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Proc Natl Acad Sci U S A. Nov 24, 2009; 106(47): 19747–19748.
Published online Nov 19, 2009. doi:  10.1073/pnas.0910273106
PMCID: PMC2785235

A cornucopia of Helitrons shapes the maize genome

Transposable elements (TEs) are mobile segments of DNA first discovered in corn by Barbara McClintock in the 1940s. Her studies revealed that the movement of TEs could cause chromosomal breakage and affect phenotypes in dramatic or subtle ways. Her findings met with some disbelief because they challenged the tenet of the static genome by showing that the genome could be much more dynamic than previously appreciated. It is difficult to imagine what McClintock would have to say if she were to learn that the genome of her beloved corn is, in fact, mostly composed of TEs whose capacity to alter the genome seems limited only by the sensitivity of the techniques available to detect the alterations they engender. In this issue of PNAS (1, 2) two groups have deployed innovative computational approaches to scan the nearly complete genome sequence of maize (21) to establish a comprehensive catalog of Helitrons, a colorful group of TEs with a peculiar predisposition to restructure genes and genomes.

Helitrons Enter the Dance

Much has been learned in the ensuing 60+ years since the discovery of the first TEs in corn. Perhaps it would come as no surprise to McClintock that many important TE discoveries were made in plants. Among recent milestones lies the discovery of an exotic group of eukaryotic TEs coined Helitrons (3) or rolling-circle transposons by virtue of their resemblance, both in terms of coding and structural properties, to bacterial transposons, plasmids, and viruses that replicate via a rolling-circle mechanism. Despite their abundance in some species (3, 4) and the fact that their movement has caused spontaneous mutations (5, 6), Helitrons were not discovered until 1999 when the first (partial) copies were computationally identified in the mustard weed Arabidopsis thaliana (7). It took another 2 years before the sequences of protein-encoding and presumably autonomous (i.e., self-sufficient) Helitrons could be unearthed from the Arabidopsis genome, but also from rice and nematode (3). These long (>5 kb) and potentially complete Helitrons all were found to encode a putative protein with rolling-circle initiator motif and PIF1-like DNA helicase domains. In addition, plant Helitrons were predicted to encode from one to three homologs of replication protein A (RPA)-like ssDNA binding proteins, which are necessary for the rolling-circle replication of certain plasmids, bacteriophages, and plant geminiviruses, but are normally supplied by the host. Because of the restricted distribution of the RPA-like genes to plant Helitrons, it was hypothesized that they had been kidnapped from their host and retained because of their function in transposition (3). This suspicion was later reinforced by the characterization of the first maize Helitrons, which were found to be particularly corpulent (>10 kb) because of the capture of gene fragments from multiple chromosomal locations (5, 810).

Maize Helitrons have transduplicated and reshuffled a staggering amount of sequences.

Highway to Helitrons

Helitrons have been particularly recalcitrant to automated computational identification because of their lack of terminal repeats and propensity for gene capture, which creates extreme heterogeneity in size and sequence. This hurdle has now been cleared by the development of two new programs designed to recognize the peculiar terminal sequence features of Helitrons: HelitronFinder by the Dooner group (11) and HelSearch by Yang and Bennetzen, recently benchmarked in another PNAS article (12). These programs are welcome additions to the expanding toolbox for automated annotation of TEs (13).

Using the new programs to scan the ≈2 Gb of genome sequences generated for the maize inbred line B73 (21), the two groups identified a largely overlapping set of ≈2,000 Helitrons predicted with high confidence (1, 2). Using this library to query the rest of the B73 sequence, many more pieces of Helitrons were identified (>20,000 according to ref. 2), amounting to at least 2% of the nuclear genome. The high level of fragmentation is likely an indicator of the patchy nature of the current genome assembly but also of the Helitrons themselves. Thus, these numbers should be regarded as conservative and remind us that measuring the actual content of Helitron-derived sequences is a challenging and uncertain exercise.

The classification of Helitrons into traditional TE families is also problematic. Typically, members of the same TE family share high levels (>80%) of sequence identity across all or most of their length, reflecting their descent from one or a few active ancestors. However, the sequence and length heterogeneity of maize Helitrons coupled with our limited knowledge of the transposition mechanism made it necessary to adopt a specific set of rules for Helitron classification based on the most terminal regions (2, 14). Yang and Bennetzen (2) considered elements as belonging to the same family when the last 30 bp are >80% identical and the same subfamily when they also share >80% sequence identity in the first 30 bp. Based on these criteria, they subdivided 1,930 intact Helitrons into eight families and 62 subfamilies. Du et al. (1) used a similar system of classification but with a more stringent criterion for the definition of a subfamily, thereby clustering the elements into more subfamilies (see refs. 1 and 2 for correspondence between their subfamilies). Both studies found that nearly all maize Helitrons (>98% in ref. 2) belong to a single (super)family called Hip (2) or Hel1 (1, 14) that includes several elements that have spread very efficiently, reaching several thousand copies in the B73 genome.

Exon Shuffling Machines

The premise that Helitrons act as “exon shuffling machines” (15) gained support with the discovery of the first maize Helitrons, which were found to have transduced and often recombined exons from multiple genes leading to novel transcriptional units (5, 810). However, it remained unclear whether this phenomenon was a peculiarity of a subset of Helitrons or a mere reflection of the remarkable plasticity of the maize genome (9, 16, 17). The new studies (1, 2) provide some resolution to this question. First, they confirm that maize Helitrons have transduplicated and reshuffled a staggering amount of sequences, probably exceeding 20,000 gene fragments in the B73 genome (2). Of 272 Helitrons individually annotated by Du et al. (1), 258 (94%) carry host sequences captured from a total of 376 different genes. Although the majority of maize Helitrons carry one or two gene fragments, some carry exons from as many as nine different genes. Not many Helitrons seem to have transduced a complete gene and few have reached high copy numbers. One intriguing exception is the abundant Hip1 (Hel1–105) subfamily (>1,000 copies) that carries a chunk of the maize phosphatase 2C-like gene. The functional consequence of this amplification is unknown, but the captured exons display the signature of purifying selection (2).

Although most of the capture events involved the Hip family, the predominant group of Helitrons in the maize genome, many of the other families include members with transduced gene fragments. Relatives of the Hip family are also among the most abundant Helitrons in rice and sorghum. Yet, very few Helitron-mediated transduction events have been reported in these other grasses (12, 17). Together these observations suggest that the propensity for maize Helitrons to capture gene fragments has more to do with some peculiar properties of the maize genome (e.g., replication/repair fidelity) or the population genetics of the species than with the elements themselves.

How Are Genes Captured?

In the absence of an experimental system to study the movement of Helitrons, inferences of the mechanisms underlying Helitron gene transduction have been made by comparison with bacterial rolling-circle transposons. The latter have been shown to transduce flanking host sequences either via misrecognition of initiation sites upstream of the element's origin of replication (ori) or downstream through bypass of its termination (ter) signal (18). Although the existence and position of ori and ter remain to be determined for Helitrons, previous studies in maize (8), Aspergillus nidulans (19), and bats (4) suggested that Helitrons can capture flanking sequences in either direction, and that the acquisition of new terminal sequences is a potent mechanism for the evolution of new Helitrons. The new studies (1, 2) confirm and further these observations through several striking examples illustrating the structural plasticity of maize Helitrons and their ability to form new subfamilies via the accretion of new 5′ and 3′ termini.

A less understood aspect of the transduction mechanism is the strong bias (estimated as 14 to 1 in ref. 2) for captured gene fragments to be in the same (or “sense”) orientation as the Rep/Helicase gene encoded by autonomous Helitrons. One explanation would be that when transcribed, exons captured in the antisense orientation are more likely to interfere with expression of the parental gene, for example, via the RNA interference pathway. Consistent with this hypothesis, Yang and Bennetzen (2) found that the few Helitrons containing gene fragments in antisense orientation are all of very recent origin, whereas those with fragments in sense orientation have a broader age distribution. This pattern is consistent with natural selection acting to remove Helitrons carrying gene fragments that interfere with parental gene expression.

Forecast

Although the genomes of many organisms are known to harbor substantial populations of Helitrons (3, 4, 12), none are known to be as recent as those of maize. Yang and Bennetzen (2) estimated that most of the elements identified in the B73 genome inserted <250,000 years ago. In addition, experiments conducted by Du et al. (1), and those published previously (5, 9, 10, 16), have shown that most Helitron insertions are polymorphic among maize inbred lines. These results yield two important conclusions. First, it is likely that Helitrons are still transposing at an appreciable frequency in some lines (see also ref. 20), making corn the species of choice to isolate and analyze the behavior of an autonomous Helitron in the near future. In fact, both of the new studies (1, 2) identified a few seemingly intact Helitrons in the B73 genome that are prime contenders as autonomous elements. Second, when one considers the sheer amount of DNA and the plethora of gene fragments carried by maize Helitrons it becomes evident that these transposons are responsible for much of the haplotype diversity exhibited by this model grass species (10, 16). How much of this dazzling genomic plasticity is correlated to phenotypic variation remains the burning question, and one indeed that will certainly receive further attention.

Footnotes

The authors declare no conflict of interest.

See companion articles on pages 19916 and 19922.

References

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