Human L1 Retrotransposition: <em>cis</em> Preference versus <em>trans</em> Complementation

Wei W

N Gilbert

S Ooi

J Lawler

Departments of Human Genetics and Internal Medicine, The University of Michigan Medical School, Ann Arbor, Michigan 48109^{; Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, Maryland 21205}^{; and Department of Genetics, The University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104}³

^{Corresponding author. Mailing address: Departments of Human Genetics and Internal Medicine, The University of Michigan Medical School, Ann Arbor, MI 48109. Phone: (734) 615-0456. Fax: (734) 763-3784. E-mail:}ude.hcimu@jnarom.

Received 2000 Aug 21; Revisions requested 2000 Oct 18; Accepted 2000 Nov 6.

Abstract

Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the ∼400,000 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.

Abstract

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 5′ 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 5′ untranslated region (UTR) harboring an internal promoter (43), two nonoverlapping open reading frames (open reading frame 1 [ORF1] and ORF2) (7, 41), and a 3′ 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 3′ 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 retrotransposition in cultured human HeLa cells, we demonstrated that a wide variety of site-directed point mutations in conserved domains of the ORF1- and ORF2-encoded proteins essentially abolish L1 retrotransposition (10, 37).

L1 retrotransposition can be mutagenic and has resulted in various genetic disorders (23, 24). The characterization of mutagenic L1 insertions in humans and mice yielded the unexpected finding that each insertion is derived from a progenitor L1 containing intact ORFs (7, 19, 25, 38). Thus, despite the vast majority of RD-L1s in the genome, it appears that only RNAs derived from RC-L1s efficiently retrotranspose (i.e., the L1 proteins demonstrate an apparent cis preference) (7, 8, 37). Paradoxically, it also is proposed that the proteins encoded by RC-L1s function in trans to promote both processed pseudogene formation and the retrotransposition of certain short interspersed nuclear elements (SINEs) (1, 6, 8, 21, 23, 30, 44).

Here, we use a two-plasmid complementation assay to demonstrate that the RC-L1 proteins preferentially mobilize the transcript from which they are encoded. This cis-preference mechanism likely allows RC-L1s to persist despite the presence of overwhelming numbers of nonfunctional elements. We further show that the RC-L1 proteins can function at a low level in trans to retrotranspose both mutant L1 RNAs and cellular mRNAs, resulting in the formation of processed pseudogenes.

ACKNOWLEDGMENTS

We thank Anne Marie DesLauriers at the University of Michigan Flow Core for help with flow cytometry, Robert Lyons at the University of Michigan DNA Sequencing Core for help with oligonucleotide synthesis and DNA sequencing, and Ali Lotia for help with generating computer graphics. We thank David Ginsburg for providing pAI1 cDNAs. We thank Alice Telesnitsky, John Goodier, Eline Luning Prak, Tom Glaser, Dennis Hartigan-O'Connor, and current members of the Moran Lab for critical reading of the manuscript and for helpful discussions during the course of this work.

This work was supported in part by a Damon Runyon Scholar Award (J.V.M.) and National Institutes of Health grants GM60518 (J.V.M.) and CA16519 (J.D.B.).

ACKNOWLEDGMENTS

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