LINE-1 ribonucleoprotein particle retrotransposition intermediates.

Abstract

LINE-1s (L1s) are non-LTR retrotransposons that mobilize (i.e. , retrotranspose) using an RNA intermediate. There are ∼517,000 L1s in the average human genome, ∼80-100 of which are active. Full length active L1s contain a 5' UTR, two open reading frames (ORF1 and ORF2), and a 3' UTR. ORF1 encodes an RNA-binding protein (ORF1p), whereas ORF2 encodes a protein (ORF2p) with endonuclease (EN) and reverse transcriptase (RT) activities. It is hypothesized that only one molecule of ORF2p is synthesized per L1 transcript, whereas ORF1p is produced at greater quantities to coat the L1 RNA. The L1-encoded proteins demonstrate a cis-preference; i.e., they preferentially associate with their encoding transcript to form a ribonucleoprotein particle (RNP), which is hypothesized to be the cytoplasmic retrotransposition intermediate. The L1 RNP forms in the cytoplasm and only after it gains access to the nucleus is the L1 cDNA synthesized by target primed reverse transcription (TPRT). Here, I developed a system to follow the fate of wild type and mutant ORF1p against a background of endogenously expressed L1s in cultured human cells. Using this system, wild type ORF1p and L1 RNA were shown to co-localize in a cytoplasmic RNP, and two classes of mutants in the ORF1p nucleic acid binding domain were discovered that prove RNP formation to be necessary but not sufficient for L1 retrotransposition. I developed a biochemical assay to detect specifically the L1 ORF2p in RNPs via its RT activity, called LEAP, for L&barbelow;ine E&barbelow;lement A&barbelow;mplification P&barbelow;rotocol. Using the LEAP assay, I demonstrated biochemical properties of the L1 RT that are congruent with analysis of genomic integration sites in vivo. Consistent with cis-preference, the LEAP assay also showed the L1 RT prefers its own template and does not promiscuously utilize other cellular RNAs in trans. In sum, these data strongly support the hypothesis that RNPs are bona fide cytoplasmic intermediates, and by the co-localization of ORF2p to the RNP, offers major mechanistic advancements to L1 biology.Ph.D.Biological SciencesGeneticsMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125414/2/3192686.pd