Deanna A Kulpa | Search PubMed

PubMed2006

Cis-preferential LINE-1 reverse transcriptase activity in ribonucleoprotein particles.

LINE-1 retrotransposons (L1s) constitute approximately 17% of human DNA, and their activity continues to affect genome evolution. Retrotransposition-competent human L1s encode two proteins required for their mobility (ORF1p and ORF2p); however, biochemical activities associated with ORF2p have been difficult to detect in cells. Here, we show for the first time the colocalization of L1 RNA, ORF1p and ORF2p to a putative ribonucleoprotein retrotransposition intermediate. We further demonstrate that ORF2p preferentially uses its encoding RNA as a template for reverse transcription. Thus, our data provide the first biochemical evidence supporting the cis-preferential action of the L1 reverse transcriptase.

Base Sequence↗

PubMed2005

Ribonucleoprotein particle formation is necessary but not sufficient for LINE-1 retrotransposition.

Long interspersed elements (LINE-1s or L1s) are abundant non-LTR retrotransposons that mobilize through an RNA intermediate by target site primed reverse transcription. The L1-encoded proteins (ORF1p and ORF2p) preferentially associate with their encoding transcript to form a ribonucleoprotein particle (RNP), which is a proposed retrotransposition intermediate. Here, we have used epitope tagging to discriminate the proteins encoded by engineered L1s from those encoded by endogenously expressed L1s. We demonstrate that an L1 containing an epitope tag at the carboxyl terminus of ORF1p remains retrotransposition-competent and that tagged ORF1p and its encoding RNA localize to cytoplasmic RNPs. We also identified two classes of ORF1p mutants, one that severely decreased RNP formation and blocked retrotransposition, and another that allows RNP formation but reduces retrotransposition by 100-fold. Thus, these data indicate that RNP formation is important but not sufficient for L1 retrotransposition and suggest that ORF1p also may function at downstream steps in the L1 retrotransposition pathway.

Blotting, Western↗