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

Probing genuine strong interactions and post-translational modifications in the heterogeneous yeast exosome protein complex.

The characterization of heterogeneous multicomponent protein complexes, which goes beyond identification of protein subunits, is a challenging task. Here we describe and apply a comprehensive method that combines a mild affinity purification procedure with a multiplexed mass spectrometry approach for the in-depth characterization of the exosome complex from Saccharomyces cerevisiae expressed at physiologically relevant levels. The exosome is an ensemble of primarily 3' --> 5' exoribonucleases and plays a major role in RNA metabolism. The complex has been reported to consist of 11 proteins in molecular mass ranging from 20 to 120 kDa. By using native macromolecular mass spectrometry we measured accurate masses (around 400 kDa) of several (sub)exosome complexes. Combination of these data with proteolytic peptide LC tandem mass spectrometry using a linear ion trap coupled to a FT-ICR mass spectrometer and intact protein LC mass spectrometry provided us with the identity of the different exosome components and (sub)complexes, including the subunit stoichiometry. We hypothesize that the observed complexes provide information about strongly and weakly interacting exosome-associated proteins. In our analysis we also identified for the first time phosphorylation sites in seven different exosome subunits. The phosphorylation site in the Rrp4 subunit is fully conserved in the human homologue of Rrp4, which is the only previously reported phosphorylation site in any of the human exosome proteins. The described multiplexed mass spectrometry-based procedure is generic and thus applicable to many different types of cellular molecular machineries even if they are expressed at endogenous levels

Characterization of the fast and promiscuous macrocyclase from plant PCY1 enables the use of simple substrates

Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization, but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and nonamino acids, highlighting PCY1's potential in biocatalysis

The proteome signatures of fibroblasts from patients with severe, intermediate and mild spinal muscular atrophy show limited overlap

Most research to characterise the molecular consequences of spinal muscular atrophy (SMA) have focused on SMA I. Here, proteomic profiling of skin fibroblasts from severe (SMA I), intermediate (SMA II), and mild (SMA III) patients, alongside age-matched controls was conducted using SWATH mass spectrometry analysis. Differentially expressed proteome profiles showed limited overlap across each SMA type, and variability was greatest within SMA II fibroblasts which was not explained by SMN2 copy number. Despite limited proteomic overlap, enriched canonical pathways common to two of three SMA severities with at least one differentially expressed pro-tein from the third included mTOR signaling, regulation of eIF2 and eIF4 signaling, and protein ubiquitination. Network expression clustering analysis identified protein profiles that may dis-criminate or correlate with SMA severity. From these clusters, the differential expression of PYGB (SMA I), RAB3B (SMA II), and IMP1 and STAT1 (SMA III) was verified by western blot. All SMA fibroblasts were transfected with an SMN-enhanced construct but only RAB3B expression in SMA II fibroblasts demonstrated an SMN-dependent response. The diverse proteome profiles and pathways identified here pave the way for studies to determine their utility as biomarkers for patient stratification or monitoring treatment efficacy, and for identification of severity-specific treatments

Multivalent peptide and protein dendrimers using native chemical ligation

A wide variety of well-defined multivalent peptides and proteins can be made by conjugating synthetic peptides and recombinantly expressed proteins to cysteine-functionalized dendrimers using native chemical ligation (see picture). This modular approach provides access to dendrimers that are attractive both for understanding fundamental issues of multivalency in biological interactions as well as for biomedical applications

Purification and Reconstitution of the S. cerevisiae TRAMP and Ski Complexes for Biochemical and Structural Studies

The RNA exosome is a macromolecular machine that degrades a large variety of RNAs from their 3'-end. It comprises the major 3'-to-5' exonuclease in the cell, completely degrades erroneous and overly abundant RNAs, and is also involved in the precise processing of RNAs. To degrade transcripts both specifically and efficiently the exosome functions together with compartment-specific cofactors. In the yeast S. cerevisiae, the exosome associates with the Ski complex in the cytoplasm and with Mtr4 alone or with Mtr4 as part of the TRAMP complex in the nucleus. Here we describe how to produce, purify, and assemble the Ski and TRAMP complexes from S. cerevisiae

A strategy for dissecting the architectures of native macromolecular assemblies

Despite the central role of large multi-protein complexes in many biological processes, it remains challenging to elucidate their structures and particularly problematic to define the structures of native macromolecular assemblies, which are often of low abundance. Here, we present a strategy for isolating such complexes and for extracting distance restraints that allow the determination of their molecular architectures. The method was optimized to allow facile use of the extensive global resources of GFP-tagged transgenic cells and animals