RuBisCO in Non-Photosynthetic Alga <i>Euglena longa</i>: Divergent Features, Transcriptomic Analysis and Regulation of Complex Formation

<div><p><i>Euglena longa</i>, a close relative of the photosynthetic model alga <i>Euglena gracilis</i>, possesses an enigmatic non-photosynthetic plastid. Its genome has retained a gene for the large subunit of the enzyme RuBisCO (<i>rbcL</i>). Here we provide new data illuminating the putative role of RuBisCO in <i>E</i>. <i>longa</i>. We demonstrated that the <i>E</i>. <i>longa</i> RBCL protein sequence is extremely divergent compared to its homologs from the photosynthetic relatives, suggesting a possible functional shift upon the loss of photosynthesis. Similarly to <i>E</i>. <i>gracilis</i>, <i>E</i>. <i>longa</i> harbors a nuclear gene encoding the small subunit of RuBisCO (RBCS) as a precursor polyprotein comprising multiple RBCS repeats, but one of them is highly divergent. Both RBCL and the RBCS proteins are synthesized in <i>E</i>. <i>longa</i>, but their abundance is very low compared to <i>E</i>. <i>gracilis</i>. No RBCS monomers could be detected in <i>E</i>. <i>longa</i>, suggesting that processing of the precursor polyprotein is inefficient in this species. The abundance of RBCS is regulated post-transcriptionally. Indeed, blocking the cytoplasmic translation by cycloheximide has no immediate effect on the RBCS stability in photosynthetically grown <i>E</i>. <i>gracilis</i>, but in <i>E</i>. <i>longa</i>, the protein is rapidly degraded. Altogether, our results revealed signatures of evolutionary degradation (becoming defunct) of RuBisCO in <i>E</i>. <i>longa</i> and suggest that its biological role in this species may be rather unorthodox, if any.</p></div

Expression of the <i>RbcS</i> and <i>rbcL</i> genes in <i>Euglena gracilis</i> and <i>Euglena longa</i>.

<p>Expression levels of <i>RbcS</i> and <i>rbcL</i> mRNAs were analyzed by quantitative RT-PCR and normalized over the 18S ribosomal RNA. Cultivation conditions and species are denoted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158790#pone.0158790.g002" target="_blank">Fig 2</a>.</p

Abundance of the RBCS and RBCL proteins in <i>Euglena gracilis</i> and <i>Euglena longa</i>.

<p>Protein immunodetection was performed using anti-RBCS, anti-RBCL, and anti-Tubulin antibodies. Three bands with different molecular weights were observed in anti-RBCS immunoblotting. The ~130 kDa band (marked *1) corresponds to polyprotein synthesized in the nucleus. The ~15 kDa band (marked *3) corresponds to the processed monomer after cleavage of the signal sequence and excision of decapeptides. The ~22 kDa band (marked *2) possibly corresponds to a monomer still attached to the transit peptide. The identity of the RBCL protein (arrowhead in the anti-RBCL panel) was confirmed by mass-spectrometry. Tubulin served as a loading control. Molecular weights in kDa are indicated on the left. EG-, <i>E</i>. <i>gracilis</i> cultivated photosynthetically (without ethanol); EG+, <i>E</i>. <i>gracilis</i> cultivated mixotrophically (with ethanol); EL, <i>E</i>. <i>longa</i>.</p

Stability of RBCS and RBCL proteins in <i>Euglena gracilis</i> and <i>Euglena longa</i>.

<p>Cell cultures were treated with 20 μg/ml of cycloheximide, aliquots were taken at 0, 1, 4, 8, and 24 h post treatment, and analyzed by western blotting using anti-RBCS, anti-RBCL and anti-Tubulin antibodies. Molecular weights (in kDa) are indicated on the left of each panel. The identity of the RBCL protein (arrowhead in the anti-RBCL panel) was confirmed by mass-spectrometry. Tubulin served as a loading control. Cultivation conditions and species are denoted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158790#pone.0158790.g002" target="_blank">Fig 2</a>.</p

Phylogenetic tree of RBCL protein sequences.

<p>The maximum-likelihood tree was inferred with RAxML using the LG+Γ substitution model. The bootstrap support values and posterior probabilities (from PhyloBayes) are indicated at branches when higher than 50% and 0.95, respectively. Highlighted in white boxes are non-photosynthetic species. <i>E</i>. <i>longa</i> is in bold.</p

RNA viruses in trypanosomatid parasites: a historical overview

<div><p>Viruses of trypanosomatids are now being extensively studied because of their diversity and the roles they play in flagellates’ biology. Among the most prominent examples are leishmaniaviruses implicated in pathogenesis of Leishmania parasites. Here, we present a historical overview of this field, starting with early reports of virus-like particles on electron microphotographs, and culminating in detailed molecular descriptions of viruses obtained using modern next generation sequencing-based techniques. Because of their diversity, different life cycle strategies and host specificity, we believe that trypanosomatids are a fertile ground for further explorations to better understand viral evolution, routes of transitions, and molecular mechanisms of adaptation to different hosts.</p></div

Ablation of <i>LmxM</i>.<i>22</i>.<i>0010</i> by conventional approach.

<p>A, Schematic representation of the WT and recombined alleles after replacement with Sat-, Hyg-, Neo-, and Ble-resistant genes. Annealing positions of the probes and expected fragment sizes are shown. B, Southern blot analysis of the <i>Bst</i>E II digested <i>L</i>. <i>mexicana</i> genomic DNA of the WT and BTN1 ablated strains with Sat, Hyg, Neo, Ble, 5' UTR, and <i>LmxM</i>.<i>22</i>.<i>0010</i> ORF probes.</p

Development of the WT, Cas9 and BTN1 KO strains (labeled KO) in sand flies.

<p>A, Intensity of infection was assayed on days 2–3 and 7–8 p.i. and defined as weak (less than 100 promastigotes), moderate (100–1,000 promastigotes), or heavy (over 1,000 promastigotes), depending on the number of parasites per gut. Data are summarized from five independent biological replicates, numbers above each bar indicate the total number of dissected females. B, Quantitative PCR analysis of the <i>L</i>. <i>mexicana</i> load in the insect gut 7–8 days p.i. Boxplots are from five independent biological replicates and show 1st quartile, median, 3rd quartile, and 1.5× interquartile range values. C, Localization of parasites in sand fly gut 7–8 days p.i. (SV, stomodeal valve; TH,ABM, both thoracic and abdominal midgut; ABM, abdominal midgut). Numbers above each bar indicate the number of dissected females. D, Morphological analysis of <i>Leishmania mexicana</i> cells from thoracic midgut and stomodeal valve of infected sand fly females 7–8 days p.i. (LN, long nectomonade; SN, short nectomonade; ME, metacyclic promastigote).</p

Ablation of <i>LmxM</i>.<i>22</i>.<i>0010</i> by CRISPR-Cas9.

<p>A, Schematic representation of the WT and recombined alleles after replacement with Puro resistant gene. Annealing positions of the probes and expected fragment sizes are shown. B, Southern blot analysis of the <i>Bst</i>E II digested <i>L</i>. <i>mexicana</i> genomic DNA of the WT, Cas9, and BTN1 ablated strains (labeled KO) with <i>LmxM</i>.<i>22</i>.<i>0010</i> 5' UTR, <i>LmxM</i>.<i>22</i>.<i>0010</i> 3' UTR and Puro probes.</p

DNA polymerase <b>η</b> mutational signatures are found in a variety of different types of cancer

<p>DNA polymerase (pol) η is a specialized error-prone polymerase with at least two quite different and contrasting cellular roles: to mitigate the genetic consequences of solar UV irradiation, and promote somatic hypermutation in the variable regions of immunoglobulin genes. Misregulation and mistargeting of pol η can compromise genome integrity. We explored whether the mutational signature of pol η could be found in datasets of human somatic mutations derived from normal and cancer cells. A substantial excess of single and tandem somatic mutations within known pol η mutable motifs was noted in skin cancer as well as in many other types of human cancer, suggesting that somatic mutations in A:T bases generated by DNA polymerase η are a common feature of tumorigenesis. Another peculiarity of pol ηmutational signatures, mutations in Y<u>C</u>G motifs, led us to speculate that error-prone DNA synthesis opposite methylated CpG dinucleotides by misregulated pol η in tumors might constitute an additional mechanism of cytosine demethylation in this hypermutable dinucleotide.</p