-Not complex may provide a platform contributing to dynamic interactions between the nuclear exosome and its co-factor TRAMP. Our findings connect for the first time the different players involved in nuclear and cytoplasmic RNA degradation.

Figure 1. Polyadenylated non-coding snRNAs and snoRNAs are over-expressed in specific mutants of the Ccr4-Not complex.

This figure lists all of non-coding snoRNAs and snRNAs identified in our previous micro-array experiments as being differentially expressed in any mutant of the Ccr4-Not complex growing exponentially compared to the wild-type. Black, red and green shows genes that are not affected, over-expressed or under-expressed, respectively. In the last column is indicated which category the gene belongs to either a H/ACA or C/D snoRNA, or a snRNA.

Figure 2. Synthetic growth phenotype when deletions of RRP6 and NOT5 are combined.

The indicated strains were streaked on YPD plates and let to grow for several days at 30°C.

Figure 3. Accumulation of 3′-extended and polyadenylated U14 in rrp6Δ cells is affected by the Ccr4-Not complex.

Total cellular RNA isolated from the indicated strains was analyzed by northern blot first with a probe against 3′-extended U14 (upper panel) and then with a probe for mature U14 (middle panel) and a probe for 5S rRNA (lower panel). The position of several extended U14 forms and mature U14 are indicated on each side of the blot (see the text for details). Hybridization against the 5S mature rRNA is shown as a control for loading.

Figure 4. Large complexes containing Rrp41 and Not5 are disrupted in cells lacking Caf40 or Not4.

A. Equal amounts of total protein extracts (TE) from the indicated strains expressing Tap-tagged Rrp41 were analyzed by western blot for the expression of Rrp41 with antibodies against CBP. Purified materials (E) were analyzed for the presence of Not5 and Rrp41 by western blotting with antibodies against Not5 and CBP as indicated. B and C. Wild-type, caf40Δ or not4Δ cells expressing Tap-tagged Rrp41 were grown for extract preparation. B. 2 g of extract were used to purify Rrp41 and the eluted proteins were loaded on SDS-PAGE. The gel was stained with coomassie. The nuclear exosome subunits and Not5 identified by mass spectrometry (Table S3) are indicated. Molecular weight markers are indicated on the left (in kDa). C. 5 mg of extracts were loaded on a 10–30% glycerol gradient. Proteins in the different fractions of the gradient were analyzed by western blotting for the presence of Tap-tagged Rrp41 with antibodies against CBP, and for Not5 with antibodies against Not5, as indicated.

Figure 5. Not5 co-precipitates with Csl4 and Rrp41.

A. Total protein extracts were prepared from wild-type or caf40Δ cells expressing Tap-tagged Csl4. 2 mg of total protein was incubated with (IP) or without (IP₀) antibodies against CBP. 50 µg of total extract (TE), equivalent volume of unbound extract (FT) and the immunoprecipitate were loaded on SDS-PAGE followed by western blotting with antibodies against Not5, which revealed both Not5 and Tap-tagged Csl4 as indicated. B. 2 g of total protein extract from wild-type cells expressing Tap-tagged Rrp41 treated or not with RNAse as indicated were processed for tandem affinity purification of Rrp41. Equivalent amounts of total extract (TE), flow through (FT) or one fourth of the eluate precipitated by TCA, were loaded on SDS-PAGE and analyzed with antibodies against CBP to follow Rrp41, or against Rrp4, Rrp43 and Not5 as indicated.

Figure 6. Subunits of the Ccr4-Not, exosome and TRAMP complexes co-purify.

2 g of total proteins extracted from (A) wild-type cells expressing Tap-tagged Not5 or (B) wild-type and caf40Δ cells expressing Tap-tagged Not4, Not2, or Caf130 as indicated, were subject to tandem affinity purification. After separation on SDS-PAGE, and coomassie staining, the purified proteins were identified by mass spectrometry analysis (see Table S3). The components of the Ccr4-Not, TRAMP or exosome complexes identified are indicated on the right of the gel lanes by numbers which refer to the following proteins: 1 is Not1, 2 is Caf130, 3 is Not3, 4 is Ccr4, 5 is Not5, 6 is Not4, 7 is Caf1, 8 is Caf40, 9 is Not2, 10 is Mtr4, 11 is Rrp41 and 12 is Rrp42. Molecular weight markers (M) are indicated on the left of the gels (in kDa).

Figure 7. Mtr4 co-immunoprecipitates with subunits of the Ccr4-Not complex independently of RNA.

A. Equivalent amounts of total protein extract (5 mg) treated or not (+ or −) with RNAse A as indicated, prepared from not4Δ cells expressing complementing myc₆-Not4 from an episome or caf40Δ cells expressing complementing HA₇-Caf40 from an episome, were immunoprecipitated with antibodies against Mtr4. Immunoprecipiates were analyzed by western blotting for the presence of Mtr4 and tagged Not4 or Caf40 as indicated. The efficient digestion of RNA in total extracts (TE) was verified on an agarose gel stained with ethidium bromide (see Fig. S7). B. The same experiment as in A was performed except caf40Δ not4Δ cells expressing complementing myc₆-Not4 from an episome or not4Δ caf40Δ cells expressing complementing HA₇-Caf40 were analyzed. C. 2 mg of total protein extracts from wild-type or not4Δ cells expressing Tap-tagged Trf4 were incubated with (IP) or without (IP₀) antibodies against CBP. 50 µg of total extract (TE), equivalent volume of unbound extract (FT) and the immunoprecipitate were analyzed by western blotting with antibodies against Caf40, which revealed both Caf40 and Tap-tagged Trf4 as indicated.

Figure 8. Co-purification of Mtr4 with Rrp6 depends upon the Ccr4-Not complex.

A. Total protein extracts were prepared from wild-type and mutant strains expressing Tap-tagged Rrp6. The same amount of each extract (60 mg) was incubated with IgG beads. After washing, bound proteins were eluted by TEV cleavage and analysed by western blot with anti-CBP antibodies (upper panel) or with polyclonal anti-Mtr4 antibodies (bottom panel). B. Equivalent amounts of total protein extracts from the indicated strains expressing Tap-tagged Mtr4 were analyzed by western blot for the presence of Tap-tagged Mtr4 with antibodies against Mtr4. C. 2 g of total proteins extracted from wild-type or not4Δ cells expressing Tap-tagged Rrp6 were incubated with IgG beads, and bound proteins were eluted with TEV protease. Co-purified proteins were analyzed by western blotting for the presence of Rrp43, Rrp4, or Rrp6 with antibodies against CBP. D. Total protein extracts were prepared from wild-type and caf40Δ cells expressing myc-tagged Rrp6. The same amount of extract was incubated with antibodies against Mtr4 and immunoprecipitates were analyzed by western blotting with antibodies against myc. The same blot was then analyzed with antibodies against Mtr4.

Figure 9. Model for the connections between the Ccr4-Not, TRAMP and exosome complexes.

A large(s) structure(s) containing Ccr4-Not, TRAMP, the nuclear exosome and RNA, that may function to coordinate activity of TRAMP and the nuclear exosome, is suggested by our work. Not1, the scaffold of the Ccr4-Not complex is associated with Not5, Not2, Not4 and Caf40 (shown), as well as Caf1, Ccr4, Caf130, and Not3 (not shown). Mtr4 is a subunit of TRAMP that co-immunoprecipitates with Caf40 and Not4, and is co-purified with Tap-tagged Caf40 together with the Ccr4-Not complex. Its association with Not4 (and other subunits of the Ccr4-Not complex) increases in the absence of Caf40. In contrast, Caf40 is required for the interaction between Mtr4 and Rrp6, suggesting that maybe Caf40 contributes to release Mtr4 (and TRAMP) from the Ccr4-Not complex to the nuclear exosome. Rrp41 co-purifies with Tap-tagged Not5, and Rrp41, Rrp42 and Mtr4 co-purify with Not2-Taptag or Not4-Taptag when Caf40 is deleted, probably because the tagged proteins in association with the exosome and TRAMP become accessible to purification in this mutant. Finally, the nuclear exosome becomes more accessible to purification via Rrp41-Taptag when the integrity of the Ccr4-Not complex is compromised by deleting Caf40 or Not4, or when RNA is destroyed. Not5 co-purifies with Tap-tagged Csl4, and with Tap-tagged Rrp41 when it is more accessible to purification by RNA degradation or deletion of Caf40 or Not4.