Nicolas Gilbert

Human L1 Retrotransposition: cis Preference versus trans Complementation

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 profoundcis 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.

DNA repair mediated by endonuclease-independent LINE-1 retrotransposition

Long interspersed elements (LINE-1s) are abundant retrotransposons in mammalian genomes that probably retrotranspose by target site-primed reverse transcription (TPRT). During TPRT, the LINE-1 endonuclease cleaves genomic DNA, freeing a 3′ hydroxyl that serves as a primer for reverse transcription of LINE-1 RNA by LINE-1 reverse transcriptase. The nascent LINE-1 cDNA joins to genomic DNA, generating LINE-1 structural hallmarks such as frequent 5′ truncations, a 3′ poly(A)+ tail and variable-length target site duplications (TSDs). Here we describe a pathway for LINE-1 retrotransposition in Chinese hamster ovary (CHO) cells that acts independently of endonuclease but is dependent upon reverse transcriptase. We show that endonuclease-independent LINE-1 retrotransposition occurs at near-wildtype levels in two mutant cell lines that are deficient in nonhomologous end-joining (NHEJ). Analysis of the pre- and post-integration sites revealed that endonuclease-independent retrotransposition results in unusual structures because the LINE-1s integrate at atypical target sequences, are truncated predominantly at their 3′ ends and lack TSDs. Moreover, two of nine endonuclease-independent retrotranspositions contained cDNA fragments at their 3′ ends that are probably derived from the reverse transcription of endogenous mRNA. Thus, our results suggest that LINE-1s can integrate into DNA lesions, resulting in retrotransposon-mediated DNA repair in mammalian cells.

Multiple fates of L1 retrotransposition intermediates in cultured human cells

ABSTRACT LINE-1 (L1) retrotransposons comprise ∼17% of human DNA, yet little is known about L1 integration. Here, we characterized 100 retrotransposition events in HeLa cells and show that distinct DNA repair pathways can resolve L1 cDNA retrotransposition intermediates. L1 cDNA resolution can lead to various forms of genetic instability including the generation of chimeric L1s, intrachromosomal deletions, intrachromosomal duplications, and intra-L1 rearrangements as well as a possible interchromosomal translocation. The L1 retrotransposition machinery also can mobilize U6 snRNA to new genomic locations, increasing the repertoire of noncoding RNAs that are mobilized by L1s. Finally, we have determined that the L1 reverse transcriptase can faithfully replicate its own transcript and has a base misincorporation error rate of ∼1/7,000 bases. These data indicate that L1 retrotransposition in transformed human cells can lead to a variety of genomic rearrangements and suggest that host processes act to restrict L1 integration in cultured human cells. Indeed, the initial steps in L1 retrotransposition may define a host/parasite battleground that serves to limit the number of active L1s in the genome.

Characterization of LINE-1 Ribonucleoprotein Particles

The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1–encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition.

Leucine-Rich repeat receptor kinases are sporadically distributed in eukaryotic genomes

BackgroundPlant leucine-rich repeat receptor-like kinases (LRR-RLKs) are receptor kinases that contain LRRs in their extracellular domain. In the last 15 years, many research groups have demonstrated major roles played by LRR-RLKs in plants during almost all developmental processes throughout the life of the plant and in defense/resistance against a large range of pathogens. Recently, a breakthrough has been made in this field that challenges the dogma of the specificity of plant LRR-RLKs.ResultsWe analyzed ~1000 complete genomes and show that LRR-RK genes have now been identified in 8 non-plant genomes. We performed an exhaustive phylogenetic analysis of all of these receptors, revealing that all of the LRR-containing receptor subfamilies form lineage-specific clades. Our results suggest that the association of LRRs with RKs appeared independently at least four times in eukaryotic evolutionary history. Moreover, the molecular evolutionary history of the LRR-RKs found in oomycetes is reminiscent of the pattern observed in plants: expansion with amplification/deletion and evolution of the domain organization leading to the functional diversification of members of the gene family. Finally, the expression data suggest that oomycete LRR-RKs may play a role in several stages of the oomycete life cycle.ConclusionsIn view of the key roles that LRR-RLKs play throughout the entire lifetime of plants and plant-environment interactions, the emergence and expansion of this type of receptor in several phyla along the evolution of eukaryotes, and particularly in oomycete genomes, questions their intrinsic functions in mimicry and/or in the coevolution of receptors between hosts and pathogens.

Ancient repeat sequence derived from U6 snRNA in primate genomes.

LINE-1 (L1) is the most represented sequence of the human genome (17% of the total genomic mass). Moreover, it has been proposed for many years and demonstrated more recently that L1 has contributed to the mobilization of pseudogenes, small non-coding RNAs, such as tRNAs or snRNAs, and SINEs. In fact, it is estimated that L1 is responsible for at least 30% of our genome. The mobilization of non-L1 RNAs can occur in different ways and at different steps of the retrotransposition cycle. Here, by looking at U6 snRNA sequences mobilized by L1, we have observed an ancient repeat sequence derived from U6, present in all primate genomes. We were able to trace its origin in Euarchota genomes, most likely during the divergence of the four orders; Scandentia, Dermoptera, Plesiadapiform (extinct) and Primates.