Novel retrotransposon analysis reveals multiple mobility pathways dictated by hosts

Abstract

Autonomous non-long-terminal-repeat retrotransposons (NLRs) proliferate by retrotransposition via coordinated reactions of target DNA cleavage and reverse transcription by a mechanism called target-primed reverse transcription (TPRT). Whereas this mechanism guarantees the covalent attachment of the NLR and its target site at the 3Ј junction, mechanisms for the joining at the 5Ј junction have been conjectural. To better understand the retrotransposition pathways, we analyzed target-NLR junctions of zebrafish NLRs with a new method of identifying genomic copies that reside within other transposons, termed "target analysis of nested transposons" (TANT). Application of the TANT method revealed various features of the zebrafish NLR integrants; for example, half of the integrants carry extra nucleotides at the 5Ј junction, which is in stark contrast to the major human NLR, LINE-1. Interestingly, in a cell culture assay, retrotransposition of the zebrafish NLR in heterologous human cells did not bear extra 5Ј nucleotides, indicating that the choice of the 5Ј joining pathway is affected by the host. Our results suggest that several pathways exist for NLR retrotransposition and argue in favor of host protein involvement. With genomic sequence information accumulating exponentially, our data demonstrate the general applicability of the TANT method for the analysis of a wide variety of retrotransposons.

A new mechanism to ensure integration during LINE retrotransposition: A suggestion from analyses of the 5′ extra nucleotides

“…To elucidate the mechanism by which the 5′ extra nucleotides were generated, we here analyzed the sequences at the 5′ junction of the integrants. Consistent with our previous data (Ichiyanagi et al, 2007), only 3 of 20 integrants (10%) in human HeLa cells accompanied extra nucleotides at their 5′ junctions (Fig. 1B, HeLa).…”

“…One of these, ZfL2-2, also called CR1-2_DR (Kapitonov and Jurka, 2003), is retrotransposition-competent in cultured cells (Sugano et al, 2006;Suzuki et al, 2009). Approximately half of ZfL2-2 elements in zebrafish have 5′ extra nucleotides (Ichiyanagi et al, 2007), suggesting that the 5′ integration accompanying the extra nucleotides is a predominant pathway in the homologous system. On the other hand, the extra nucleotides are rarely observed when ZfL2-2 is retrotransposed in HeLa cells (Ichiyanagi et al, 2007).…”

“…Approximately half of ZfL2-2 elements in zebrafish have 5′ extra nucleotides (Ichiyanagi et al, 2007), suggesting that the 5′ integration accompanying the extra nucleotides is a predominant pathway in the homologous system. On the other hand, the extra nucleotides are rarely observed when ZfL2-2 is retrotransposed in HeLa cells (Ichiyanagi et al, 2007). Thus, the generation of the 5′ extra nucleotides appears to be dependent on host factors, although their precise role has not been revealed.…”

Epigenetic regulation of transcription and possible functions of mammalian short interspersed elements, SINEs

“…Recognition of the RNA template is mediated via the LINE homology region present in the 3′ region of the SINE. This figure represent the main mobility pathway, and all the other proposed pathways also involve the reverse transcriptase-template RNA recognition (Ichiyanagi et al, 2007). Table 1.…”

Genomic alterations upon integration of zebrafish L1 elements revealed by the TANT method

“…5B; P ≅ 10 − 18 by a χ 2 test with 6 degrees of freedom), suggesting that the MH regions were not created by chance but rather by a mechanism involved in retrotransposition (Schwarz-Sommer et al, 1987). It has been proposed that the upstream target and the LINE cDNA are joined via annealing of these two DNA strands in the MH region at the 5′ junction (Voliva et al, 1984;Feng et al, 1998;Martin et al, 2005b;Zingler et al, 2005;Ichiyanagi et al, submitted for publication). L1_DRs presumably utilize a similar mechanism for the 5′ joining in a substantial fraction of their retrotransposition events.…”