Immunopurification of Polyribosomal Complexes of Arabidopsis for Global Analysis of Gene Expression

Zanetti, Margherita; Chang, Ing-Feng; Gong, Fangcheng; Galbraith, David W.; Bailey‐Serres, Julia

doi:10.1104/pp.105.059477

Cited by 226 publications

(251 citation statements)

References 57 publications

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“…It has been reported that the percentage of particular mRNAs bound to polysomes in A. thaliana varies typically from 35 to 85% (30). For the AtFtsH10 transcript, this value was 59% (30).…”

Section: Identification Of Atftsh3 and Atftsh10mentioning

confidence: 90%

“…For the AtFtsH10 transcript, this value was 59% (30). Taking into consideration the above and the fact that the steady-state level of the FtsH10 transcript is similar in ftsh3 and wild type plants, but almost twice as many molecules of this transcript are bound to polysomes in ftsh3, we conclude that practically all AtFtsH10 transcripts are associated with polysomes in the ftsh3 mutant.…”

Section: Identification Of Atftsh3 and Atftsh10mentioning

confidence: 96%

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Identification and Characterization of High Molecular Weight Complexes Formed by Matrix AAA Proteases and Prohibitins in Mitochondria of Arabidopsis thaliana

Piechota

Kołodziejczak

Juszczak

et al. 2010

Journal of Biological Chemistry

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We identify and characterize two matrix (m)-AAA proteases (AtFtsH3 and AtFtsH10) present in the mitochondria of Arabidopsis thaliana. AtFtsH3 is the predominant protease in leaves of wild type plants. Both proteases assemble with prohibitins (PHBs) into high molecular weight complexes (ϳ2 MDa), similarly to their yeast counterparts. A smaller PHB complex (ϳ1 MDa), without the m-AAA proteases, was also detected. Unlike in yeast, stable prohibitin-independent high molecular weight assemblies of m-AAA proteases could not be identified in A. thaliana. AtFtsH3 and AtFtsH10 form at least two types of m-AAA-PHB complexes in wild type plants. The one type contains PHBs and AtFtsH3, and the second one is composed of PHBs and both AtFtsH3 and AtFtsH10. Complexes composed of PHBs and AtFtsH10 were found in an Arabidopsis mutant lacking AtFtsH3 (ftsh3). Thus, both AtFtsH3 and AtFtsH10 may form hetero-and homo-oligomeric complexes with prohibitins. The increased level of AtFtsH10 observed in ftsh3 suggests that functions of the homo-and hetero-oligomeric complexes containing AtFtsH3 can be at least partially substituted by AtFtsH10 homo-oligomers. The steady-state level of the AtFtsH10 transcripts did not change in ftsh3 compared with wild type plants, but we found that almost twice more of the AtFtsH10 transcripts were associated with polysomes in ftsh3. Based on this result, we assume that the AtFtsH10 protein is synthesized at a higher rate in the ftsh3 mutant. Our results provide the first data on the composition of m-AAA and PHB complexes in plant mitochondria and suggest that the abundance of m-AAA proteases is regulated not only at the transcriptional but also at the translational level.It is well established now that mitochondria have their own system for protein degradation. In the case of membrane proteins, this system is mainly based on AAA proteases (AAA stands for ATPases associated with diverse cellular activities) (1-3). AAA proteases, also called FtsH proteases, belong to a conserved family of ATP-dependent metallopeptidases with members found in bacteria, yeast, plants, and humans. The FtsH proteases are bifunctional enzymes, in which a proteolytic domain is accompanied by an ATPase domain with a chaperone-like activity. The location of FtsH proteases in eukaryotic cells is restricted to mitochondria and chloroplasts. In mitochondria, two types of AAA proteases have been identified. Both of them are anchored in the inner mitochondrial membrane, but their active centers are directed to opposite membrane surfaces as follows: the m-AAA 2 proteases face the matrix, whereas the i-AAA proteases are oriented toward the intermembrane space (1, 4).Mutations in m-AAA proteases cause severe defects in yeast (5), mouse (6), and humans (7, 8). They have been shown to degrade misfolded and unassembled membrane proteins (1, 9 -11) and are also responsible for proteolytic activation and maturation of several mitochondrial proteins. Substrates that are cleaved rather than degraded by the m-AAA proteases include mitochondrial...

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“…It has been reported that the percentage of particular mRNAs bound to polysomes in A. thaliana varies typically from 35 to 85% (30). For the AtFtsH10 transcript, this value was 59% (30).…”

Section: Identification Of Atftsh3 and Atftsh10mentioning

confidence: 90%

Section: Identification Of Atftsh3 and Atftsh10mentioning

confidence: 96%

Identification and Characterization of High Molecular Weight Complexes Formed by Matrix AAA Proteases and Prohibitins in Mitochondria of Arabidopsis thaliana

Piechota

Kołodziejczak

Juszczak

et al. 2010

Journal of Biological Chemistry

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“…The transgenic A. thaliana 35S:HF-RPL18B line 12-2-4 (ecotype Col-0) was used in this study (1). Seedlings were grown on solid media [0.43% (wt/vol) Murashige and Skoog salts, 1% (wt/vol) sucrose] at 23°C under a 16-h light (80 μE·m −2 ·s −1 ) and 8-h dark cycle for 7 d. Hypoxia was imposed as described previously (15) for 2 h under low light (7 μE·m −2 ·s −1 ).…”

Section: Methodsmentioning

confidence: 99%

“…A typical way to assess translation is to isolate transcripts in association with one or more ribosomes (i.e., polysomes) by differential centrifugation. To facilitate the capture of ribosome-associated mRNA, we developed a method for translating ribosome affinity immunopurification (TRAP) incorporating FLAG-tagged ribosomal protein L18 (RPL18) into functional ribosomes in the model plant Arabidopsis thaliana (1). Two advantages of TRAP are that it reduces contamination of polysome preparations with mRNA-ribonucleoprotein (mRNP) complexes of similar density and can be used to obtain mRNAs from subpopulations of cells of a multicellular organ or tissue in plants (2,3), as well as in mammals and insects (4,5).…”

mentioning

confidence: 99%

Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis

Juntawong

Girke

Bazin

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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361

415

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Translational regulation contributes to plasticity in metabolism and growth that enables plants to survive in a dynamic environment. Here, we used the precise mapping of ribosome footprints (RFs) on mRNAs to investigate translational regulation under control and sublethal hypoxia stress conditions in seedlings of Arabidopsis thaliana. Ribosomes were obtained by differential centrifugation or immunopurification and were digested with RNase I to generate footprint fragments that were deep-sequenced. Comparison of RF number and position on genic regions with fragmented total and polysomal mRNA illuminated numerous aspects of posttranscriptional and translational control under both growth conditions. When seedlings were oxygen-deprived, the frequency of ribosomes at the start codon was reduced, consistent with a global decline in initiation of translation. Hypoxia-up-regulated gene transcripts increased in polysome complexes during the stress, but the number of ribosomes per transcript relative to normoxic conditions was not enhanced. On the other hand, many mRNAs with limited change in steady-state abundance had significantly fewer ribosomes but with an overall similar distribution under hypoxia, consistent with restriction of initiation rather than elongation of translation. RF profiling also exposed the inhibitory effect of upstream ORFs on the translation of downstream protein-coding regions under normoxia, which was further modulated by hypoxia. The data document translation of alternatively spliced mRNAs and expose ribosome association with some noncoding RNAs. Altogether, we present an experimental approach that illuminates prevalent and nuanced regulation of protein synthesis under optimal and energy-limiting conditions. ribosome profiling | uORF | alternative splicing | long intergenic noncoding RNA | translational efficiency

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“…To date, analyses of the Arabidopsis mitochondrial proteome have only identified five mitochondrial r-proteins (S4, L3, L7/L12, L22, and L25) (Heazlewood et al 2004;Heazlewood and Millar 2005). An approach that may unambiguously answer this question would be to epitope tag S15a proteins (Types I and II) in a manner that facilitated immunoprecipitation of the ribosome complex as shown for cytosolic RPL18 and RPL23 (Zanetti et al 2005). The coimmunoprecipitated proteins could then be subsequently analyzed by mass spectrometry to determine the coassociated proteins.…”

Section: Subcellular Location Of Type II S15amentioning

confidence: 99%

Analysis of RPS15aE, an Isoform of a Plant-Specific Evolutionarily Distinct Ribosomal Protein in Arabidopsis thaliana, Reveals its Potential Role as a Growth Regulator

Szick-Miranda

Zanial

et al. 2009

Plant Mol Biol Rep

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There is increasing evidence for ribosome heterogeneity in biological systems. In Arabidopsis thaliana, the ribosomal protein S15a is encoded by six separate genes, which fall into two evolutionarily distinct categories (Type I and Type II). Type I S15a is a universally conserved component of cytosolic ribosomes, whereas there is ambiguity as to the specific subcellular location of Type II S15a (cytosolic and/or mitochondrial ribosomes). In this study, we investigated the functional significance of the distinct form of ribosomal protein S15a (Type II) in Arabidopsis by examining: the evolutionary relationship of eukaryotic S15a proteins with respect to organellar homologs, the expression of individual Type II S15a genes during various developmental stages by RT-PCR, and the phenotypes of an insertional mutation into the RPS15aE gene. The Type II S15a proteins are plant specific, and the duplication event that gave rise to the Type II S15a genes appears to have occurred during the evolution of land plants. The genes encoding Type II S15a in Arabidopsis are differentially expressed, and mutant plants in which the gene encoding S15aE is knocked down produce larger leaves, longer roots, and possess larger cells than wild-type plants suggesting that the RPS15aE isoform of Type II S15a may act as a regulator of translational activity. Our results add significantly to the understanding of the protein constitution of plant ribosomes and the functional significance of ribosome heterogeneity.

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Immunopurification of Polyribosomal Complexes of Arabidopsis for Global Analysis of Gene Expression

Cited by 226 publications

References 57 publications

Identification and Characterization of High Molecular Weight Complexes Formed by Matrix AAA Proteases and Prohibitins in Mitochondria of Arabidopsis thaliana

Identification and Characterization of High Molecular Weight Complexes Formed by Matrix AAA Proteases and Prohibitins in Mitochondria of Arabidopsis thaliana

Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis

Analysis of RPS15aE, an Isoform of a Plant-Specific Evolutionarily Distinct Ribosomal Protein in Arabidopsis thaliana, Reveals its Potential Role as a Growth Regulator

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