Elsevier

Gene

Volume 767, 30 January 2021, 145186
Gene

Research paper
Functional chimeric genes in ciliates: An instructive case from Euplotes raikovi

https://doi.org/10.1016/j.gene.2020.145186Get rights and content

Highlights

  • Chimeric genes are generated by ciliates during the macronuclear development.

  • In Euplotes raikovi, a pheromone-coding gene, mac-er-1, coexists with a chimeric copy, mac-er-1*.

  • mac-er-1* originates from the mac-er-1 assembly with an extraneous sequence.

  • mac-er-1* is functional and practically duplicates the mac-er-1 activity.

Abstract

In ciliates, with every sexual event the transcriptionally active genes of the sub-chromosomic somatic genome that resides in the cell macronucleus are lost. They are de novo assembled starting from ‘Macronuclear Destined Sequences’ that arise from the fragmentation of transcriptionally silent DNA sequences of the germline chromosomic genome enclosed in the cell micronucleus. The RNA-mediated epigenetic mechanism that drives the assembly of these sequences is subject to errors which result in the formation of chimeric genes. Studying a gene family that in Euplotes raikovi controls the synthesis of protein signal pheromones responsible for a self/not-self recognition mechanism, we identified the chimeric structure of an 851-bp macronuclear gene previously known to specify soluble and membrane-bound pheromone molecules through an intron-splicing mechanism. This chimeric gene, designated mac-er-1*, conserved the native pheromone-gene structure throughout its coding and 3′ regions. Instead, its 5′ region is completely unrelated to the pheromone gene structure at the level of a 360-bp sequence, which derives from the assembly with a MDS destined to compound a 2417-bp gene encoding a 696-amino acid protein with unknown function. This mac-er-1* gene characterization provides further evidence that ciliates rely on functional chimeric genes that originate in non-programmed phenomena of somatic MDS recombination to increase the species genetic variability independently of gene reshuffling phenomena of the germline genome.

Introduction

Ciliates are a monophyletic group of single-celled organisms that, unlike all the other eukaryotes, evolved two nuclear genomes distinct in both structure and function that coexist in the same cytoplasm. A chromosomic and diploid germline genome enclosed in the cell micronucleus (MIC) is transcriptionally silent, while a sub-chromosomic and polyploid somatic genome inside the cell macronucleus (MAC) is transcriptionally active (Prescott, 1994). With every sexual event, manifested as conjugation or autogamy, the MAC genome is de novo generated starting from a mitotic product of the synkaryon and developing a sub-chromosomic organization through phenomena that are particularly impressive in the large group of spirotrich ciliates (Gong et al., 2020, Jiang et al., 2019, Juranek and Lipps, 2007). In a rapid sequence, they involve chromosome polytenization, chromosome fragmentation, DNA elimination and gene amplification (Chalker and Yao, 2011, Coyne et al., 1996, Jahn and Klobutcher, 2002, Yao et al., 2002).

In Oxytricha trifallax, in which these phenomena are best described (Bowen et al., 2013, Chen et al., 2014), the fragmentation of polytene chromosomes results in a DNA loss of nearly 90% with the production of an estimated number of 225,000 fragments known as ‘Macronuclear Destined Sequences’ (MDSs). Under the control of noncoding RNA templates synthesized by the maternal MAC before being destroyed, these MDSs are assembled into sub-chromosomic, or ‘gene-size’ DNA molecules (also reported as ‘nano-chromosomes’) destined to compound the new transcriptionally active MAC (Nowacki et al., 2008, Nowacki et al., 2011, Swart et al., 2013, Chen et al., 2014). Ranging in size from approximately only 400 to 20,000 bp, these DNA molecules are de facto individual MAC genes that usually include a single protein-coding region flanked by short telomere-capped 5′ and 3′ regions (Prescott, 1994, Swart et al., 2013). Although so simple in structure, the MAC genes are subject to assembly errors arising from MDSs that, irrespective of their final gene destination, recombine with the sequence of another gene that is not their home gene. The result of these errors is the formation of chimeric genes that come to be stably integrated into the transcriptionally active MAC genome of the cell, in which they can be transmitted faithfully across the next generations through the RNA-mediated epigenetic control imposed by the old maternal MAC onto the new one (Chalker et al., 2013, Maurer-Alcalá and Nowacki, 2019, Neeb and Nowacki, 2018, Nowacki and Landweber, 2009, Nowacki et al., 2011).

In another spirotrich ciliate, Euplotes raikovi, we report here the chimeric nature of a MAC gene, designated mac-er-1* given its kinship with the family of MAC pheromone genes designated mac-er-1, mac-er-2, mac-er-3, and so forth. These genes are the expressed versions of transcriptionally silent high-multiple alleles that segregate in Mendelian fashion at the mat (mating-type) locus of the MIC germline genome, and encode diffusible signaling protein pheromones, designated Er-1, Er-2, Er-3 and so forth, each conferring a chemical specificity (phenotype) to otherwise morphologically identical conspecific cells. In practice, the mac-er-1 gene encodes pheromone Er-1 distinctive of type-I cells, the mac-er-2 gene encodes pheromone Er-2 distinctive of type-II cells, the mac-er-3 gene encodes pheromone Er-3 distinctive of type-III cells, and so forth (Luporini et al., 2005, Luporini et al., 2014, Vallesi et al., 2016).

The mac-er-1* structure was originally reconstructed from two mRNA sequences generated in type-I cells through a mechanism of intron splicing and used to synthesize soluble and membrane-bound Er-1 molecules (Miceli et al., 1989, Miceli et al., 1992). Its chimeric origin was suggested by comparing a set of eight MAC pheromone coding genes cloned from a group of interbreeding cell types and observing that the mac-er-1* 5′ region upstream of the ATG codon of the open reading frame was structurally unique, unrelated to the 5′ region of the other MAC pheromone coding genes (Ricci et al., 2019). We show that the mac-er-1* chimeric structure derives from the assembly with an MDS destined to a 2417-bp MAC gene that is extraneous to the pheromone system.

Section snippets

Cell cultures and Rapid Amplification of Telomeric Ends (RATE)-PCR

Clonal cultures of E. raikovi cell types I, II and XI were cultivated following routine procedures (Luporini et al., 1986) and used for DNA extraction as previously described (Ricci et al., 2019, Vallesi et al., 2014).

The RATE-PCR procedure was applied using the C4A4 repeats, distinctive of the 5′ and 3′ telomeric ends of Euplotes MAC genes (Klobutcher et al., 1981), as primer target in combination with oligonucleotides internal to the gene sequences. Amplification reactions were run in an

mac-er-1* gene cloning

As outlined in the Introduction, the mac-er-1* gene was originally identified and characterized following a cDNA library screening , which resulted in a partial 604-bp sequence (Miceli et al., 1992). The initial experimental step was thus directed to clone the mac-er-1* full-length sequence from the type-I cells, and compare this sequence with the 1033-bp sequence of the mac-er-1 gene recently cloned from the same type-I cells (Ricci et al., 2019). A 385-bp amplicon equivalent to the gene 5′

Discussion

Chimeric genes are widespread in nature, and mostly result from errors in DNA replication or repair that determine chance combinations between pieces of different genes, as well as from aberrant phenomena of ectopic recombination between unrelated genomic portions, or retro-transposition in which a transcript is accidentally copied by a retrotransposon and inserted into a new genome site (Long et al., 2003, Long et al., 2013). While many are predictably dysfunctional and subject to elimination

Declaration of Competing Interest

The authors declare that they have no conflicts of interest with the contents of this article.

Acknowledgements

This work was financially supported by the Ministero dell’Università e Ricerca, PRIN research project no. 20109XZEPR. We would like to thank Sheila Beatty for editing the English usage in the manuscript.

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