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Discovering the gene expression signature associated with a cellular state is one of the basic quests in majority of biological studies. For most of the clinical and cellular manifestations, these molecular differences may be exhibited across multiple layers of gene regulation like genomic variations, gene expression, protein translation and post-translational modifications. These system wide variations are dynamic in nature and their crosstalk is overwhelmingly complex, thus analyzing them separately may not be very informative. This necessitates the integrative analysis of such multiple layers of information to understand the interplay of the individual components of the biological system. Recent developments in high throughput RNA sequencing and mass spectrometric (MS) technologies to probe transcripts and proteins made these as preferred methods for understanding global gene regulation. Subsequently, improvements in "big-data" analysis techniques enable novel conclusions to be drawn from integrative transcriptomic-proteomic analysis. The unified analyses of both these data types have been rewarding for several biological objectives like improving genome annotation, predicting RNA-protein quantities, deciphering gene regulations, discovering disease markers and drug targets. There are different ways in which transcriptomics and proteomics data can be integrated; each aiming for different research objectives. Here, we review various studies, approaches and computational tools targeted for integrative analysis of these two high-throughput omics methods.
© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.
The wheel-lock (WL) model for depressed ambulatory activity in rats has shown metabolic maladies ensuing within 53-173 h after WL begins. We sought to determine if WL beginning after 21-23 days of voluntary running in growing female Wistar rats affected the mRNA profile in the polyribosomal fraction from plantaris muscle shortly following WL. In experiment 1, WL occurred at 0200 and muscles were harvested at 0700 daily at 5 h (WL5h, n = 4), 29 h (WL29h, n = 4), or 53 h (WL53h, n = 4) after WL. Affymetrix Rat Gene 1.0 ST Arrays were used to test the initial question as to whether WL affects mRNA occupancy on skeletal muscle polyribosomes. Using a false discovery rate of 15%, no changes in mRNAs in the polyribosomal fraction were observed at WL29h and eight mRNAs (of over 8,200 identified targets) were altered at WL53h compared with WL5h. Interestingly, two of the six downregulated genes included ankyrin repeat domain 2 (Ankrd2) and cysteine-rich protein 3/muscle LIM protein (Csrp3), both of which encode mechanical stretch sensors and RT-PCR verified their WL-induced decline. In experiment 2, whole muscle mRNA and protein levels were analyzed for Ankrd2 and Csrp3 from the muscles of WL5h (4 original samples + 2 new), WL29h (4 original), WL53h (4 original + 2 new), as well as WL173 h (n = 6 new) and animals that never ran (SED, 4-5 new). Relative to WL5h controls, whole tissue Ankrd2 and Csrp3 mRNAs were lower (P < 0.05) at WL53h, WL173h, and SED; Ankrd2 protein tended to decrease at WL53h (P = 0.054) and Csrp3 protein was less in WL173h and SED rats (P < 0.05). In summary, unique early declines in Ankrd2 and Csrp3 mRNAs were identified with removal of voluntary running, which was subsequently followed by declines in Csrp3 protein levels during longer periods of wheel lock.
Aspergillus fumigatus is the most common and deadly pulmonary fungal infection worldwide. In the lung, the fungus usually forms a dense colony of filaments embedded in a polymeric extracellular matrix. To identify candidate genes involved in this biofilm (BF) growth, we used RNA-Seq to compare the transcriptomes of BF and liquid plankton (PL) growth. Sequencing and mapping of tens of millions sequence reads against the A. fumigatus transcriptome identified 3,728 differentially regulated genes in the two conditions. Although many of these genes, including the ones coding for transcription factors, stress response, the ribosome, and the translation machinery, likely reflect the different growth demands in the two conditions, our experiment also identified hundreds of candidate genes for the observed differences in morphology and pathobiology between BF and PL. We found an overrepresentation of upregulated genes in transport, secondary metabolism, and cell wall and surface functions. Furthermore, upregulated genes showed significant spatial structure across the A. fumigatus genome; they were more likely to occur in subtelomeric regions and colocalized in 27 genomic neighborhoods, many of which overlapped with known or candidate secondary metabolism gene clusters. We also identified 1,164 genes that were downregulated. This gene set was not spatially structured across the genome and was overrepresented in genes participating in primary metabolic functions, including carbon and amino acid metabolism. These results add valuable insight into the genetics of biofilm formation in A. fumigatus and other filamentous fungi and identify many relevant, in the context of biofilm biology, candidate genes for downstream functional experiments.
The myotonic dystrophy type 2 protein ZNF9/CNBP is a small nucleic acid binding protein proposed to act as a regulator of transcription and translation. The precise functions and activity of this protein are poorly understood. Previous studies suggested that ZNF9 regulates translation and facilitates the process of cap-independent translation through interactions with mRNA and the translating ribosome. To help determine the role played by ZNF9 in the activation of translation initiation, we combined genetic and biochemical analysis of the putative ZNF9 ortholog GIS2, in the budding yeast Saccharomyces cerevisiae. Purification of the Gis2p protein followed by mass spectrometry based-proteomic analysis identified a large number of co-purifying ribosomal subunits and translation factors, strongly suggesting that Gis2p interacts with the protein translation machinery. Polysome profiling and ribosome isolation experiments confirm that Gis2p physically interacts with the translating ribosome. Interestingly, expression of yeast Gis2p in HEK293T cells activates cap-independent translation driven by the 5'UTR of the ODC gene. These data suggest that Gis2 is functionally orthologous to ZNF9 and acts as a cap-independent translation factor.
Copyright © 2011 Elsevier Inc. All rights reserved.
Myotonic dystrophy types 1 and 2 (DM1 and DM2) are forms of muscular dystrophy that share similar clinical and molecular manifestations, such as myotonia, muscle weakness, cardiac anomalies, cataracts, and the presence of defined RNA-containing foci in muscle nuclei. DM2 is caused by an expansion of the tetranucleotide CCTG repeat within the first intron of ZNF9, although the mechanism by which the expanded nucleotide repeat causes the debilitating symptoms of DM2 is unclear. Conflicting studies have led to two models for the mechanisms leading to the problems associated with DM2. First, a gain-of-function disease model hypothesizes that the repeat expansions in the transcribed RNA do not directly affect ZNF9 function. Instead repeat-containing RNAs are thought to sequester proteins in the nucleus, causing misregulation of normal cellular processes. In the alternative model, the repeat expansions impair ZNF9 function and lead to a decrease in the level of translation. Here we examine the normal in vivo function of ZNF9. We report that ZNF9 associates with actively translating ribosomes and functions as an activator of cap-independent translation of the human ODC mRNA. This activity is mediated by direct binding of ZNF9 to the internal ribosome entry site sequence (IRES) within the 5'UTR of ODC mRNA. ZNF9 can activate IRES-mediated translation of ODC within primary human myoblasts, and this activity is reduced in myoblasts derived from a DM2 patient. These data identify ZNF9 as a regulator of cap-independent translation and indicate that ZNF9 activity may contribute mechanistically to the myotonic dystrophy type 2 phenotype.
The 5'-untranslated region of the ornithine decarboxylase (ODC) mRNA contains an internal ribosomal entry site (IRES). Mutational analysis of the ODC IRES has led to the identification of sequences necessary for cap-independent translation of the ODC mRNA. To discover novel IRES trans-acting factors (ITAFs), we performed a proteomics screen for proteins that regulate ODC translation using the wild-type ODC mRNA and a mutant version with an inactive IRES. We identified two RNA-binding proteins that associate with the wild-type ODC IRES but not the mutant IRES. One of these RNA-binding proteins, PCBP2, is an established activator of viral and cellular IRESs. The second protein, ZNF9 (myotonic dystrophy type 2 protein), has not been shown previously to bind IRES-like elements. Using a series of biochemical assays, we validated the interaction of these proteins with ODC mRNA. Interestingly ZNF9 and PCBP2 biochemically associated with each other and appeared to function as part of a larger holo-ITAF ribonucleoprotein complex. Our functional studies showed that PCBP2 and ZNF9 stimulate translation of the ODC IRES. Importantly these results may provide insight into the normal role of ZNF9 and why ZNF9 mutations cause myotonic dystrophy.
Translation regulation is a critical means by which cells control growth, division, and apoptosis. To gain further insight into translation and related processes, we performed multifaceted mass spectrometry-based proteomic screens of yeast ribosomal complexes and discovered an association of 77 uncharacterized yeast proteins with ribosomes. Immunoblotting revealed an EDTA-dependent cosedimentation with ribosomes in sucrose gradients for 11 candidate translation-machinery-associated (TMA) proteins. Tandem affinity purification linked one candidate, LSM12, to the RNA processing proteins PBP1 and PBP4. A second candidate, TMA46, interacted with RBG1, a GTPase that interacts with ribosomes. By adapting translation assays to high-throughput screening methods, we showed that null yeast strains harboring deletions for several of the TMA genes had alterations in protein synthesis rates (TMA7 and TMA19), susceptibility to drugs that inhibit translation (TMA7), translation fidelity (TMA20), and polyribosome profiles (TMA7, TMA19, and TMA20). TMA20 has significant sequence homology with the oncogene MCT-1. Expression of human MCT-1 in the Deltatma20 yeast mutant complemented translation-related defects, strongly implying that MCT-1 functions in translation-related processes. Together these findings implicate the TMA proteins and, potentially, their human homologs, in translation related processes.
Mass spectrometry-based identification of the components of multiprotein complexes often involves solution-phase proteolytic digestion of the complex. The affinity purification of individual protein complexes often yields nanogram to low-microgram amounts of protein, which poses several challenges for enzymatic digestion and protein identification. We tested different solvent systems to optimize trypsin digestions of samples containing limited amounts of protein for subsequent analysis by LC-MS-MS. Data collected from digestion of 10-, 2-, 1-, and 0.2-microg portions of a protein standard mixture indicated that an organic-aqueous solvent system containing 80% acetonitrile consistently provided the most complete digestion, producing more peptide identifications than the other solvent systems tested. For example, a 1-h digestion in 80% acetonitrile yielded over 52% more peptides than the overnight digestion of 1 microg of a protein mixture in purely aqueous buffer. This trend was also observed for peptides from digested ribosomal proteins isolated from Rhodopseudomonas palustris. In addition to improved digestion efficiency, the shorter digestion times possible with the organic solvent also improved trypsin specificity, resulting in smaller numbers of semitryptic peptides than an overnight digestion protocol using an aqueous solvent. The technique was also demonstrated for an affinity-isolated protein complex, GroEL. To our knowledge, this report is the first using mass spectrometry data to show a linkage between digestion solvent and trypsin specificity.
Algorithmic search engines bridge the gap between large tandem mass spectrometry data sets and the identification of proteins associated with biological samples. Improvements in these tools can greatly enhance biological discovery. We present a new scoring scheme for comparing tandem mass spectra with a protein sequence database. The MASPIC (Multinomial Algorithm for Spectral Profile-based Intensity Comparison) scorer converts an experimental tandem mass spectrum into a m/z profile of probability and then scores peak lists from potential candidate peptides using a multinomial distribution model. The MASPIC scoring scheme incorporates intensity, spectral peak density variations, and m/z error distribution associated with peak matches into a multinomial distribution. The scoring scheme was validated on two standard protein mixtures and an additional set of spectra collected on a complex ribosomal protein mixture from Rhodopseudomonas palustris. The results indicate a 5-15% improvement over Sequest for high-confidence identifications. The performance gap grows as sequence database size increases. Additional tests on spectra from proteinase-K digest data showed similar performance improvements demonstrating the advantages in using MASPIC for studying proteins digested with less specific proteases. All these investigations show MASPIC to be a versatile and reliable system for peptide tandem mass spectral identification.