The \u3cem\u3eChlamydomonas\u3c/em\u3e Genome Reveals the Evolution of Key Animal and Plant Functions

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella

In vivo analysis of the 5' untranslated leader sequence of mitochondrial cytochrome b transcripts in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae the production of respiratory competent mitochondria requires the activity of several hundred proteins, the vast majority of which are encoded by nuclear genes and imported into mitochondria. A subset of these nuclear gene products are required for the expression of individual mitochondrial genes. Expression of the mitochondrial gene cob, encoding cytochrome b, requires the activity of at least seven nuclear gene products, including CBP1, CBS1 and CBS2. CBP1 is required for the stability of cob transcripts, while CBS1 and CBS2 are required for translation of cob mRNA. Previous analyses of mitochondrial rearrangements that suppress cbp1, cbs1, or cbs2 mutations have led to the hypothesis that CBP1, CBS1 and CBS2 interact with the 5' untranslated leader (UTR) of cob transcripts. To further define cob sequence important for stability or translation of cob transcripts, the expression of mutant cob genes, in which portions of the cob coding sequence or 5' UTR have been deleted, has been analyzed in vivo. Quantitation of steady-state levels of cob transcripts in wild-type CBP1 and mutant cbp1 strains carrying deletion mitochondrial genomes leads to the conclusion that a 63 nucleotide sequence is sufficient for the CBP1-dependent stability of cob transcripts. This sequence encompasses the site of cleavage that produces the mature 5' end of cob mRNA from precursor transcripts. Determination of the 5' ends of mature cob mRNAs in the deletion strains indicated that this sequence is also sufficient for correct positioning of the cleavage. The data suggest that the cleavage is CBP1-dependent and occurs at a specific distance 5' of a recognition site located within the defined 63 nucleotides. In addition, the data suggest that the stability of the mature cob mRNA produced by the cleavage is dependent on sequence at its 5' end. Lastly, an analysis of the structure of a high molecular weight cob transcript present in a respiratory incompetent deletion strain leads to the hypothesis that 5' UTR sequence between -200 and -4 is required for interaction with factors, such as CBS1 or CBS2, required for translation of cob transcripts

Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

WOS: 000323756100010PubMed ID: 23873865Like many algae, Chlamydomonas reinhardtii is phototactic, using two anterior flagella to swim toward light optimal for photosynthesis. The flagella are responsive to signals initiated at the photosensory eyespot, which comprises photoreceptors in the plasma membrane and layers of pigment granules in the chloroplast. Phototaxis depends on placement of the eyespot at a specific asymmetric location relative to the flagella, basal bodies, and bundles of two or four highly acetylated microtubules, termed rootlets, which extend from the basal bodies toward the posterior of the cell. Previous work has shown that the eyespot is disassembled prior to cell division, and new eyespots are assembled in daughter cells adjacent to the nascent four-membered rootlet associated with the daughter basal body (D4), but the chronology of these assembly events has not been determined. Here we use immunofluorescence microscopy to follow assembly and acetylation of the D4 rootlet, localization of individual eyespot components in the plasma membrane or chloroplast envelope, and flagellar emergence during and immediately following cell division. We find that the D4 rootlet is assembled before the initiation of eyespot assembly, which occurs within the same time frame as rootlet acetylation and flagellar outgrowth. Photoreceptors in the plasma membrane are correctly localized in eyespot mutant cells lacking pigment granule layers, and chloroplast components of the eyespot assemble in mutant cells in which photoreceptor localization is retarded. The data suggest that plasma membrane and chloroplast components of the eyespot are independently responsive to a cytoskeletal positioning cue.National Science FoundationNational Science Foundation (NSF) [MCB 1157795]Support for this research was provided by the National Science Foundation, award no. MCB 1157795

The DEAD-box RNA helicase Ded1 has a role in the translational response to TORC1 inhibition

Ded1 is a DEAD-box RNA helicase with essential roles in translation initiation. It binds to the eukaryotic initiation factor 4F (eIF4F) complex and promotes 48S preinitiation complex assembly and start-site scanning of 5' untranslated regions of mRNAs. Most prior studies of Ded1 cellular function were conducted in steady-state conditions during nutrient-rich growth. In this work, however, we examine its role in the translational response during target of rapamycin (TOR)C1 inhibition and identify a novel function of Ded1 as a translation repressor. We show that C-terminal mutants of DED1 are defective in down-regulating translation following TORC1 inhibition using rapamycin. Furthermore, following TORC1 inhibition, eIF4G1 normally dissociates from translation complexes and is degraded, and this process is attenuated in mutant cells. Mapping of the functional requirements for Ded1 in this translational response indicates that Ded1 enzymatic activity and interaction with eIF4G1 are required, while homo-oligomerization may be dispensable. Our results are consistent with a model wherein Ded1 stalls translation and specifically removes eIF4G1 from translation preinitiation complexes, thus removing eIF4G1 from the translating mRNA pool and leading to the codegradation of both proteins. Shared features among DED1 orthologues suggest that this role is conserved and may be implicated in pathologies such as oncogenesis.American Cancer Society [RSG-13-263-01-RMC]; Arizona Biomedical Research Commission [ADHS14-082993]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Correction: Fernandes et al. Stress Granule Assembly Can Facilitate but Is Not Required for TDP-43 Cytoplasmic Aggregation. Biomolecules 2020, 10, 1367

In the original article [...

C2 Domain Protein MIN1 Promotes Eyespot Organization in Chlamydomonas reinhardtii▿ †

Assembly and asymmetric localization of the photosensory eyespot in the biflagellate, unicellular green alga Chlamydomonas reinhardtii requires coordinated organization of photoreceptors in the plasma membrane and pigment granule/thylakoid membrane layers in the chloroplast. min1 (mini-eyed) mutant cells contain abnormally small, disorganized eyespots in which the chloroplast envelope and plasma membrane are no longer apposed. The MIN1 gene, identified here by phenotypic rescue, encodes a protein with an N-terminal C2 domain and a C-terminal LysM domain separated by a transmembrane sequence. This novel domain architecture led to the hypothesis that MIN1 is in the plasma membrane or the chloroplast envelope, where membrane association of the C2 domain promotes proper eyespot organization. Mutation of conserved C2 domain loop residues disrupted association of the MIN1 C2 domain with the chloroplast envelope in moss cells but did not abolish eyespot assembly in Chlamydomonas. In min1 null cells, channelrhodopsin-1 (ChR1) photoreceptor levels were reduced, indicating a role for MIN1 in ChR1 expression and/or stability. However, ChR1 localization was only minimally disturbed during photoautotrophic growth of min1 cells, conditions under which the pigment granule layers are disorganized. The data are consistent with the hypothesis that neither MIN1 nor proper organization of the plastidic components of the eyespot is essential for localization of ChR1