Molecular Biology of Euglenids

@article{GUMINSKA2024126033,

title = {Circular extrachromosomal DNA in Euglena gracilis under normal and stress conditions},

author = {Natalia Gumińska and Paweł Hałakuc and Bożena Zakryś and Rafał Milanowski},

url = {https://www.sciencedirect.com/science/article/pii/S1434461024000257},

doi = {https://doi.org/10.1016/j.protis.2024.126033},

issn = {1434-4610},

year  = {2024},

date = {2024-04-03},

urldate = {2024-01-01},

journal = {Protist},

volume = {175},

number = {3},

pages = {126033},

abstract = {Extrachromosomal circular DNA (eccDNA) enhances genomic plasticity, augmenting its coding and regulatory potential. Advances in high-throughput sequencing have enabled the investigation of these structural variants. Although eccDNAs have been investigated in numerous taxa, they remained understudied in euglenids. Therefore, we examined eccDNAs predicted from Illumina sequencing data of Euglena gracilis Z SAG 1224–5/25, grown under optimal photoperiod and exposed to UV irradiation. We identified approximately 1000 unique eccDNA candidates, about 20% of which were shared across conditions. We also observed a significant enrichment of mitochondrially encoded eccDNA in the UV-irradiated sample. Furthermore, we found that the heterogeneity of eccDNA was reduced in UV-exposed samples compared to cells that were grown in optimal conditions. Hence, eccDNA appears to play a role in the response to oxidative stress in Euglena, as it does in other studied organisms. In addition to contributing to the understanding of Euglena genomes, our results contribute to the validation of bioinformatics pipelines on a large, non-model genome.},

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}

@article{Haakuc2022,

title = {Typical structure of rRNA coding genes in diplonemids points to two independent origins of the bizarre rDNA structures of euglenozoans},

author = {Paweł Hałakuc and Anna Karnkowska and Rafał Milanowski},

url = {https://doi.org/10.1186/s12862-022-02014-9

https://bmcevolbiol.biomedcentral.com/articles/10.1186/s12862-022-02014-9},

doi = {10.1186/s12862-022-02014-9},

issn = {2730-7182},

year  = {2022},

date = {2022-05-09},

journal = {BMC Ecology and Evolution},

volume = {22},

number = {1},

pages = {59},

abstract = {Members of Euglenozoa (Discoba) are known for unorthodox rDNA organization. In Euglenida rDNA is located on extrachromosomal circular DNA. In Kinetoplastea and Euglenida the core of the large ribosomal subunit, typically formed by the 28S rRNA, consists of several smaller rRNAs. They are the result of the presence of additional internal transcribed spacers (ITSs) in the rDNA. Diplonemea is the third of the main groups of Euglenozoa and its members are known to be among the most abundant and diverse protists in the oceans. Despite that, the rRNA of only one diplonemid species, Diplonema papillatum, has been examined so far and found to exhibit continuous 28S rRNA. Currently, the rDNA organization has not been researched for any diplonemid. Herein we investigate the structure of rRNA genes in classical (Diplonemidae) and deep-sea diplonemids (Eupelagonemidae), representing the majority of known diplonemid diversity. The results fill the gap in knowledge about diplonemid rDNA and allow better understanding of the evolution of the fragmented structure of the rDNA in Euglenozoa.},

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pubstate = {published},

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}

@article{GUMINSKA2021166758,

title = {A New Type of Circular RNA derived from Nonconventional Introns in Nuclear Genes of Euglenids},

author = {Natalia Gumińska and Bożena Zakryś and Rafał Milanowski},

url = {https://www.sciencedirect.com/science/article/pii/S0022283620306835?via%3Dihub},

doi = {https://doi.org/10.1016/j.jmb.2020.166758},

year  = {2021},

date = {2021-02-05},

journal = {Journal of Molecular Biology},

volume = {433},

number = {3},

pages = {166758},

abstract = {Nuclear protein-coding genes of euglenids (Discoba, Euglenozoa, Euglenida) contain conventional (spliceosomal) and nonconventional introns. The latter have been found only in euglenozoans. A unique feature of nonconventional introns is the ability to form a stable and slightly conserved RNA secondary structure bringing together intron ends and placing adjacent exons in proximity. To date, little is known about the mechanism of their excision (e.g. whether it involves the spliceosome or not). The tubA gene of Euglena gracilis harbors three conventional and three nonconventional introns. While the conventional introns are excised as lariats, nonconventional introns are present in the cell solely as circular RNAs with full-length ends. Based on this discovery as well as on previous observations indicating that nonconventional introns are observed frequently at unique positions of genes, we suggest that this new type of intronic circRNA might play a role in intron mobility.},

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pubstate = {published},

tppubtype = {article}

}

@article{Guminska2018,

title = {Culture purification and DNA extraction procedures suitable for next-generation sequencing of euglenids},

author = {Natalia Gumińska and Magdalena Płecha and Halszka Walkiewicz and Paweł Hałakuc and Bożena Zakryś and Rafał Milanowski},

url = {https://doi.org/10.1007/s10811-018-1496-0},

doi = {10.1007/s10811-018-1496-0},

issn = {1573-5176},

year  = {2018},

date = {2018-01-01},

journal = {Journal of Applied Phycology},

volume = {30},

number = {6},

pages = {3541--3549},

abstract = {In the present study, five different DNA extraction procedures were examined to determine their effectiveness for extracting DNA suitable for NGS applications. This included two silica-membrane spin column kits, phenol:chloroform, and two CTAB-based methods. Spectrophotometric and fluorimetric measurements as well as standard gel electrophoresis were used as criteria for evaluating the quantity and quality of the isolated DNA prior to the sequencing. Herein, the method of establishing and maintaining axenic Euglena cultures is also presented. The modified CTAB-based method proved to be highly efficient. In terms of DNA quantity and purity (according to the absorbance ratios), the chosen method resulted in DNA of high molecular weight and quality, which fulfills the library construction requirements. Genomic DNA of Euglena hiemalis (CCAP 1224/35) and E. longa (CCAP 1204-17a) isolated using the suggested protocol had been successfully sequenced on the Illumina HiSeq platform. A modified, rapid CTAB-based method of total DNA isolation from Euglena has been described. In terms of the DNA quantity and quality, the protocol devised involving the washing step with DMSO:acetonitrile proved superior to the commonly used, commercially manufactured kits and isolation with phenol:chloroform. The method is also less labor-intensive and time-consuming than the traditional CTAB-based protocol.},

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}

@article{Guminska2018b,

title = {Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis},

author = {Natalia Gumińska and Magdalena Płecha and Bożena Zakryś and Rafał Milanowski},

doi = {10.1371/journal.pgen.1007761},

issn = {15537404},

year  = {2018},

date = {2018-01-01},

journal = {PLoS Genetics},

volume = {14},

number = {10},

pages = {e1007761},

abstract = {Nuclear genes of euglenids and marine diplonemids harbor atypical, nonconventional introns which are not observed in the genomes of other eukaryotes. Nonconventional introns do not have the conserved borders characteristic for spliceosomal introns or the sequence complementary to U1 snRNA at the 5' end. They form a stable secondary structure bringing together both exon/intron junctions, nevertheless, this conformation does not resemble the form of self-splicing or tRNA introns. In the genes studied so far, frequent nonconventional introns insertions at new positions have been observed, whereas conventional introns have been either found at the conserved positions, or simply lost. In this work, we examined the order of intron removal from Euglena gracilis transcripts of the tubA and gapC genes, which contain two types of introns: nonconventional and spliceosomal. The relative order of intron excision was compared for pairs of introns belonging to different types. Furthermore, intermediate products of splicing were analyzed using the PacBio Next Generation Sequencing system. The analysis led to the main conclusion that nonconventional introns are removed in a rapid way but later than spliceosomal introns. Moreover, the observed accumulation of transcripts with conventional introns removed and nonconventional present may suggest the existence of a time gap between the two types of splicing.},

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pubstate = {published},

tppubtype = {article}

}

@article{Milanowski2016,

title = {Intermediate introns in nuclear genes of euglenids – are they a distinct type?},

author = {Rafał Milanowski and Natalia Gumińska and Anna Karnkowska and Takao Ishikawa and Bożena Zakryś},

url = {https://bmcecolevol.biomedcentral.com/articles/10.1186/s12862-016-0620-5},

doi = {10.1186/s12862-016-0620-5},

issn = {1471-2148},

year  = {2016},

date = {2016-12-01},

journal = {BMC Evolutionary Biology},

volume = {16},

number = {1},

pages = {49},

abstract = {textcopyright 2016 Milanowski et al. Background: Nuclear genes of euglenids contain two major types of introns: Conventional spliceosomal and nonconventional introns. The latter are characterized by variable non-canonical borders, RNA secondary structure that brings intron ends together, and an unknown mechanism of removal. Some researchers also distinguish intermediate introns, which combine features of both types. They form a stable RNA secondary structure and are classified into two subtypes depending on whether they contain one (intermediate/nonconventional subtype) or both (conventional/intermediate subtype) canonical spliceosomal borders. However, it has been also postulated that most introns classified as intermediate could simply be special cases of conventional or nonconventional introns. Results: Sequences of tubB, hsp90 and gapC genes from six strains of Euglena agilis were obtained. They contain four, six, and two or three introns, respectively (the third intron in the gapC gene is unique for just one strain). Conventional introns were present at three positions: Two in the tubB gene (at one position conventional/intermediate introns were also found) and one in the gapC gene. Nonconventional introns are present at ten positions: T wo in the tubB gene (at one position intermediate/nonconventional introns were also found), six in hsp90 (at four positions intermediate/nonconventional introns were also found), and two in the gapC gene. Conclusions: Sequence and RNA secondary structure analyses of nonconventional introns confirmed that their most strongly conserved elements are base pairing nucleotides at positions +4, +5 and +6/-8,-7 and-6 (in most introns CAG/CTG nucleotides were observed). It was also confirmed that the presence of the 5' GT/C end in intermediate/nonconventional introns is not the result of kinship with conventional introns, but is due to evolutionary pressure to preserve the purine at the 5' end. However, an example of a nonconventional intron with GC-AG ends was shown, suggesting the possibility of intron type conversion between nonconventional and conventional. Furthermore, an analysis of conventional introns revealed that the ability to form a stable RNA secondary structure by some introns is probably not a result of their relationship with nonconventional introns. It was also shown that acquisition of new nonconventional introns is an ongoing process and can be observed at the level of a single species. In the recently acquired intron in the gapC gene an extended direct repeats at the intron-exon junctions are present, suggesting that double-strand break repair process could be the source of new nonconventional introns.},

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pubstate = {published},

tppubtype = {article}

}

@article{Milanowski2014,

title = {Distribution of Conventional and Nonconventional Introns in Tubulin (alpha and beta) Genes of Euglenids},

author = {Rafał Milanowski and Anna Karnkowska and Takao Ishikawa and Bożena Zakryś},

url = {https://academic.oup.com/mbe/article-lookup/doi/10.1093/molbev/mst227},

doi = {10.1093/molbev/mst227},

issn = {1537-1719},

year  = {2014},

date = {2014-03-01},

journal = {Molecular Biology and Evolution},

volume = {31},

number = {3},

pages = {584--593},

abstract = {The nuclear genomes of euglenids contain three types of introns: conventional spliceosomal introns, nonconventional introns for which a splicing mechanism is unknown (variable noncanonical borders, RNA secondary structure bringing together intron ends), and so-called intermediate introns, which combine features of conventional and nonconventional introns. Analysis of two genes, tubA and tubB, from 20 species of euglenids reveals contrasting distribution patterns of conventional and nonconventional introns - positions of conventional introns are conserved, whereas those of the nonconventional ones are unique to individual species or small groups of closely related taxa. Moreover, in the group of phototrophic euglenids, 11 events of conventional intron loss versus 15 events of nonconventional intron gain were identified. A comparison of all nonconventional intron sequences highlighted the most conserved elements in their sequence and secondary structure. Our results led us to put forward two hypotheses. 1) The first one posits that mutational changes in intron sequence could lead to a change in their excision mechanism - intermediate introns would then be a transitional form between the conventional and nonconventional introns. 2) The second hypothesis concerns the origin of nonconventional introns - because of the presence of inverted repeats near their ends, insertion of MITE-like transposon elements is proposed as a possible source of new introns. textcopyright 2013 The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.},

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}