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. 2012 Mar;190(3):965-75.
doi: 10.1534/genetics.111.136176. Epub 2011 Dec 14.

Gene Capture by Helitron Transposons Reshuffles the Transcriptome of Maize

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Free PMC article

Gene Capture by Helitron Transposons Reshuffles the Transcriptome of Maize

Allison M Barbaglia et al. Genetics. .
Free PMC article

Abstract

Helitrons are a family of mobile elements that were discovered in 2001 and are now known to exist in the entire eukaryotic kingdom. Helitrons, particularly those of maize, exhibit an intriguing property of capturing gene fragments and placing them into the mobile element. Helitron-captured genes are sometimes transcribed, giving birth to chimeric transcripts that intertwine coding regions of different captured genes. Here, we perused the B73 maize genome for high-quality, putative Helitrons that exhibit plus/minus polymorphisms and contain pieces of more than one captured gene. Selected Helitrons were monitored for expression via in silico EST analysis. Intriguingly, expression validation of selected elements by RT-PCR analysis revealed multiple transcripts not seen in the EST databases. The differing transcripts were generated by alternative selection of splice sites during pre-mRNA processing. Selection of splice sites was not random since different patterns of splicing were observed in the root and shoot tissues. In one case, an exon residing in close proximity but outside of the Helitron was found conjoined with Helitron-derived exons in the mature transcript. Hence, Helitrons have the ability to synthesize new genes not only by placing unrelated exons into common transcripts, but also by transcription readthrough and capture of nearby exons. Thus, Helitrons have a phenomenal ability to "display" new coding regions for possible selection in nature. A highly conservative, minimum estimate of the number of new transcripts expressed by Helitrons is ~11,000 or ~25% of the total number of genes in the maize genome.

Figures

Figure 1
Figure 1
Sequence alignment of the terminal ends of maize Helitrons. (Left) Names of the Helitrons: sh2-7527 (Lal et al. 2003), bal-Ref (Gallavotti et al. 2004), RplB73 (Gupta et al. 2005a), ZeinBSSS53 (Song and Messing 2003), P450B73 (Jameson et al. 2008), HelA-1 (Lai et al. 2005), HelA-2 (Lai et al. 2005), GHIJKLM9002 (Morgante et al. 2005), NOPQ9002 (Morgante et al. 2005), NOPQB73_14578 (Brunner et al. 2005), NOPQMo17_14594 (Brunner et al. 2005), NOPQB73_9002 (Brunner et al. 2005), Mo17NOPQ_14577 (Brunner et al. 2005), RST9002 (Morgante et al. 2005), U9002 (Morgante et al. 2005), HI9002 (Morgante et al. 2005), Hel-BSSS53-Zici (Xu and Messing 2006), Hel1-4 (Wang and Dooner 2006), and Hel1-5 (Wang and Dooner 2006). (Center and right) Multiple sequence alignment of the conserved 5′ and 3′ termini of the Helitrons, respectively. (Bottom) Consensus sequence used for the database search for other Helitron family members.
Figure 2
Figure 2
Strategy used to discover maize Helitrons and analysis of their captured gene expression. (Top) Structure of nonautonomous maize Helitrons. The exons captured by nonautonomous Helitrons are represented by colored blocks. The terminal ends of the Helitrons are displayed by pattern filled boxes, and the loop near the 3′ terminus represents the palindrome sequence. The A and T nucleotides immediately flanking the insertion site of the Helitron are indicated.
Figure 3
Figure 3
Genomic and RT–PCR analysis of Helitron Hel1-331. (A) PCR product amplified from genomic DNA extracted from different maize inbred lines using primers, H31-1F and H31-1R, flanking the 5′ and 3′ sequence of the Helitron insertion, respectively. (B) RT–PCR products amplified from root and shoot tissues of maize inbred lines B73 and Mo17 using primers, H31E1F and H31E7R. (C) Splice alignment of the sequences of the RT–PCR products shown in B with the Helitron Hel1-331 sequence. The exons of a captured hypothetical gene, gi: 212721678, and an uncharacterized gene, are color coded in orange and yellow, respectively. In the alignment, boxes and lines denote exons and introns, respectively. Alternative donor and acceptor splice sites are joined by dashed lines and * marks the position of the retained introns. The size of the transcripts and the A and T nucleotides flanking the insertion site of the Helitron are indicated.
Figure 4
Figure 4
Protein alignment of alternatively spliced transcripts of Hel1-331. Alignment of the deduced protein sequences of Helitron Hel1-331 transcripts are displayed in Figure 3C. The solid area marks the positions at which the same residue occurs in >60% of the sequences. The red line spans the conserved hnRNP-U1 domain.
Figure 5
Figure 5
Expression analysis of Helitron Hel1-332a. (A) RT–PCR products resolved on a 1% agarose gel amplified from maize roots and shoots using primers E32E1F and E32E6R. (B) Splice alignment of the Hel1-332a sequence with RT–PCR products shown in A. The boxes and lines denote exons and introns, respectively. Dashed lines join alternative donor and acceptor sites and * denotes a retained intron. The sizes of the RT–PCR products are indicated on the right. The captured gene fragments of proteins, gi: 212275660, gi: 242041151, and gi: 195657737 are displayed in green, blue, and violet, respectively.
Figure 6
Figure 6
Molecular and sequence analysis of Helitron Hel1-333. (A) Pairwise sequence alignment of HTG sequence flanking the Hel1-333 insertion (top sequence) with the paralogous locus. An arrow marks the putative insertion site of the Helitron. (B) RT–PCR products from maize roots and shoots amplified using primers H33E1F and H33E14R. The splice alignment of the RT–PCR products in A with the Hel1-333 sequence is shown in C. The boundaries of the Helitron and the predicted length of the RT–PCR products are indicated. The * marks the retained intron and alternative donor and acceptor sites are joined by dashed lines. The gene fragments of proteins, gi: 242043402, 242094646, and 29333527, are color coded in red, fuchsia, and pink, respectively. The fuchsia-shaded regions of the exons of the alternatively spliced transcripts represent the ORFs spanning the conserved peptidase domain.
Figure 7
Figure 7
Genomic and RT–PCR analysis of Helitron Hel1-334. (A) Pairwise sequence alignment of the flanking HTGS (top sequence) without the Helitron insertion and the sequence of the paralogous locus. The putative insertion site of the Helitron is marked by an arrow. (B) RT–PCR products amplified from root and shoot tissues using primers H34E1F and H34E6R. (C) Schematic representation of the exon and intron junction of the alternatively spliced products in B. Exons of the captured genes, gi: 242080485 and gi: 226528348, are color coded in lime green and aqua, respectively. The dashed lines join alternative donor and acceptor sites. The predicted sizes of the transcripts are indicated.

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