Abstract
BackgroundCcr4-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 FindingsIn 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/SignificanceWe 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.
Highlights
In eukaryotic cells RNAs are synthesized by 3 different RNA polymerases and they are extensively processed to reach their mature forms
In this study we demonstrate genetic and biochemical interactions between the Ccr4-Not complex and the nuclear RNA degradation machinery provided by TRAMP and the nuclear exosome
To determine whether the synthetic growth phenotypes might correlate with synthetic defects in expression of nuclear snoRNAs, we studied the expression of one snoRNA identified in our microarrays, namely U14, in single or double mutant cells
Summary
In eukaryotic cells RNAs are synthesized by 3 different RNA polymerases and they are extensively processed to reach their mature forms. The eukaryotic exosome is made up of 9 core subunits, with 6 subunits carrying the fold of bacterial RNase PH, a phosphorolytic RNase, namely Rrp, Rrp, Rrp, Rrp, Rrp and Mtr, and 3 putative RNA-binding proteins, Csl, Rrp and Rrp40 [2] It is constitutively associated with a processive hydrolytic exoribonuclease, Rrp, which is the only active nuclease of the yeast core exosome [3]. Ccr4-Not is a highly conserved multi-protein complex consisting in yeast of 9 subunits, including Not 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. There has been no evidence so far that this complex is important for RNA degradation in the nucleus
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