Michaela Rode

Proteome Organization in a Genome-Reduced Bacterium

Simply Mycoplasma The bacterium Mycoplasma pneumoniae, a human pathogen, has a genome of reduced size and is one of the simplest organisms that can reproduce outside of host cells. As such, it represents an excellent model organism in which to attempt a systems-level understanding of its biological organization. Now three papers provide a comprehensive and quantitative analysis of the proteome, the metabolic network, and the transcriptome of M. pneumoniae (see the Perspective by Ochman and Raghavan). Anticipating what might be possible in the future for more complex organisms, Kühner et al. (p. 1235) combine analysis of protein interactions by mass spectrometry with extensive structural information on M. pneumoniae proteins to reveal how proteins work together as molecular machines and map their organization within the cell by electron tomography. The manageable genome size of M. pneumoniae allowed Yus et al. (p. 1263) to map the metabolic network of the organism manually and validate it experimentally. Analysis of the network aided development of a minimal medium in which the bacterium could be cultured. Finally, G‡ell et al. (p. 1268) applied state-of-the-art sequencing techniques to reveal that this “simple” organism makes extensive use of noncoding RNAs and has exon- and intron-like structure within transcriptional operons that allows complex gene regulation resembling that of eukaryotes. The simplified proteome of a bacterium provides insight into the organization of proteins into molecular machines. The genome of Mycoplasma pneumoniae is among the smallest found in self-replicating organisms. To study the basic principles of bacterial proteome organization, we used tandem affinity purification–mass spectrometry (TAP-MS) in a proteome-wide screen. The analysis revealed 62 homomultimeric and 116 heteromultimeric soluble protein complexes, of which the majority are novel. About a third of the heteromultimeric complexes show higher levels of proteome organization, including assembly into larger, multiprotein complex entities, suggesting sequential steps in biological processes, and extensive sharing of components, implying protein multifunctionality. Incorporation of structural models for 484 proteins, single-particle electron microscopy, and cellular electron tomograms provided supporting structural details for this proteome organization. The data set provides a blueprint of the minimal cellular machinery required for life.

Transcriptome Complexity in a Genome-Reduced Bacterium

Simply Mycoplasma The bacterium Mycoplasma pneumoniae, a human pathogen, has a genome of reduced size and is one of the simplest organisms that can reproduce outside of host cells. As such, it represents an excellent model organism in which to attempt a systems-level understanding of its biological organization. Now three papers provide a comprehensive and quantitative analysis of the proteome, the metabolic network, and the transcriptome of M. pneumoniae (see the Perspective by Ochman and Raghavan). Anticipating what might be possible in the future for more complex organisms, Kühner et al. (p. 1235) combine analysis of protein interactions by mass spectrometry with extensive structural information on M. pneumoniae proteins to reveal how proteins work together as molecular machines and map their organization within the cell by electron tomography. The manageable genome size of M. pneumoniae allowed Yus et al. (p. 1263) to map the metabolic network of the organism manually and validate it experimentally. Analysis of the network aided development of a minimal medium in which the bacterium could be cultured. Finally, G‡ell et al. (p. 1268) applied state-of-the-art sequencing techniques to reveal that this “simple” organism makes extensive use of noncoding RNAs and has exon- and intron-like structure within transcriptional operons that allows complex gene regulation resembling that of eukaryotes. Sequencing of a tiny bacterium’s RNA reveals many noncoding RNAs and complex gene regulation reminiscent of eukaryotes. To study basic principles of transcriptome organization in bacteria, we analyzed one of the smallest self-replicating organisms, Mycoplasma pneumoniae. We combined strand-specific tiling arrays, complemented by transcriptome sequencing, with more than 252 spotted arrays. We detected 117 previously undescribed, mostly noncoding transcripts, 89 of them in antisense configuration to known genes. We identified 341 operons, of which 139 are polycistronic; almost half of the latter show decaying expression in a staircase-like manner. Under various conditions, operons could be divided into 447 smaller transcriptional units, resulting in many alternative transcripts. Frequent antisense transcripts, alternative transcripts, and multiple regulators per gene imply a highly dynamic transcriptome, more similar to that of eukaryotes than previously thought.

Impact of Genome Reduction on Bacterial Metabolism and Its Regulation

Simply Mycoplasma The bacterium Mycoplasma pneumoniae, a human pathogen, has a genome of reduced size and is one of the simplest organisms that can reproduce outside of host cells. As such, it represents an excellent model organism in which to attempt a systems-level understanding of its biological organization. Now three papers provide a comprehensive and quantitative analysis of the proteome, the metabolic network, and the transcriptome of M. pneumoniae (see the Perspective by Ochman and Raghavan). Anticipating what might be possible in the future for more complex organisms, Kühner et al. (p. 1235) combine analysis of protein interactions by mass spectrometry with extensive structural information on M. pneumoniae proteins to reveal how proteins work together as molecular machines and map their organization within the cell by electron tomography. The manageable genome size of M. pneumoniae allowed Yus et al. (p. 1263) to map the metabolic network of the organism manually and validate it experimentally. Analysis of the network aided development of a minimal medium in which the bacterium could be cultured. Finally, G‡ell et al. (p. 1268) applied state-of-the-art sequencing techniques to reveal that this “simple” organism makes extensive use of noncoding RNAs and has exon- and intron-like structure within transcriptional operons that allows complex gene regulation resembling that of eukaryotes. Reconstruction of a bacterial metabolic network reveals strategies for metabolic control with a genome of reduced size. To understand basic principles of bacterial metabolism organization and regulation, but also the impact of genome size, we systematically studied one of the smallest bacteria, Mycoplasma pneumoniae. A manually curated metabolic network of 189 reactions catalyzed by 129 enzymes allowed the design of a defined, minimal medium with 19 essential nutrients. More than 1300 growth curves were recorded in the presence of various nutrient concentrations. Measurements of biomass indicators, metabolites, and 13C-glucose experiments provided information on directionality, fluxes, and energetics; integration with transcription profiling enabled the global analysis of metabolic regulation. Compared with more complex bacteria, the M. pneumoniae metabolic network has a more linear topology and contains a higher fraction of multifunctional enzymes; general features such as metabolite concentrations, cellular energetics, adaptability, and global gene expression responses are similar, however.

Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster

In yeast cells, the THO complex has been implicated in mitotic recombination, transcription elongation and mRNA nuclear export. The stable core of THO consists of Tho2p, Hpr1p, Mft1p and Thp2p. Whether a complex with similar functions assembles in metazoa has not yet been established. Here we report that Drosophila melanogaster THO consists of THO2, HPR1 and three proteins, THOC5–THOC7, which have no orthologs in budding yeast. Gene expression profiling in cells depleted of THO components revealed that <20% of the transcriptome was regulated by THO. Nonetheless, export of heat-shock mRNAs under heat stress was strictly dependent on THO function. Notably, 8% of upregulated genes encode proteins involved in DNA repair. Thus, although THO function seems to be conserved, the vast majority of mRNAs are transcribed and exported independently of THO in D. melanogaster.

Overexpression of TAP/p15 Heterodimers Bypasses Nuclear Retention and Stimulates Nuclear mRNA Export

Human TAP and its yeast orthologue Mex67p are members of the multigene family of NXF proteins. A conserved feature of NXFs is a leucine-rich repeat domain (LRR) followed by a region related to the nuclear transport factor 2 (the NTF2-like domain). The NTF2-like domain of metazoan NXFs heterodimerizes with a protein known as p15 or NXT. A C-terminal region related to ubiquitin-associated domains (the UBA-like domain) is present in most, but not all NXF proteins.Saccharomyces cerevisiae Mex67p and Caenorhabditis elegans NXF1 are essential for the export of messenger RNA from the nucleus. Human TAP mediates the export of simian type D retroviral RNAs bearing the constitutive transport element, but the precise role of TAP and p15 in mRNA nuclear export has not yet been established. Here we show that overexpression of TAP/p15 heterodimers bypasses nuclear retention and stimulates the export of mRNAs that are otherwise exported inefficiently. This stimulation of mRNA export is strongly reduced by removing the UBA-like domain of TAP and abolished by deleting the LRR domain or the NTF2-like domain. Similar results are obtained when TAP/p15 heterodimers are directly tethered to the RNA export cargo. Our data indicate that formation of TAP/p15 heterodimers is required for TAP-mediated export of mRNA and show that the LRR domain of TAP plays an essential role in this process.

The Yeast Ski Complex: Crystal Structure and RNA Channeling to the Exosome Complex

The Ski complex is a conserved multiprotein assembly required for the cytoplasmic functions of the exosome, including RNA turnover, surveillance, and interference. Ski2, Ski3, and Ski8 assemble in a tetramer with 1:1:2 stoichiometry. The crystal structure of an S. cerevisiae 370 kDa core complex shows that Ski3 forms an array of 33 TPR motifs organized in N-terminal and C-terminal arms. The C-terminal arm of Ski3 and the two Ski8 subunits position the helicase core of Ski2 centrally within the complex, enhancing RNA binding. The Ski3 N-terminal arm and the Ski2 insertion domain allosterically modulate the ATPase and helicase activities of the complex. Biochemical data suggest that the Ski complex can thread RNAs directly to the exosome, coupling the helicase and the exoribonuclease through a continuous RNA channel. Finally, we identify a Ski8-binding motif common to Ski3 and Spo11, rationalizing the moonlighting properties of Ski8 in mRNA decay and meiosis.

Nuclear Export of mRNA by TAP/NXF1 Requires Two Nucleoporin-Binding Sites but Not p15

ABSTRACT Metazoan NXF1/p15 heterodimers promote export of bulk mRNA through nuclear pore complexes (NPC). NXF1 interacts with the NPC via two distinct structural domains, the UBA-like domain and the NTF2-like scaffold, which results from the heterodimerization of the NTF2-like domain of NXF1 with p15. Both domains feature a single nucleoporin-binding site, and they act synergistically to promote NPC translocation. Whether the NTF2-like scaffold (and thereby p15) contributes only to NXF1/NPC association or is also required for other functions, e.g., to impart directionality to the export process by regulating NXF1/NPC or NXF1/cargo interactions, remains unresolved. Here we show that a minimum of two nucleoporin-binding sites is required for NXF1-mediated export of cellular mRNA. These binding sites can be provided by an NTF2-like scaffold followed by a UBA-like domain (as in the wild-type protein) or by two NTF2-like scaffolds or two UBA-like domains in tandem. In the latter case, the export activity of NXF1 is independent of p15. Thus, as for the UBA-like domain, the function of the NTF2-like scaffold is confined to nucleoporin binding. More importantly, two copies of either of these domains are sufficient to promote directional transport of mRNA cargoes across the NPC.

The crystal structure of S. cerevisiae Ski2, a DExH helicase associated with the cytoplasmic functions of the exosome

Ski2 is a cytoplasmic RNA helicase that functions together with the exosome in the turnover and quality control of mRNAs. Ski2 is conserved in eukaryotes and is related to the helicase Mtr4, a cofactor of the nuclear exosome involved in the processing and quality control of a variety of structured RNAs. We have determined the 2.4 Å resolution crystal structure of the 113 kDa helicase region of Saccharomyces cerevisiae Ski2. The structure shows that Ski2 has an overall architecture similar to that of Mtr4, with a core DExH region and an extended insertion domain. The insertion is not required for the formation of the Ski2-Ski3-Ski8 complex, but is instead an RNA-binding domain. While this is reminiscent of the Mtr4 insertion, there are specific structural and biochemical differences between the two helicases. The insertion of yeast Mtr4 consists of a β-barrel domain that is flexibly attached to a helical stalk, contains a KOW signature motif, and binds in vitro-transcribed tRNA(i)(Met), but not single-stranded RNA. The β-barrel domain of yeast Ski2 does not contain a KOW motif and is tightly packed against the helical stalk, forming a single structural unit maintained by a zinc-binding site. Biochemically, the Ski2 insertion has broad substrate specificity, binding both single-stranded and double-stranded RNAs. We speculate that the Ski2 and Mtr4 insertion domains have evolved with different properties tailored to the type of transcripts that are the substrates of the cytoplasmic and nuclear exosome.

The structure of the Pan2–Pan3 core complex reveals cross-talk between deadenylase and pseudokinase

Pan2–Pan3 is a conserved complex involved in the shortening of mRNA poly(A) tails, the initial step in eukaryotic mRNA turnover. We show that recombinant Saccharomyces cerevisiae Pan2–Pan3 can deadenylate RNAs in vitro without needing the poly(A)-binding protein Pab1. The crystal structure of an active ~200-kDa core complex reveals that Pan2 and Pan3 interact with an unusual 1:2 stoichiometry imparted by the asymmetric nature of the Pan3 homodimer. An extended region of Pan2 wraps around Pan3 and provides a major anchoring point for complex assembly. A Pan2 module formed by the pseudoubiquitin-hydrolase and RNase domains latches onto the Pan3 pseudokinase with intertwined interactions that orient the deadenylase active site toward the A-binding site of the interacting Pan3. The molecular architecture of Pan2–Pan3 suggests how the nuclease and its pseudokinase regulator act in synergy to promote deadenylation.