Rolling-circle transposons catalyze genomic innovation in a mammalian lineage

Abstract

Rolling-circle transposons (Helitrons) are a newly discovered group of mobile DNA widespread in plant and invertebrate genomes but limited to the bat family Vespertilionidae among mammals. Little is known about the long-term impact of Helitron activity because the genomes where Helitron activity has been extensively studied are predominated by young families. Here, we report a comprehensive catalog of vetted Helitrons from the 7× Myotis lucifugus genome assembly. To estimate the timing of transposition, we scored presence/absence across related vespertilionid genome sequences with estimated divergence times. This analysis revealed that the Helibat family has been a persistent source of genomic innovation throughout the vespertilionid diversification from approximately 30-36 Ma to as recently as approximately 1.8-6 Ma. This is the first report of persistent Helitron transposition over an extended evolutionary timeframe. These findings illustrate that the pattern of Helitron activity is akin to the vertical persistence of LINE retrotransposons in primates and other mammalian lineages. Like retrotransposition in primates, rolling-circle transposition has generated lineage-specific variation and accounts for approximately 110 Mb, approximately 6% of the genome of M. lucifugus. The Helitrons carry a heterogeneous assortment of host sequence including retroposed messenger RNAs, retrotransposons, DNA transposons, as well as introns, exons and regulatory regions (promoters, 5'-untranslated regions [UTRs], and 3'-UTRs) of which some are evolving in a pattern suggestive of purifying selection. Evidence that Helitrons have contributed putative promoters, exons, splice sites, polyadenylation sites, and microRNA-binding sites to transcripts otherwise conserved across mammals is presented, and the implication of Helitron activity to innovation in these unique mammals is discussed.

Keywords: Helitron; Vespertilionidae bat family; gene capture; gene duplication; retrogene; transposable element.

Fig. 1.—

The abundance of Helitron generated DNA in different organisms. The front (blue) row represents percent of the genome composed of Helitrons and the back (red) row indicates the amount of DNA (in Mb) contributed by Helitrons. The different organisms include Arabidopsis thaliana (1.3%, 1.56 Mb), Medicago trunculata (1.3%, 4.1 Mb), Oryza sativa spp. japonica (2.1%, 9 Mb), Sorghum bicolor (3%, 22.2 Mb), Zea mays (2.2%, 45.4 Mb), Caenorhabditis elegans (2.3%, 2.3 Mb) (Yang and Bennetzen 2009b), Nematostella vectensis (3%, 8.9 Mb) (Putnam et al. 2007), Bombyx mori (4.2%, 19.7 Mb), Heliconius melpomene (6.6%, 17.8 Mb) (Han et al. 2013), and Myotis lucifugus (5.8%, 109.8 Mb).

Fig. 2.—

Helitrons carry different kinds of genic fragments in variable copy numbers. A representative of the Helitron containing the gene fragment was compared with the corresponding human gene to identify the nature of the gene fragment captured. Numbers above the bars denote the number of genes from which the respective genic region (x axis) was captured. The x axis corresponds to the type of genic region captured by the Helitron and y axis corresponds to the copy number of Helitrons containing the genic regions. A detailed list of the captured genes and their copy numbers are provided in supplementary table S2, Supplementary Material online.

Fig. 3.—

Continuous activity and sequential capture of gene fragments by Helitrons. (A) The phylogenetic relationship of the three vespertilionid bat genome sequences publicly available (used in this study) and the sister family Miniopteridae. Activity of Helitrons is limited to the vespertilionid bat lineage and is estimated to have begun around 30–36 Ma (red lighting bolt) (Pritham and Feschotte 2007). The timing of divergence is represented at each node (in Myr) (Miller-Butterworth et al. 2007; Stadelmann et al. 2007; Gu et al. 2008). The number within the parenthesis represents the lineage-specific Helitron copies with host sequences. The lineage-specific insertions in the M. davidii and Eptesicus genome are underestimates as we did not thoroughly analyze the Helitron content of those genomes. (B) A cartoon structure of the Helitron containing TMBIM4 (involved in apoptosis inhibition (Saraiva et al. 2013) gene fragment, HelibatN217.1. (C) An alignment of the TMBIM4 fragment within HelibatN217.1 to that of the host fragment from the mouse lemur, bat (parental), and human genome. The captured region shares 87% identity over 223 bp (excluding gaps) with the TMBIM4 parental gene and occurs in the reverse orientation relative to the Helitron. (D) A cartoon representation of the structure of the Helitron with TMBIM4 fragment and the sequentially captured TACC3 (involved in stabilizing spindle microtubules [for review, Gergely 2002]) retrogene, HelibatN22. (E) An alignment of the TACC3 retrogene fragment within HelibatN22 to the sequence of a cDNA from human and the retrogene from bat (not parental) and horse. The captured region spanning the last four exons and the 3′-UTR of the gene shared approximately 78% sequence identity over 265-bp excluding gaps. The color of the lines above the alignment indicates the region of the gene (e.g., red line shows 5′-UTR, blue line shows the coding exons, purple line shows the 3′-UTR, and pink line denotes the intron).

Fig. 4.—

A recently active Helitron in M. lucifugus. (A) The structure of HelibatN541, the Helitron unique to the M. lucifugus genome, which carries a small piece of an exon and the complete 3′-UTR of the TACC3 retrogene fragment. (B) An alignment of the each copy of HelibatN541 family to its consensus (made from five copies by majority rule). The 5′ and 3′ terminal 30 bp and internal 30 bp are aligned. (C) Alignment of the insertion sites of HelibatN541 copies in M. lucifugus genome to the corresponding empty sites in the M. davidii genome.

Fig. 5.—

RPLP0 mRNA retroposed into the exemplar HelibatN211. (A) The structure of the HelibatN211 exemplar, which contains SRPK1 gene fragment. (B) Alignment of the SRPK1 (a single copy human gene known to play a regulatory role in intron splicing [for review, Giannakouros et al. 2011]) gene fragment within Helitron to human and walrus. Yellow and orange lines show the 30 bp at the 5′-end and 3′-end, respectively. Cyan line corresponds to the promoter regions. The black triangle shows the position of the retroposition event. (C) The structure of HelibatN424, which represents HelibatN211 after the retroposition of the RPLP0 (a component of the 60S subunit of the ribosome) mRNA. (D) An alignment of RPLP0 cDNA carried by Helitron to the human, mouse RPLP0 cDNAs, and RPLP0 gene (without introns) from M. lucifugus. The TSDs generated during retroposition are marked in green. The colors of the lines above the alignment indicate the corresponding regions: 5′-UTR (red), exons (blue) and 3′-UTR (purple), and poly-As (brown).

Fig. 6.—

The subset of adult salivary gland transcript containing Helitrons. (A) Categorization of transcripts that have human homologs. In X/Y%, X corresponds to the number of transcripts that fall in that category (alternative transcripts containing the same Helitron are counted only once). Y denotes the percent of the total transcripts (excluding the alternate transcripts) altered by a Helitron in each category. Note that if a transcript can be included in multiple categories, it is counted as a member of all applicable categories. (B) to (I) are examples of various categories. (B) A Helitron is part of the 5′-UTR. It is inserted in the intron in the 5′-UTR of the DEDD gene, interrupting the splicing of the intron. (C) Helitron introduces a premature stop codon (shown as a star). It is inserted in the intron before the 3′-UTR of YWHAQ gene and interrupts the splicing of the intron. (D) A Helitron provides a cryptic splice site and fuses with a transcript from the ZMYM4 gene. The Helitron provides an initiation codon and a few codons in the predicted ORF of the alternative transcript of ZMYM4 gene. (E) A gene fragment within Helitron provides a cryptic splice site and fuses with a transcript from SGSM3 gene. The Helitron contains the promoter, 5′-UTR and exon1 and part of the intron1 of the NUBPL gene and introduces a premature stop codon in the transcript. (F) A Helitron contributes to an exon present in two long intergenic noncoding RNAs (lincRNAS) (TCONS_00001429 and TCONS_00002307). (G) A Helitron introduces predicted miRNA-binding sites to the 3′-UTR of POFUT1 transcript. The Helitron also introduces fragments of the 5′-UTR and exon1 from the DENND5B gene into the 3′-UTR. (H) A Helitron is inserted in the 3′-UTR of the UFSP2 gene and introduces a Mushashi protein-binding motif. (I) A Helitron adds a novel polyadenylation site to OTUD3 gene transcripts.