Evolution of introns in nuclear genes of euglenids

Nuclear genes of euglenids contain two types of introns: conventional spliceosomal introns with GT/C-AG borders and nonconventional introns. Nonconventional introns have non-canonical borders and form a stable RNA secondary structure bringing together both ends. Conventional introns are present at conserved positions, while nonconventional ones at positions unique to individual species or clades grouping closely related taxa. Some introns (intermediate introns) have features of both types and it is expected that they could be a transitional form between the conventional and nonconventional introns. Knowledge on the acquisition of new nonconventional introns is limited to data for individual genes. The best source of larger data sets seems to be sequencing of nuclear genomes of different representatives of euglenids. Therefore the main objective of the project is sequencing of nuclear genomes of Euglena hyemalis (the species most closely related to Euglena gracilis, for which the nuclear genome sequencing project is almost finished) and Euglena longa (the secondarily heterotrophic species). The analysis of introns’ sequences and introns’ distribution in homologous genes will allow for: (1) estimation of the scope of intron gains and loses, (2) estimation of the number of new nonconventional introns acquired after the split of evolutionary lineages E. longa – E. hyemalis/E. gracilis and E. hyemalis – E. gracilis, (3) estimation of most conserved elements within nonconventional introns based on the large amount of data, (4) comparision of the sequence/structure of old nonconventional introns (present in all species) and newly acquired ones (unique for E. hyemalis or E. gracilis), which could help to identify elements characteristic for the source sequence of nonconventional introns and also for mechanism of introns acquisition, (5) identification of intermediate introns excised by two different mechanisms, (6) identification of introns for which the change of the type/mechanism of excision took place.

Two next generation sequencing technologies will be applied. Illumina sequencing technology with pair-end and mate-pairs libraries will be used to obtain contigs with high coverage and PacBio method for sequencing of long fragments required for scaffolding. Obtained and available transcriptomic data will be used to support the process of genes prediction. Predicted gene models will be compared for two genomes obtained in the project and also for genome of E. gracilis. Homologues will be identified, then comparative analyses of introns distribution, as well as analyses of the sequence and structure of nonconventional introns will be carried out.

The analysis of nuclear genomes of euglenids will allow for global comparison of introns distribution in different species and for searching of intermediate introns. Genomic data will also enable more detailed analysis of conserved features of nonconventional introns, especially those that have been inserted relatively recently. It would help to identify their origin and the mechanism of insertions. The obtained data on evolution of nonconventional introns in nuclear genes of euglenids will contribute to expanding our knowledge about the role of intervening sequences in eukaryotic genes, which is still not completely understood.

Conferences

• Semik A., Hałakuc P., Mikina W., Milanowski R. (2022). Statistical machine learning aiding the recognition of noncanonical introns in three Euglena genomes. 41th Jirovec’s Protozoological Days by Czech Society for Parasitology, 21-24 June, Svratka, Czech Republic.
• Hałakuc P., Jagielska M., Płecha M., Maciszewski K., Zakryś B., Milanowski R., Karnkowska A. (2022). Structure and evolution of mitochondrial genomes in phototrophic Euglenids. 41th Jirovec’s Protozoological Days by Czech Society for Parasitology, 21-24 June, Svratka, Czech Republic.
• Jagielska M., Płecha M., Hałakuc P., Gumińska N., Karnkowska A., Zakryś B., Milanowski R. (2022). The analysis of whole genome assemblies (WGA) reveals a bizzare structure of euglenids mitochondrial genomes. 41th Jirovec’s Protozoological Days by Czech Society for Parasitology, 21-24 June, Svratka, Czech Republic.
• Mikina W., Hałakuc P. Milanowski R. (2021). Manual annotation as a key reference for automatic prediction of nonconventional introns. 1st Annual International Congress on Euglenoids. 8-11 November, Online.
• Hałakuc P., Semik A., Mikina W., Różycka J., Płecha M., Gumińska N., Karnkowska A., Milanowski R. (2021). Canonical and nonconventional introns in three new Euglena genomes. 1st Annual International Congress on Euglenoids. 8-11 November, Online.
• Płecha M., Hałakuc P., Jagielska M., Karnkowska A., Milanowski R. (2021). The analysis of whole genome assemblies (WGA) reveals a bizarre structure of euglenids mitochondrial genomes. 1st Annual International Congress on Euglenoids. 8-11 November, Online.
• Płecha M., Hałakuc P., Jagielska M., Karnkowska A., Milanowski R. (2021). Mitochondrial genomes of Euglena gracilis , E. hiemalis and E. longa obtained from the whole genome assemblies (WGA). EMBO Young Scientists Forum. 21-22 October, Warsaw, Poland.
• Hałakuc P., Płecha M., Różycka J., Milanowski R., Karnkowska A. (2020). Nonconventional genomics of Euglena species. (2020). 4th EMBO Workshop on Computational and Structural Biology and Chemistry; 28-29.02, Waplewo, Poland
• Hałakuc P., Gumińska N., Karnkowska A., Milanowski R. (2019) Exception within exceptions – unusual organization of rRNA genes in Euglena longa – poster, VIII European Congress of Protistology – ISOP joint meeting; 28 lipca – 2 sierpnia, Rzym, Włochy
• Hałakuc P., Karnkowska A., Milanowski R. (2018). Tracing evolutionary changes in rRNA genes in Euglenozoa. The Sixth Polish Evolutionary Conference. 26-28 September, Warsaw, Poland.
• Płecha M., Walkiewicz H., Gumińska N., Karnkowska A., Zakryś B., Milanowski R. (2018). Evolution of introns in the Euglena hiemalis genes based on the draft genome assembly analysis. The Sixth Polish Evolutionary Conference. 26-28 September, Warsaw, Poland.
• Płecha M., Walkiewicz H., Gumińska N., Karnkowska A., Zakryś B., Milanowski R. (2018). Annotation-directed draft genome of the non-model species Euglena hiemalis. XXII meeting of the International Society for Evolutionary Protistology, 27 May – 1 June, Droushia village, Cyprus.
• Hałakuc P., Karnkowska A., Milanowski R. (2018). Tracing evolutionary changes in rRNA genes in Euglenozoa. 48^th Jirovec’s Protozoological Days by Czech Society for Parasitology, 1-4 May, Ostrava, Czech Republic.
• Walkiewicz H., Płecha M., Karnkowska A., Gawryluk R., Keeling P.J., Milanowski R. (2018). Characteristics of nonconventional introns in genomes of marine diplonemids. 48^th Jirovec’s Protozoological Days by Czech Society for Parasitology, 1-4 May, Ostrava, Czech Republic.
• Wysocka-Korzun H., Płecha M., Karnkowska A., Gawryluk R., Keeling P.J., Milanowski R. (2017). Analysis of nonconventional introns in genomes of marine diplonemids. 15^th International Congress of Protistology, 30 July – 4 August, Prague, Czech Republic.
• Płecha M., Wysocka-Korzun H., Gumińska N., Karnkowska A., Zakryś B., Milanowski R. (2017). Whole genome studies of two non-model species of euglenids: Euglena longa and E. hiemalis. 15^th International Congress of Protistology, 30 July – 4 August, Prague, Czech Republic.
• Hałakuc P., Karnkowska A., Milanowski R. (2017). Evolution of ribosomal RNA genes in Euglenozoa. 15^th International Congress of Protistology, 30 July – 4 August, Prague, Czech Republic.

Diploma thesis

• Weronika Mikina (2023). Analiza zdarzeń ewolucyjnych kształtujących strukturę genomów jądrowych euglenin na podstawie manualnej adnotacji wybranych genów. Master thesis, Biotechnology.
• Maria Jagielska (2022). Analiza genomów mitochondrialnych wybranych gatunków euglenin. Master thesis, Biologia.
• Anna Semik (2021). Analiza intronów niekonwencjonalnych w genach jądrowych euglenin. Bachelor thesis, Bioinformatics and Systems Biology.
• Antonina Ignatenko (2019). Mechanizmy utraty i nabywania intronów spliceosomalnych w genach jądrowych eukariontów. Bachelor thesis, Biotechnology.
• Paweł Hałakuc (2018). Tracing evolutionary changes in rRNA genes of Euglenozoa. Master thesis, Biotechnology, MISMaP.

Publications

@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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Soukal2021,

title = {Heterotrophic euglenid Rhabdomonas costata resembles its phototrophic relatives in many aspects of molecular and cell biology},

author = {Petr Soukal and Štěpánka Hrdá and Anna Karnkowska and Rafał Milanowski and Jana Szabová and Miluše Hradilová and Hynek Strnad and Čestmír Vlček and Ivan Čepička and Vladimír Hampl},

url = {https://doi.org/10.1038/s41598-021-92174-3 

http://www.nature.com/articles/s41598-021-92174-3},

doi = {10.1038/s41598-021-92174-3},

issn = {2045-2322},

year  = {2021},

date = {2021-12-01},

journal = {Scientific Reports},

volume = {11},

number = {1},

pages = {13070},

abstract = {Euglenids represent a group of protists with diverse modes of feeding. To date, only a partial genomic sequence of Euglena gracilis and transcriptomes of several phototrophic and secondarily osmotrophic species are available, while primarily heterotrophic euglenids are seriously undersampled. In this work, we begin to fill this gap by presenting genomic and transcriptomic drafts of a primary osmotroph, Rhabdomonas costata . The current genomic assembly length of 100 Mbp is 14× smaller than that of E. gracilis . Despite being too fragmented for comprehensive gene prediction it provided fragments of the mitochondrial genome and comparison of the transcriptomic and genomic data revealed features of its introns, including several candidates for nonconventional types. A set of 39,456 putative R. costata proteins was predicted from the transcriptome. Annotation of the mitochondrial core metabolism provides the first data on the facultatively anaerobic mitochondrion of R. costata , which in most respects resembles the mitochondrion of E. gracilis with a certain level of streamlining. R. costata can synthetise thiamine by enzymes of heterogenous provenances and haem by a mitochondrial-cytoplasmic C4 pathway with enzymes orthologous to those found in E. gracilis . The low percentage of green algae-affiliated genes supports the ancestrally osmotrophic status of this species.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ignatenko2019,

title = {Mechanizmy utraty i nabywania intronów spliceosomalnych},

author = {Antonina Ignatenko and Natalia Gumińska and Rafał Milanowski},

url = {https://postepybiochemii.ptbioch.edu.pl/index.php/PB/article/view/292},

doi = {10.18388/pb.2019_292},

issn = {00325422},

year  = {2019},

date = {2019-01-01},

journal = {Postepy biochemii},

volume = {65},

number = {4},

pages = {289--298},

abstract = {Introny to wewnątrzgenowe sekwencje niekodujące. Dawniej były uznawane za „śmieciowy” DNA, jednak obecnie uważa się, że są ważnymi elementami wpływającymi na funkcjonowanie genomu. Udowodniono, że introny zwiększają różnorodność transkryptomu i proteomu, spełniają w komórce role regulatorowe, wpływają na ekspresję genów oraz obróbkę, translację i degradację mRNA. Ze względu na sposób powstawania dzielą się na trzy główne kategorie: spliceosomalne, samowycinające się oraz introny tRNA. Introny spliceosomalne są charakterystyczne dla organizmów eukariotycznych. Analizy sekwencji genów ortologicznych w rożnych grupach eukariontów pozwoliły zidentyfikować wiele przypadków nabywania oraz utraty intronów. Niektóre z tych zdarzeń miały miejsce w dalekiej przeszłości, do innych doszło stosunkowo niedawno. Uważa się, że procesy te mogą działać jako jedna z sił napędowych w ewolucji genów eukariotycznych.},

keywords = {},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}