Human L1 Retrotransposition: cisPreference versus trans Complementation

Abstract

Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the ϳ400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.Retrotransposons are DNA sequences that can move (i.e., retrotranspose) to different genomic locations via an RNA intermediate. They are present in the genomes of virtually all eukaryotes and can be subdivided into two general structural classes. Long terminal repeat (LTR) retrotransposons resemble simple retroviruses but lack a functional envelope (Env) gene (2). Non-LTR retrotransposons lack LTRs and generally terminate in a polyadenylic acid [poly(A)] tail (20,23).L1s are the most abundant non-LTR retrotransposons in the human genome and comprise approximately 17% of nuclear DNA (42). The overwhelming majority of L1s are retrotransposition defective (RD-L1s) and cannot retrotranspose because they are 5Ј truncated, internally rearranged, or mutated (23); however, an estimated 30 to 60 human L1s remain retrotransposition competent (RC-L1s) (40). RC-L1s are 6.0 kb in length and contain a 5Ј untranslated region (UTR) harboring an internal promoter (43), two nonoverlapping open reading frames (open reading frame 1 [ORF1] and ORF2) (7, 41), and a 3Ј UTR ending in an unorthodox poly(A) tail (20,46). In addition, these elements are flanked by variable-length target site duplications, which are hallmarks of the retrotransposition process (20).Non-LTR retrotransposons encode endonuclease activities, which can generate either site-specific (4, 11, 47) or relatively non-site-specific nicks in chromosomal DNA (5, 10). The liberated 3Ј hydroxyl residue then acts as a primer for reverse transcription of the retrotransposon RNA by the retrotransposon-encoded reverse transcriptase (RT) by a mechanism termed target site-primed reverse transcription (TPRT) (28, 29). Thus, the processes of integration and reverse transcription are coupled for non-LTR retrotransposons.Biochemical studies revealed that ORF1 encodes a 40-kDa RNA binding protein that colocalizes with L1 RNA in cytoplasmic ribonucleoprotein particles (RNPs) (17, 18). ORF2 encodes a multifunctional protein containing endonuclease and RT activities (10, 34) and also has a carboxyl-terminal cysteine-rich domain (C) of unknown function (9). Using an assay to monitor L1 ret...

Diverse cis factors controlling Alu retrotransposition: What causes Alu elements to die?

“…1B) driven by a cotransfected full-length L1 element (Dewannieux and Heidmann 2005), but the level of variation observed would not provide much explanation of the differential efficiency of genomic Ya5 amplification relative to Sx elements. Our own studies confirm this observation when using either a cotransfected wildtype L1 (JM101/L1.3 no tag; Wei et al 2001) or an ORF2-only expression plasmid to drive Alu retrotransposition (data not shown). However, we were concerned that the increased expression of L1 proteins in transient transfection experiments might lead to unusually high concentrations of ORF2p and artificially augment its interaction with shorter A-tail Alus.…”

“…Surprisingly, the A3T construct, although highly disrupted, can function almost as well as the control under these overexpressed ORF2p conditions, but decreases to ;50% activity under endogenous conditions, indicating that overexpression of ORF2p may drive relatively inactive Alus in this assay system. Experiments using wild-type L1 (JM101/L1.3 no tag; Wei et al 2001) as the driver (Supplemental Fig. 1S) showed an intermediate level of Alu activity between those observed when using the endogenous L1 and the exogenously supplied ORF2p conditions.…”

DNA methylation in cancer: too much, but also too little

“…LINE-1 repeats constitute about 15% of the human genome, but of the *4610 5 copies of LINE-1 elements in the human genome, only about 30 -60 are estimated to be competent for transposition (Sassaman et al, 1997). There have been occasional reports of cancer-associated retrotransposition-like insertions involving LINE-1 sequences (Miki et al, 1992;Morse et al, 1988), and they may mobilize cellular RNAs at low frequencies (Wei et al, 2001). Their activation can also lead to transcriptional interference involving neighboring genes (Whitelaw and Martin, 2001).…”

Origin and evolution of SINEs in eukaryotic genomes

“…Currently, we know two systems protecting LINE RTs from processing foreign templates: sequence recognition of the RNA encoding the enzyme and cis-preference, when the RNA molecule used for RT translation is used by the translated enzyme as the template for reverse transcription (Esnault et al, 2000;Wei et al, 2001;Kajikawa and Okada, 2002). Overcoming this protection is an essential step in SINE formation.…”