Gene Expression Profiling of Flagellar Disassembly in <i>Chlamydomonas reinhardtii</i>

Chamberlain, Kara Lista; Miller, Steven H.; Keller, Laura R.

doi:10.1534/genetics.107.082149

Cited by 15 publications

(30 citation statements)

References 54 publications

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“…ICL1, which is a metabolic enzyme (EC 4.1.3.1) that catalyzes the first step in the glyoxylate bypass in bacteria, fungi, and plants, was the only polypeptide not rescued. The abundance of mRNA encoding ICL1 appears to be increased during flagellar regeneration (44); however, it is controversial whether ICL1 is a consistent structural component of the axoneme.…”

Section: Comparative Proteomic Analysis Of Drc Mutants Identifies Knomentioning

confidence: 99%

Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella

Lin

Tritschler

Song

et al. 2011

Journal of Biological Chemistry

145

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The directional flow generated by motile cilia and flagella is critical for many processes, including human development and organ function. Normal beating requires the control and coordination of thousands of dynein motors, and the nexin-dynein regulatory complex (N-DRC) has been identified as an important regulatory node for orchestrating dynein activity. The nexin link appears to be critical for the transformation of dynein-driven, linear microtubule sliding to flagellar bending, yet the molecular composition and mechanism of the N-DRC remain largely unknown. Here, we used proteomics with special attention to protein phosphorylation to analyze the composition of the N-DRC and to determine which subunits may be important for signal transduction. Two-dimensional electrophoresis and MALDI-TOF mass spectrometry of WT and mutant flagellar axonemes from Chlamydomonas identified 12 N-DRC-associated proteins, including all seven previously observed N-DRC components. Sequence and PCR analyses identified the mutation responsible for the phenotype of the sup-pf-4 strain, and biochemical comparison with a radial spoke mutant revealed two components that may link the N-DRC and the radial spokes. Phosphoproteomics revealed eight proteins with phosphorylated isoforms for which the isoform patterns changed with the genotype as well as two components that may play pivotal roles in N-DRC function through their phosphorylation status. These data were assembled into a model of the N-DRC that explains aspects of its regulatory function.

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Section: Comparative Proteomic Analysis Of Drc Mutants Identifies Knomentioning

confidence: 99%

Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella

Lin

Tritschler

Song

et al. 2011

Journal of Biological Chemistry

145

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“…During the cell cycle and under certain physiological conditions (e.g., stress), ciliary genes are under direct transcriptional control in both unicellular (16) and multicellular (15,17) unikont and nonunikont organisms. In multicellular organisms, some ciliary genes are differentially regulated for cell-specific expression patterns (18).…”

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confidence: 99%

Regulatory Factor X (RFX)-mediated transcriptional rewiring of ciliary genes in animals

Piasecki

Burghoorn

Swoboda

2010

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

103

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Cilia were present in the last eukaryotic common ancestor (LECA) and were retained by most organisms spanning all extant eukaryotic lineages, including organisms in the Unikonta (Amoebozoa, fungi, choanoflagellates, and animals), Archaeplastida, Excavata, Chromalveolata, and Rhizaria. In certain animals, including humans, ciliary gene regulation is mediated by Regulatory Factor X (RFX) transcription factors (TFs). RFX TFs bind X-box promoter motifs and thereby positively regulate >50 ciliary genes. Though RFX-mediated ciliary gene regulation has been studied in several bilaterian animals, little is known about the evolutionary conservation of ciliary gene regulation. Here, we explore the evolutionary relationships between RFX TFs and cilia. By sampling the genome sequences of >120 eukaryotic organisms, we show that RFX TFs are exclusively found in unikont organisms (whether ciliated or not), but are completely absent from the genome sequences of all nonunikont organisms (again, whether ciliated or not). Sampling the promoter sequences of 12 highly conserved ciliary genes from 23 diverse unikont and nonunikont organisms further revealed that phylogenetic footprints of X-box promoter motif sequences are found exclusively in ciliary genes of certain animals. Thus, there is no correlation between cilia/ciliary genes and the presence or absence of RFX TFs and X-box promoter motifs in nonanimal unikont and in nonunikont organisms. These data suggest that RFX TFs originated early in the unikont lineage, distinctly after cilia evolved. The evolutionary model that best explains these observations indicates that the transcriptional rewiring of many ciliary genes by RFX TFs occurred early in the animal lineage.cilia | transcription factor | X-box | Unikont

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“…Fragments of 278 bp (variant 1) and 143 bp (variant 2) were expected and actually obtained in the RT-PCR (Figure 6 and Additional file 1 : Table S3); it should be mentioned that in addition to the correct fragments, some non-specific side products were amplified (Figure 6 ). Subsequently, the relative expression levels of both splice variants were measured by quantitative real-time RT-PCR, which is a sensitive method for analyzing relative expression levels of alternative splicing variants [ 49 , 73 – 75 ]. As a reference gene for the quantitative real-time RT-PCRs the actin gene was used.…”

Section: Resultsmentioning

confidence: 99%

Genome-wide analysis of alternative splicing in Volvox carteri

et al. 2014

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BackgroundAlternative splicing is an essential mechanism for increasing transcriptome and proteome diversity in eukaryotes. Particularly in multicellular eukaryotes, this mechanism is involved in the regulation of developmental and physiological processes like growth, differentiation and signal transduction.ResultsHere we report the genome-wide analysis of alternative splicing in the multicellular green alga Volvox carteri. The bioinformatic analysis of 132,038 expressed sequence tags (ESTs) identified 580 alternative splicing events in a total of 426 genes. The predominant type of alternative splicing in Volvox is intron retention (46.5%) followed by alternative 5′ (17.9%) and 3′ (21.9%) splice sites and exon skipping (9.5%). Our analysis shows that in Volvox at least ~2.9% of the intron-containing genes are subject to alternative splicing. Considering the total number of sequenced ESTs, the Volvox genome seems to provide more favorable conditions (e.g., regarding length and GC content of introns) for the occurrence of alternative splicing than the genome of its close unicellular relative Chlamydomonas. Moreover, many randomly chosen alternatively spliced genes of Volvox do not show alternative splicing in Chlamydomonas. Since the Volvox genome contains about the same number of protein-coding genes as the Chlamydomonas genome (~14,500 protein-coding genes), we assumed that alternative splicing may play a key role in generation of genomic diversity, which is required to evolve from a simple one-cell ancestor to a multicellular organism with differentiated cell types (Mol Biol Evol 31:1402-1413, 2014). To confirm the alternative splicing events identified by bioinformatic analysis, several genes with different types of alternatively splicing have been selected followed by experimental verification of the predicted splice variants by RT-PCR.ConclusionsThe results show that our approach for prediction of alternative splicing events in Volvox was accurate and reliable. Moreover, quantitative real-time RT-PCR appears to be useful in Volvox for analyses of relationships between the appearance of specific alternative splicing variants and different kinds of physiological, metabolic and developmental processes as well as responses to environmental changes.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1117) contains supplementary material, which is available to authorized users.

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Gene Expression Profiling of Flagellar Disassembly in Chlamydomonas reinhardtii

Cited by 15 publications

References 54 publications

Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella

Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella

Regulatory Factor X (RFX)-mediated transcriptional rewiring of ciliary genes in animals

Genome-wide analysis of alternative splicing in Volvox carteri

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