Article pubs.acs.org/jpr
Proteomic Analysis of Yeast Mutant RNA Exosome Complexes Rogério F. Lourenço,† Adriana F. P. Leme,‡ and Carla C. Oliveira†,* †
Department of Biochemistry, Chemistry Institute, University of São Paulo, Av. Prof. Lineu Prestes 748, 05508-000 São Paulo, Brazil Mass Spectrometry Laboratory, Brazilian Biosciences National Laboratory- CNPEM, R. Giuseppe Máximo Scolfaro 10000, 13083-970 Campinas, Brazil
‡
S Supporting Information *
ABSTRACT: The yeast exosome is a conserved multiprotein complex essential for RNA processing and degradation. The complex is formed by a nine-subunit core that associates with two hydrolytic 3′-5′ exoribonucleases. Although catalytically inert, the assembly of this nine-subunit core seems to be essential for the exosome activity, as mutations in regions that do not directly bind RNA or are not in the active sites of the exonucleases impair the function of the complex. Previously isolated mutations in the exosome core subunit Rrp43p have been shown to negatively affect the function of the complex. With the aim of investigating the effect of these mutations on the complex stability and activity, Rrp43p and its mutant forms were purified by means of the TAP method. Mass spectrometry analyses showed that lower amounts of the exosome subunits are copurified with the mutant Rrp43p proteins. Additionally, by decreasing the stability of the exosome, other nonspecific protein interactions are favored (the data have been deposited to the ProteomeXchange with identifier PXD000580). Exosome copurified with mutant Rrp43p exhibited increased exonuclease activity, suggesting higher dissociation constants for these mutant complexes. Therefore, data reported here indicate that complexes containing a mutant Rrp43p exhibit decreased stability and provide information on additional protein interactions. KEYWORDS: protein-protein interaction, yeast exosome, RNA processing, Rrp43
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INTRODUCTION The RNA exosome complex is formed by eleven protein subunits, involved in processing and degradation of all types of RNAs in eukaryotic cells, both in the nucleus and in the cytoplasm. The composition of the yeast exosome has been determined by the purification of the protein complex and functional studies showing that all the components were involved in the same pathways.1,2 In the cytoplasm, the exosome is composed of ten subunits (Rrp4p, Rrp40p, Csl4p, Rrp41p, Rrp42p, Rrp43p, Rrp44p, Rrp45p, Rrp46p, Mtr3p), whereas, in the nucleus, the exosome has an additional subunit, Rrp6p.2−4 The core of the exosome is formed by six subunits containing inactive RNase PH domains (Rrp41p, Rrp42p, Rrp43p, Rrp45p, Rrp46p, Mtr3p) and three subunits containing RNA binding domains (Rrp4p, Rrp40p, Csl4p).5 These nine subunits are considered important for controlling the catalytic activities of the hydrolytic RNases Rrp44p6,7 and Rrp6p.8 The exosome is evolutionarily conserved, and the structure of the complex was first obtained for the archaeal counterpart, including the determination of the amino acid residues involved in the interaction with the substrate and in the catalysis.9−12 The structures of the eukaryotic complexes were subsequently determined, first of the human complex, and after that, the © 2013 American Chemical Society
subunit organization and structure of the yeast exosome was analyzed by mass spectrometry, electron microscopy, and crystallography.7,13−15 In addition to the interactions within the complex, the exosome subunits interact with other cellular factors, adding to the complexity of the protein conformations during the control of the activity of the exosome in the different RNA processing pathways in which it participates. One of the protein complexes interacting with the exosome is the Ski complex, which activates the exosome during mRNA degradation.16 In the nucleus, the exosome has been shown to interact with the TRAMP complex, which is responsible for the addition of short poly-A tails to RNAs that are subsequently degraded by the exosome, promoting therefore the exosome activity.17 In this cell compartment, the exosome also interacts with Rrp47p, a cofactor required for the processing of stable RNAs.18 Two other nucleolar proteins have been shown to interact with the exosome and affect pre-rRNA processing. Nop53p interacts with Rrp6p and stimulates the activity of the exosome during maturation of 5.8S rRNA.19 Nop8p has also been shown to interact with Rrp6p, but instead of activating it, Nop8p shows a Received: September 24, 2013 Published: November 18, 2013 5912
dx.doi.org/10.1021/pr400972x | J. Proteome Res. 2013, 12, 5912−5922
Journal of Proteome Research
Article
8.0, 150 mM NaCl, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride (PMSF), and disrupted using a ball mill device (Mill MM 200, Retsch). Extract was prepared by centrifugation at 45,000 rpm for 1 h at 4 °C. The supernatant was incubated for 2 h at 4 °C with IgG-Sepharose beads (GE Healthcare, Little Chalfont, U.K.), followed by extensive washing with buffer containing 50 mM Tris pH 8.0, 500 mM NaCl, and 10% glycerol. Proteins were eluted from beads by incubating the resin with 20 U of Tobacco Etch Virus protease (Invitrogen, Carlsbad, CA, USA) for 16 h at 4 °C.
negative effect upon the exosome activity during pre-rRNA processing.20 Despite all the functional and structural information on the yeast RNA exosome already available, details on how mutations in specific subunits affect the function of the complex are still lacking. We have previously isolated and functionally characterized three mutants of the RNase PH domaincontaining subunit Rrp43p.21 Those Rrp43p mutants cause a temperature sensitive phenotype, which suggested that the point mutations could interfere with the stability of the exosome complex. Rrp43p is a 394 amino acids long protein, and the amino acid substitutions in the three isolated mutants are located in positions 162, 212 (rrp43-1, Ser162Pro, Val212Ala), 230, 274, 276 (rrp43-2, Cys230Tyr, Ile274Thr, Cys276Tyr), and 78, 133, 162, 246 (rrp43-3, Asn78Asp, Val133Met, Ser162Phe, Ala246Thr). As demonstrated previously,21 yeast cells expressing either of these Rrp43p mutants show pre-rRNA processing defects, as well as defective degradation of mRNAs, suggesting that both nuclear and cytoplasmic exosomes are not fully functional. These observations could suggest that the interaction between the exosome core and the active subunits Rrp44p and Rrp6p may be less stable in these mutant strains. Based on these considerations, in this work, we investigated the possible effects of mutations in Rrp43p on the exosome stability and the activity of the complex. We report here that those mutations in Rrp43p reduce the stability of the complex, shedding light on the functional effects of amino acid changes in this particular exosome subunit.
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Mass Spectrometry and Data Analysis
For identification of proteins, 5 μg of total protein obtained from wild type cells was resolved in SDS-PAGE and gels were stained with silver nitrate. Bands of high intensity were removed by cutting gels in pieces of around 1 mm, reduced, alkylated, and digested with trypsin.23 The digestion was also performed in solution, in which the protein amounts from the different samples were set to have equivalent levels of either wild type or mutant Rrp43p (samples obtained from the wild type cells cultured at 25 °C had about 1 μg of proteins). To remove the glycerol, the protein mixtures were precipitated with acetone and resuspended in 50 mM Tris-HCl pH 8.0 and 150 mM NaCl, before following the in solution digestion protocol. Three samples, each purified from an independent culture, were used for each condition analyzed by the in solution digestion approach. The resulting peptides (4.5 μL) were separated by C18 (100 μm × 100 mm) RP-nanoUPLC (nanoAcquity, Waters) coupled with a Q-Tof PREMIER mass spectrometer (Waters) with nanoelectrospray source at a flow rate of 0.6 μL/min. The gradient was 2−90% acetonitrile in 0.1% formic acid over 45 min. The nanoelectrospray voltage was set to 3.5 kV, the cone voltage was 50 V, and the source temperature was 80 °C. The instrument was operated in the “top three” mode, in which one MS spectrum is acquired followed by MS/MS of the top three most-intense peaks detected. After MS/MS fragmentation, the ion was placed on the exclusion list for 60 s. The spectra were acquired using software MassLynx v.4.1, and the raw data files were converted to a peak list format (mgf) by the software Mascot Distiller v.2.3.2.0, 2009 (Matrix Science Ldt.) and searched against the yeast database (6702 sequences; 3018299 residues; release data April, 2013) using Mascot engine v.2.3.01 (Matrix Science Ltd.), with carbamidomethylation as fixed modifications, oxidation of methionine as variable modification, one trypsin missed cleavage, and a tolerance of 0.1 Da for both precursor and fragment ions. Peptides were considered unique when they differed in at least 1 amino acid residue. Among these, covalently modified peptides, including N- or C-terminal elongation (i.e., missed cleavages), were counted as unique, but different charge states of the same peptide and modifications were not considered as a criterion to differentiate peptides. Unique peptides with a minimum of five amino acid residues and displaying a significant threshold (p < 0.05) in the Mascot-based score were considered in the results. Data obtained using Mascot are shown in Table S1 of the Supporting Information. For protein quantitation, the .dat files from the Mascot output were analyzed in Scaffold Q+ (version 3.4.5, Proteome Software) and the quantitative value (normalized spectral counts) was obtained. Proteins differentially copurified from the mutant strains were identified by applying the Fischer’s exact test, and p values lower than 0.05 were assumed as
EXPERIMENTAL PROCEDURES
Yeast Strains and Growth Conditions
Saccharomyces cerevisiae strains were cultured in synthetic medium (YNB) supplemented with 2% glucose and appropriated nitrogen bases and amino acids. Cells were either continuously kept at 25 °C or shifted to 37 °C after a previous growth at 25 °C and cultured for additional 6 h. For all experiments, cells were harvest from cultures at exponential growth phase (optical density at 600 nm around 1.0). Construction of Strain Expression TAP-Tagged Proteins
RRP43 mutants were previously obtained by random mutagenic PCR, followed by in vivo recombination, and screening for a temperature sensitive phenotype.21 To individually express TAP-tagged proteins in yeast cells, a fragment corresponding to the TAP tag was amplified using the primer set Rrp43TAPfor (5′-CGATTTGTCAACAGGATCCATGGAA AAGAAGAGAAGATGC-3′) and Rrp43TAPrev (5′-ACGCCCTCGACCAAGCTTCAGGTTGACTT-3′), digested with BamHI and SalI and cloned into pMET25.22 Genes RRP43, rrp43-1, rrp43-2, and rrp43-3 were isolated from the corresponding pC-FUS vectors21 with BamHI and HincII and cloned into pMET25 upstream from the TAP sequence (Goldfeder and Oliveira, unpublished results). The resulting constructs were transferred to the YCO43 strain.21 CWC24 ORF was cloned into pMET25-TAP after digestion with BamHI-NcoI, for excision of the RRP43 ORF (Perona and Oliveira, unpublished results). Co-immunoprecipitation by the TAP-Tag Strategy
Co-purification of proteins by means of Rrp43-TAP (wild type and mutant forms of the protein) or Cwc24-TAP was performed as previously described.6,7 Briefly, yeast cells were harvest by centrifugation, resuspended in 50 mM Tris-HCl pH 5913
dx.doi.org/10.1021/pr400972x | J. Proteome Res. 2013, 12, 5912−5922
Journal of Proteome Research
Article
Table 1. Proteins Differentially Copurified with Mutant Versions of Rrp43-TAP rrp43-1 × RRP43
spectral countc identification
a
function
b
RRP43
rrp43-1
rrp43-3
fold change
d
rrp43-3 × RRP43 e
Fisher’s exact test
fold changed
Fisher’s exact teste
0.267 0.361 0.143 0.452 0.083 0.111
0.011
