31 research outputs found
Not4 and Not5 modulate translation elongation by Rps7A ubiquitination, Rli1 moonlighting, and condensates that exclude eIF5A
The CCR4-NOT Complex Physically and Functionally Interacts with TRAMP and the Nuclear Exosome
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
The role of the E3 ligase Not4 in cotranslational quality control
Cotranslational quality control (QC) is the mechanism by which the cell checks the integrity of newly synthesized proteins and mRNAs. In the event of mistakes these molecules are degraded. The Ccr4-Not complex has been proposed to play a role in this process. It contains both deadenylation and ubiquitination activities, thus it may target both aberrant proteins and mRNAs. Deadenylation is the first step in mRNA degradation. In yeast it is performed by the Ccr4 subunit of the Ccr4-Not complex. Another complex subunit, namely Not4, is a RING E3 ligase and it provides the ubiquitination activity of the complex. It was found associated with translating ribosomes. Thus, it has been suggested that Not4 is involved in ribosome-associated ubiquitination and degradation of aberrant peptides. However, several other E3 ligases have been associated with peptide ubiquitination on the ribosome and the relevance of Not4 in this process remains unclear. In this review we summarize the recent data and suggest a role for Not4 in cotranslational protein QC
Ribosome Association and Stability of the Nascent Polypeptide-Associated Complex Is Dependent Upon Its Own Ubiquitination
In this work we addressed the role of ubiquitination in the function of the nascent polypeptide-associated complex (NAC), named EGD in the yeast Saccharomyces cerevisiae. To this end, we first identified the lysines residues required for ubiquitination of EGD/NAC. While simultaneous mutation of many lysines in the α-subunit of NAC (Egd2p) was required to abolish its ubiquitination, for the β-subunit of NAC (Egd1p), mutation of K29 and K30 was sufficient. We determined that the ubiquitination of the two EGD subunits was coordinated, occurring during growth first on Egd1p and then on Egd2p. Egd2p was ubiquitinated earlier during growth if Egd1p could not be ubiquitinated. The use of mutants revealed the importance of EGD ubiqutination for its ribosome association and stability. Finally, our study demonstrated an interaction of EGD/NAC with the proteasome and revealed the importance of the Not4p E3 ligase, responsible for EGD/NAC ubiquitination, in this association
The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control
<div><p>Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome.</p></div
Proteasome was defective in <i>not4Δ</i> cells but not in <i>ccr4Δ</i> cells.
<p><b>A.</b> Total cellular extracts were prepared from wild-type, <i>caf1Δ</i>, <i>ccr4Δ</i>, and <i>not4Δ</i> cells and loaded on 3.5% native gels. After electrophoresis gels were incubated with Suc-LLVY-AMC to analyze the proteasome activity in the absence (-SDS) and then in the presence (+SDS) of 0.02% SDS to detect the latent CP activity. The positions of double (RP<sub>2</sub>-CP) and single (RP<sub>1</sub>-CP) capped proteasomes and CP alone are indicated on the left. <b>B.</b> RPs were purified from wild-type, <i>caf1Δ</i>, <i>ccr4Δ</i>, and <i>not4Δ</i> cells, loaded on a gradient 3–12% native gel and then analyzed for activity (upper panel). The same purified material was analyzed by SDS-PAGE and western blot with antibodies against the RP subunit (Rpt1) and with antibodies against CP subunits (α1-7) (lower panel).</p
Not4 accumulates in polysomes in response to AZC and high temperature.
<p><b>A.</b> Polysome profiles from the cells expressing Not4-ProteinA. Cells were exponentially grown on YPD media at 30°C or 37°C, as indicated, and collected at OD<sub>600</sub> of 1.0. When indicated, cells were treated with 0.4 mg/ml of AZC. AZC was added at OD<sub>600</sub> of 0.15 and cells were grown till OD<sub>600</sub> of 1.0 and collected. Extracts, containing 3 mg of total proteins, were subjected to 7–47% sucrose gradient centrifugation and analyzed by UV reading at 254 nm. Fraction numbers and the positions of 40S, 60S, 80S, and polysomes are indicated. <b>B.</b> Fractions were collected and analyzed by western blot with PAP and Rpl35 antibodies. <b>C.</b> Not4 content in polysomes was quantified. For this the Not4 signal in polysomes (fraction 10) was quantified with ImageQuant TL software (GE Healthcare) and normalized on the Rpl35 signal.</p
Deletions of the E3 ligase Not4, and the deadenylase subunits Ccr4 and Caf1, have different phenotypes.
<p>The indicated strains were grown to exponential phase and diluted to the same OD<sub>600</sub> of 0.5. 10-fold serial dilutions were spotted on the YPD plates containing, when indicated, HygB 0.1 mg/ml; CHX 0.05 µg/ml; AZC 0.5 mg/ml, and left to grow for 4 days (A, except 16°C), for 17 days (A, 16°C) or for 6 days (B).</p