Absence of farnesol salvage in Candida albicans and probably in other fungi.

Farnesol salvage, a two-step pathway converting farnesol to farnesyl pyrophosphate (FPP), occurs in bacteria, plants, and animals. This paper investigates the presence of this pathway in fungi. Through bioinformatics, biochemistry, and physiological analyses, we demonstrate its absence in the yeasts Saccharomyces cerevisiae and Candida albicans, suggesting a likely absence across fungi. We screened 1,053 fungal genomes, including 34 from C. albicans, for potential homologs to four genes (Arabidopsis thaliana AtFOLK, AtVTE5, AtVTE6, and Plasmodium falciparum PfPOLK) known to accomplish farnesol/prenol salvage in other organisms. Additionally, we showed that 3H-farnesol was not converted to FPP or any other phosphorylated prenol, and exogenous farnesol was not metabolized within 90 minutes at any phase of growth and did not rescue cells from the toxic effects of atorvastatin, but it did elevate the levels of intracellular farnesol (Fi). All these experiments were conducted with C. albicans. In sum, we found no evidence for farnesol salvage in fungi. IMPORTANCE: The absence of farnesol salvage constitutes a major difference in the metabolic capabilities of fungi. In terms of fungal physiology, the lack of farnesol salvage pathways relates to how farnesol acts as a quorum-sensing molecule in Candida albicans and why farnesol should be investigated for use in combination with other known antifungal antibiotics. Its absence is essential for a model (K. W. Nickerson et al., Microbiol Mol Biol Rev 88:e00081-22, 2024), wherein protein farnesylation, protein chaperones, and the unfolded protein response are combined under the unifying umbrella of a cell's intracellular farnesol (Fi). In terms of human health, farnesol should have at least two different modes of action depending on whether those cells have farnesol salvage. Because animals have farnesol salvage, we can now see the importance of dietary prenols as well as the potential importance of farnesol in treating neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis.

Pan-cancer analysis reveals multifaceted roles of retrotransposon-fusion RNAs.

Transposon-derived transcripts are abundant in RNA sequences, yet their landscape and function, especially for fusion transcripts derived from unannotated or somatically acquired transposons, remains underexplored. Here, we developed a new bioinformatic tool to detect transposon-fusion transcripts in RNA-sequencing data and performed a pan-cancer analysis of 10,257 cancer samples across 34 cancer types as well as 3,088 normal tissue samples. We identified 52,277 cancer-specific fusions with ~30 events per cancer and hotspot loci within transposons vulnerable to fusion formation. Exonization of intronic transposons was the most prevalent genic fusions, while somatic L1 insertions constituted a small fraction of cancer-specific fusions. Source L1s and HERVs, but not Alus showed decreased DNA methylation in cancer upon fusion formation. Overall cancer-specific L1 fusions were enriched in tumor suppressors while Alu fusions were enriched in oncogenes, including recurrent Alu fusions in EZH2 predictive of patient survival. We also demonstrated that transposon-derived peptides triggered CD8+ T-cell activation to the extent comparable to EBV viruses. Our findings reveal distinct epigenetic and tumorigenic mechanisms underlying transposon fusions across different families and highlight transposons as novel therapeutic targets and the source of potent neoantigens.

Small RNAs >26 nt in length associate with AGO1 and are upregulated by nutrient deprivation in the alga Chlamydomonas.

Small RNAs (sRNAs) associate with ARGONAUTE (AGO) proteins forming effector complexes with key roles in gene regulation and defense responses against molecular parasites. In multicellular eukaryotes, extensive duplication and diversification of RNA interference (RNAi) components have resulted in intricate pathways for epigenetic control of gene expression. The unicellular alga Chlamydomonas reinhardtii also has a complex RNAi machinery, including 3 AGOs and 3 DICER-like proteins. However, little is known about the biogenesis and function of most endogenous sRNAs. We demonstrate here that Chlamydomonas contains uncommonly long (>26 nt) sRNAs that associate preferentially with AGO1. Somewhat reminiscent of animal PIWI-interacting RNAs, these >26 nt sRNAs are derived from moderately repetitive genomic clusters and their biogenesis is DICER-independent. Interestingly, the sequences generating these >26-nt sRNAs have been conserved and amplified in several Chlamydomonas species. Moreover, expression of these longer sRNAs increases substantially under nitrogen or sulfur deprivation, concurrently with the downregulation of predicted target transcripts. We hypothesize that the transposon-like sequences from which >26-nt sRNAs are produced might have been ancestrally targeted for silencing by the RNAi machinery but, during evolution, certain sRNAs might have fortuitously acquired endogenous target genes and become integrated into gene regulatory networks.

Divergent evolution of extreme production of variant plant monounsaturated fatty acids.

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

A consensus-based ensemble approach to improve transcriptome assembly.

BACKGROUND: Systems-level analyses, such as differential gene expression analysis, co-expression analysis, and metabolic pathway reconstruction, depend on the accuracy of the transcriptome. Multiple tools exist to perform transcriptome assembly from RNAseq data. However, assembling high quality transcriptomes is still not a trivial problem. This is especially the case for non-model organisms where adequate reference genomes are often not available. Different methods produce different transcriptome models and there is no easy way to determine which are more accurate. Furthermore, having alternative-splicing events exacerbates such difficult assembly problems. While benchmarking transcriptome assemblies is critical, this is also not trivial due to the general lack of true reference transcriptomes. RESULTS: In this study, we first provide a pipeline to generate a set of the simulated benchmark transcriptome and corresponding RNAseq data. Using the simulated benchmarking datasets, we compared the performance of various transcriptome assembly approaches including both de novo and genome-guided methods. The results showed that the assembly performance deteriorates significantly when alternative transcripts (isoforms) exist or for genome-guided methods when the reference is not available from the same genome. To improve the transcriptome assembly performance, leveraging the overlapping predictions between different assemblies, we present a new consensus-based ensemble transcriptome assembly approach, ConSemble. CONCLUSIONS: Without using a reference genome, ConSemble using four de novo assemblers achieved an accuracy up to twice as high as any de novo assemblers we compared. When a reference genome is available, ConSemble using four genome-guided assemblies removed many incorrectly assembled contigs with minimal impact on correctly assembled contigs, achieving higher precision and accuracy than individual genome-guided methods. Furthermore, ConSemble using de novo assemblers matched or exceeded the best performing genome-guided assemblers even when the transcriptomes included isoforms. We thus demonstrated that the ConSemble consensus strategy both for de novo and genome-guided assemblers can improve transcriptome assembly. The RNAseq simulation pipeline, the benchmark transcriptome datasets, and the script to perform the ConSemble assembly are all freely available from: http://bioinfolab.unl.edu/emlab/consemble/ .

Sterol Biosynthesis in Four Green Algae: A Bioinformatic Analysis of the Ergosterol Versus Phytosterol Decision Point.

Animals and fungi produce cholesterol and ergosterol, respectively, while plants produce the phytosterols stigmasterol, campesterol, and ß-sitosterol in various combinations. The recent sequencing of many algal genomes allows the detailed reconstruction of the sterol metabolic pathways. Here, we characterized sterol synthesis in two sequenced Chlorella spp., the free-living C. sorokiniana, and symbiotic C. variabilis NC64A. Chlamydomonas reinhardtii was included as an internal control and Coccomyxa subellipsoidea as a plant-like outlier. We found that ergosterol was the major sterol produced by Chlorella spp. and C. reinhardtii, while C. subellipsoidea produced the three phytosterols found in plants. In silico analysis of the C. variabilis NC64A, C. sorokiniana, and C. subellipsoidea genomes identified 22 homologs of sterol biosynthetic genes from Arabidopsis thaliana, Saccharomyces cerevisiae, and C. reinhardtii. The presence of CAS1, CPI1, and HYD1 in the four algal genomes suggests the higher plant cycloartenol branch for sterol biosynthesis, confirming that algae and fungi use different pathways for ergosterol synthesis. Phylogenetic analysis for 40 oxidosqualene cyclases (OSCs) showed that the nine algal OSCs clustered with the cycloartenol cyclases, rather than the lanosterol cyclases, with the OSC for C. subellipsoidea positioned in between the higher plants and the eight other algae. With regard to why C. subellipsoidea produced phytosterols instead of ergosterol, we identified 22 differentially conserved positions where C. subellipsoidea CAS and A. thaliana CAS1 have one amino acid while the three ergosterol producing algae have another. Together, these results emphasize the position of the unicellular algae as an evolutionary transition point for sterols.

Complete Genome Sequence of Geobacter sp. Strain FeAm09, a Moderately Acidophilic Soil Bacterium.

A moderately acidophilic Geobacter sp. strain, FeAm09, was isolated from forest soil. The complete genome sequence is 4,099,068 bp with an average GC content of 61.1%. No plasmids were detected. The genome contains a total of 3,843 genes and 3,608 protein-coding genes, including genes supporting iron and nitrogen biogeochemical cycling.

Next-generation transcriptome assembly and analysis: Impact of ploidy.

Whole genome duplications (WGD) occur widely in plants, but the effects of these events impact all branches of life. WGD events have major evolutionary impacts, often leading to major structural changes within the chromosomes and massive changes in gene expression that facilitate rapid speciation and gene diversification. Even for species that currently have diploid genomes, the impact of ancestral duplication events is still present in the genomes, especially in the context of highly similar gene families that are retained from WGD. However, the impact of these ploidies on various bioinformatics workflows has not been studied well. In this review, we overview biological significance of polyploidy in different organisms. We describe the impact of having polyploid transcriptomes on bioinformatics analyses, especially focusing on transcriptome assembly and transcript quantification. We discuss the benefits of using simulated benchmarking data when we examine the performance of various methods. We also present an example strategy to generate simulated allopolyploid transcriptomes and RNAseq datasets and how these benchmark datasets can be used to assess the performance of transcript assembly and quantification methods. Our benchmarking study shows that all transcriptome assembly methods are affected by having polyploid genomes. Quantification accuracy is also impacted by polyploidy depending on the method. These simulated datasets can be adapted for testing, such as, read mapping, variant calling, and differential expression using biologically realistic conditions.

miRNAs in the alga Chlamydomonas reinhardtii are not phylogenetically conserved and play a limited role in responses to nutrient deprivation.

The unicellular alga Chlamydomonas reinhardtii contains many types of small RNAs (sRNAs) but the biological role(s) of bona fide microRNAs (miRNAs) remains unclear. To address their possible function(s) in responses to nutrient availability, we examined miRNA expression in cells cultured under different trophic conditions (mixotrophic in the presence of acetate or photoautotrophic in the presence or absence of nitrogen). We also reanalyzed miRNA expression data in Chlamydomonas subject to sulfur or phosphate deprivation. Several miRNAs were differentially expressed under the various trophic conditions. However, in transcriptome analyses, the majority of their predicted targets did not show expected changes in transcript abundance, suggesting that they are not subject to miRNA-mediated RNA degradation. Mutant strains, defective in sRNAs or in ARGONAUTE3 (a key component of sRNA-mediated gene silencing), did not display major phenotypic defects when grown under multiple nutritional regimes. Additionally, Chlamydomonas miRNAs were not conserved, even in algae of the closely related Volvocaceae family, and many showed features resembling those of recently evolved, species-specific miRNAs in the genus Arabidopsis. Our results suggest that, in C. reinhardtii, miRNAs might be subject to relatively fast evolution and have only a minor, largely modulatory role in gene regulation under diverse trophic states.

Identification of AGO3-associated miRNAs and computational prediction of their targets in the green alga Chlamydomonas reinhardtii.

The unicellular green alga Chlamydomonas reinhardtii harbors many types of small RNAs (sRNAs) but little is known about their role(s) in the regulation of endogenous genes and cellular processes. To define functional microRNAs (miRNAs) in Chlamydomonas, we characterized sRNAs associated with an argonaute protein, AGO3, by affinity purification and deep sequencing. Using a stringent set of criteria for canonical miRNA annotation, we identified 39 precursor miRNAs, which produce 45 unique, AGO3-associated miRNA sequences including 13 previously reported miRNAs and 32 novel ones. Potential miRNA targets were identified based on the complementarity of miRNAs with candidate binding sites on transcripts and classified, depending on the extent of complementarity, as being likely to be regulated through cleavage or translational repression. The search for cleavage targets identified 74 transcripts. However, only 6 of them showed an increase in messenger RNA (mRNA) levels in a mutant strain almost devoid of sRNAs. The search for translational repression targets, which used complementarity criteria more stringent than those empirically required for a reduction in target protein levels, identified 488 transcripts. However, unlike observations in metazoans, most predicted translation repression targets did not show appreciable changes in transcript abundance in the absence of sRNAs. Additionally, of three candidate targets examined at the protein level, only one showed a moderate variation in polypeptide amount in the mutant strain. Our results emphasize the difficulty in identifying genuine miRNA targets in Chlamydomonas and suggest that miRNAs, under standard laboratory conditions, might have mainly a modulatory role in endogenous gene regulation in this alga.

Complementarity to an miRNA seed region is sufficient to induce moderate repression of a target transcript in the unicellular green alga Chlamydomonas reinhardtii.

MicroRNAs (miRNAs) are 20-24 nt non-coding RNAs that play important regulatory roles in a broad range of eukaryotes by pairing with mRNAs to direct post-transcriptional repression. The mechanistic details of miRNA-mediated post-transcriptional regulation have been well documented in multicellular model organisms. However, this process remains poorly studied in algae such as Chlamydomonas reinhardtii, and specific features of miRNA biogenesis, target mRNA recognition and subsequent silencing are not well understood. In this study, we report on the characterization of a Chlamydomonas miRNA, cre-miR1174.2, which is processed from a near-perfect hairpin RNA. Using Gaussia luciferase (gluc) reporter genes, we have demonstrated that cre-miR1174.2 is functional in Chlamydomonas and capable of triggering site-specific cleavage at the center of a perfectly complementary target sequence. A mismatch tolerance test assay, based on pools of transgenic strains, revealed that target hybridization to nucleotides of the seed region, at the 5' end of an miRNA, was sufficient to induce moderate repression of expression. In contrast, pairing to the 3' region of the miRNA was not critical for silencing. Our results suggest that the base-pairing requirements for small RNA-mediated repression in C. reinhardtii are more similar to those of metazoans compared with the extensive complementarity that is typical of land plants. Individual Chlamydomonas miRNAs may potentially modulate the expression of numerous endogenous targets as a result of these relaxed base-pairing requirements.

Small interfering RNA-mediated translation repression alters ribosome sensitivity to inhibition by cycloheximide in Chlamydomonas reinhardtii.

Small RNAs (sRNAs; ∼20 to 30 nucleotides in length) play important roles in gene regulation as well as in defense responses against transposons and viruses in eukaryotes. Their biogenesis and modes of action have attracted great attention in recent years. However, many aspects of sRNA function, such as the mechanism(s) of translation repression at postinitiation steps, remain poorly characterized. In the unicellular green alga Chlamydomonas reinhardtii, sRNAs derived from genome-integrated inverted repeat transgenes, perfectly complementary to the 3' untranslated region of a target transcript, can inhibit protein synthesis without or with only minimal mRNA destabilization. Here, we report that the sRNA-repressed transcripts are not altered in their polyadenylation status and they remain associated with polyribosomes, indicating inhibition at a postinitiation step of translation. Interestingly, ribosomes associated with sRNA-repressed transcripts show reduced sensitivity to translation inhibition by some antibiotics, such as cycloheximide, both in ribosome run-off assays and in in vivo experiments. Our results suggest that sRNA-mediated repression of protein synthesis in C. reinhardtii may involve alterations to the function/structural conformation of translating ribosomes. Additionally, sRNA-mediated translation inhibition is now known to occur in a number of phylogenetically diverse eukaryotes, suggesting that this mechanism may have been a feature of an ancestral RNA interference machinery.