The RNA polymerase dictates ORF1 requirement and timing of LINE and SINE retrotransposition.
Mobile elements comprise close to one half of the mass of the human genome. Only LINE-1 (L1), an autonomous non-Long Terminal Repeat (LTR) retrotransposon, and its non-autonomous partners-such as the retropseudogenes, SVA, and the SINE, Alu-are currently active human retroelements. Experimental evidence shows that Alu retrotransposition depends on L1 ORF2 protein, which has led to the presumption that LINEs and SINEs share the same basic insertional mechanism. Our data demonstrate clear differences in the time required to generate insertions between marked Alu and L1 elements. In our tissue culture system, the process of L1 insertion requires close to 48 hours. In contrast to the RNA pol II-driven L1, we find that pol III transcribed elements (Alu, the rodent SINE B2, and the 7SL, U6 and hY sequences) can generate inserts within 24 hours or less. Our analyses demonstrate that the observed retrotransposition timing does not dictate insertion rate and is independent of the type of reporter cassette utilized. The additional time requirement by L1 cannot be directly attributed to differences in transcription, transcript length, splicing processes, ORF2 protein production, or the ability of functional ORF2p to reach the nucleus. However, the insertion rate of a marked Alu transcript drastically drops when driven by an RNA pol II promoter (CMV) and the retrotransposition timing parallels that of L1. Furthermore, the "pol II Alu transcript" behaves like the processed pseudogenes in our retrotransposition assay, requiring supplementation with L1 ORF1p in addition to ORF2p. We postulate that the observed differences in retrotransposition kinetics of these elements are dictated by the type of RNA polymerase generating the transcript. We present a model that highlights the critical differences of LINE and SINE transcripts that likely define their retrotransposition timing.
Kroutter
Emily N
EN
Tulane Cancer Center SL-66, Tulane University Health Sciences Center, New Orleans, Louisiana, United States of America.
Belancio
Victoria P
VP
Wagstaff
Bradley J
BJ
Roy-Engel
Astrid M
AM
D020087:Alu Elements; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020084:Long Interspersed Nucleotide Elements; D008957:Models, Genetic; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D020086:Short Interspersed Nucleotide Elements; D013997:Time Factors
D012321:DNA-Directed RNA Polymerases