A large kinome in a large cell:<i>Stentor coeruleus</i>possesses highly expanded kinase families and novel domain architectures

Sb, Reiff; Marshall, WF

doi:10.1101/168187

Cited by 8 publications

(12 citation statements)

References 78 publications

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“…A total of 103 upregulated genes were assigned as dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase (DYRK), making this the orthologous group of kinases with the highest number of genes upregulated. The high expansion of DYRKs has previously been reported in a study of the S. coeruleus kinome [11]. The DYRK genes in this orthologous group (KOG0667) were mainly upregulated at P90, P180, and P300.…”

Section: Figure 3 Heatmap With Conditions and Genes Hierarchically Csupporting

confidence: 65%

RNA Sequencing of Stentor Cell Fragments Reveals Transcriptional Changes during Cellular Regeneration

2018

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While ciliates of the genus Stentor are known for their ability to regenerate when their cells are damaged or even fragmented, the physical and molecular mechanisms underlying this process are poorly understood. To identify genes involved in the regenerative capability of Stentor cells, RNA sequencing of individual Stentor polymorphus cell fragments was performed. After splitting a cell over the anterior-posterior axis, the posterior fragment has to regenerate the oral apparatus, while the anterior part needs to regenerate the hold fast. Altogether, differential expression analysis of both posterior and anterior S. polymorphus cell fragments for four different post-split time points revealed over 10,000 upregulated genes throughout the regeneration process. Among these, genes involved in cell signaling, microtubule-based movement, and cell cycle regulation seemed to be particularly important during cellular regeneration. We identified roughly nine times as many upregulated genes in regenerating S. polymorphus posterior fragments as compared to anterior fragments, indicating that regeneration of the anterior oral apparatus is a complex process that involves many genes. Our analyses identified several expanded groups of genes, such as dual-specific tyrosine-(Y)-phosphorylation-regulated kinases and MORN domain-containing proteins that seemingly act as key regulators of cellular regeneration. In agreement with earlier morphological and cell biological studies [1, 2], our differential expression analyses indicate that cellular regeneration and vegetative division share many similarities.

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Section: Figure 3 Heatmap With Conditions and Genes Hierarchically Csupporting

confidence: 65%

RNA Sequencing of Stentor Cell Fragments Reveals Transcriptional Changes during Cellular Regeneration

2018

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“…The latest expressed genes include genes encoding components of cGMP signaling (GPCRs and guanylyl cyclases). Kinases are expressed at all stages of regeneration, consistent with the massive expansion of the kinome in Stentor [36] although specific kinase families tend to be expressed at specific stages. One abundant class of kinases observed among the upregulated genes was the dual-specificity DYRK kinases, with 17 different DYRK family members expressed among several different expression groups.…”

Section: Gene Classesmentioning

confidence: 69%

“…One abundant class of kinases observed among the upregulated genes was the dual-specificity DYRK kinases, with 17 different DYRK family members expressed among several different expression groups. However, the kinome of Stentor coeruleus has been predicted to contain 142 DYRK family members, making them one of the most highly expanded kinase families in the genome [36]. Given that the Stentor genome contains 35,000 genes, we would expect that roughly 14 DYRK would be present in any randomly chosen set of 3,000 genes.…”

Section: Gene Classesmentioning

confidence: 99%

“…These include homologs of E2F, Rb, and various cyclins. Previous analysis of the Stentor kinome identified 44 Aurora related kinases [36], of which three are found to be differentially expressed during regeneration. Figure 4D plots the expression pattern of these three Aurora kinases as a function of time during regeneration.…”

Section: Cell Cycle Regulators During Regenerationmentioning

confidence: 99%

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The Transcriptional Program of Regeneration in the Giant Single Cell,Stentor coeruleus

Sood

McGillivary

Marshall

2017

Preprint

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The giant ciliate Stentor coeruleus is a classical model system for studying regeneration and morphogenesis at the level of a single cell. Stentor are polarized cells with a complex subcellular architecture. The anterior of the cell is marked by a complex polarized array of cilia, known as the oral apparatus. This feeding organelle can be induced to shed and regenerate in a series of reproducible morphological steps, previously shown to require transcription. We used RNAseq to assay the dynamic changes in Stentor's transcriptome during regeneration with high temporal resolution, allowing us to identify five distinct waves of gene expression. We show that the oral apparatus is a model for organelle regeneration, as well many conserved genes involved in centriole assembly and ciliogenesis are induced. Additionally, we find genes involved in signaling, cell cycle regulation, transcription, and RNA binding to be expressed at distinct stages of organelle regeneration, suggesting that the morphological steps of regeneration are driven by a complex regulatory system. Materials and Methods Inducing Regeneration and Staging StentorCells were obtained from Carolina Biological Supply and cultured as previously described [20]. Briefly, cells were maintained in Pasteurized Spring Water (Carolina Biological Supply) and fed with Chlamydomonas and wheat seeds. Cells were collected from the same culture for each RNAseq experimental replicate. To induce regeneration, cells were shocked with a 15% sucrose solution for 2 minutes [11], and then washed in Carolina Spring Water thoroughly. Samples of ~20 cells were collected before shock, then at 30 minutes post shock, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours and 8 hours. At each time point, a sample of cells was lysed into RNA-stabilizing buffers specified by the extraction kit, and then stored on ice until the end of the experiment when the RNA purification was performed in parallel on all samples (see below). 4 replicates were analyzed for each time-point. Total RNA extractionRNA was extracted at each time point using the Nucleospin RNA XS kit from Clontech (cat. num. 740902.250). RNA quality was assessed using a NanoDrop and then Bioanalyzer was used to quantify RNA amount. ERCC spike ins (ThermoFisher cat. num. 4456739) were added to each sample in a dilution ranging from 1:1000 to 1:10000 depending on the initial amount of RNA extracted. RNA-Seq library preparation and sequencingRNA-seq libraries were prepared with Ovation RNA-seq system v2 kit (NuGEN). In this method, the total RNA (50 ng or less) is reverse transcribed to synthesize the first-strand cDNA using a combination of random hexamers and a poly-T chimeric primer. The RNA template is then partially degraded by heating and the second strand cDNA is synthesized using DNA polymerase. The double-stranded DNA is then amplified using single primer isothermal amplification (SPIA). SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′-end of the double-st...

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“…Protist ciliates possess remarkably high numbers of genes encoding protein kinases. The genomes of Tetrahymena thermophila , Paramecium tetraurelia , and Stentor coeruleus contain 1069, 2606, and 2057 kinase-coding genes, respectively, representing 4, 6.6, and 6% of the whole gene number (Eisen et al, 2006; Bemm et al, 2009; Reiff and Marshall, 2017). In man, in contrast, the 518 kinase-coding genes approximate to only 1.7% of all genes (Manning et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Subcellular Targeting of the Euplotes raikovi Kinase Er-MAPK1, as Revealed by Expression in Different Cell Systems

Candelori

Yamamoto

Iwamoto

et al. 2019

Front. Cell Dev. Biol.

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In the ciliate Euplotes raikovi, a 631-amino acid Er-MAPK1 protein kinase was found to localize in nucleoli of the transcriptionally active nucleus (macronucleus) and act as a key component of an autocrine, cell-growth promoting self-signaling mechanism. While its 283-amino acid N-terminal domain includes all the structural specificities of the mitogen-activated protein kinases required for a catalytic function, the 348-amino acid C-terminal domain is structurally unique with undetermined functions. By expressing the two Er-MAPK1 domains tagged with the green fluorescent protein in mammalian fibroblasts, the yeast Schizosaccharomyces pombe and the ciliate Tetrahymena thermophila, evidence was obtained that the C-terminal domain contains all the sequence information responsible for the Er-MAPK1 subcellular localization. However, in fibroblasts and S. pombe this information determined a nucleolar localization of the GFP-tagged C-terminal domain, and a ciliary localization in T. thermophila. In the light of these findings, the Er-MAPK1 localization in E. raikovi was re-examined via immunoreactions and shown to be ciliary besides that nuclear, as is the case for the mammalian intestinal cell kinase with which the Er-MAPK1 N-terminal domain shares a strong sequence identity and a catalytic function.

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A large kinome in a large cell:Stentor coeruleuspossesses highly expanded kinase families and novel domain architectures

Cited by 8 publications

References 78 publications

RNA Sequencing of Stentor Cell Fragments Reveals Transcriptional Changes during Cellular Regeneration

RNA Sequencing of Stentor Cell Fragments Reveals Transcriptional Changes during Cellular Regeneration

The Transcriptional Program of Regeneration in the Giant Single Cell,Stentor coeruleus

Subcellular Targeting of the Euplotes raikovi Kinase Er-MAPK1, as Revealed by Expression in Different Cell Systems

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