Alu elements are the most successful transposons in humans. They are 300-bp non-coding sequences transcribed by RNA polymerase III (Pol III) and are expected to retrotranspose with the aid of reverse transcriptases of cellular origin. We previously showed that human LINEs can generate cDNA copies of any mRNA transcript by means of a retroposition process involving reverse transcription and integration by the LINE-encoded endonuclease and reverse transcriptase. Here we show mobility of marked Alu sequences in human HeLa cells with the canonical features of a retrotransposition process, including splicing out of an autocatalytic intron introduced into the marked sequence, target site duplications of varying lengths and integrations into consensus A-rich sequences. We further show that the poly-A stretch at the Alu 3' end is essential for mobility, that LINEs are required for transposition and that the rate of retroposition is 100-1,000 times higher for Alu transcripts than for control mRNAs, thus accounting for the high mutational activity of these elements observed in humans.

Long interspersed elements (LINEs) are endogenous mobile genetic elements that have dispersed and accumulated in the genomes of higher eukaryotes via germline transposition, with up to 100,000 copies in mammalian genomes. In humans, LINEs are the major source of insertional mutagenesis, being involved in both germinal and somatic mutant phenotypes. Here we show that the human LINE retrotransposons, which transpose through the reverse transcription of their own transcript, can also mobilize transcribed DNA not associated with a LINE sequence by a process involving the diversion of the LINE enzymatic machinery by the corresponding mRNA transcripts. This results in the 'retroposition' of the transcribed gene and the formation of new copies that disclose features characteristic of the widespread and naturally occurring processed pseudogenes: loss of intron and promoter, acquisition of a poly(A) 3' end and presence of target-site duplications of varying length. We further show-by introducing deletions within either coding sequence of the human LINE-that both ORFs are necessary for the formation of the processed pseudogenes, and that retroviral-like elements are not able to produce similar structures in the same assay. Our results strengthen the unique versatility of LINEs as genome modellers.

RNA granules ͉ Ro ribonucleoproteins ͉ prespliceosomes T he intrinsic antiretroviral factor APOBEC3G (A3G) is highly active against HIV-1 and other retroviruses (1). Incorporation of A3G into budding HIV-1 virions promotes extensive mutation of nascent HIV-1 DNA formed by reverse transcription in the next round of infection (2-5). However, HIV-1 counters these effects of A3G with virion infectivity factor (Vif), which accelerates proteasome-mediated degradation of A3G (6-11) and partially impairs de novo synthesis of A3G (6, 12). These two actions in virus-producing cells effectively deplete intracellular A3G, making the enzyme unavailable for virion encapsidation. Resting CD4 T cells and monocytes, which are refractory to HIV-1 infection, express only the low-molecular-mass (LMM) form of A3G (13). siRNAmediated knockdown of LMM A3G expression in resting CD4 T cells renders these cells permissive for HIV-1 infection, indicating that LMM A3G functions as a potent postentry restriction factor for HIV-1 (13). Conversely, resting CD4 T cells in lymphoid tissues are permissive for HIV-1 infection, and A3G is predominantly in high-molecular-mass (HMM) complexes in these cells (14) because of the lymphoid microenvironment. Locally produced cytokines, including IL-2 and IL-15, and cell-cell interactions in lymphoid tissues stimulate assembly of the HMM A3G complexes (14) and confer permissiveness for HIV-1 infection.The genes encoding A3G and other APOBEC3 (A3) family members are clustered on human chromosome 22 (15). During mammalian evolution, this locus expanded from a single gene in mice to eight genes (A3A-H) in primates (15, 16). These genes apparently have been modulated by repeated episodes of strong Abbreviations: HMM, high-molecular-mass; LMM, low-molecular-mass; RNP, ribonucleoprotein; A3G, APOBEC3G; L1, long interspersed nucleotide elements 1; TAP, tandem affinity purification; IP, immunoprecipitation; co-IP, coimmunoprecipitation; scAlu, small cytoplasmic Alu; PB, processing body. ¶ To whom correspondence should be addressed. E-mail: wgreene@gladstone.ucsf.