Molecular Mechanisms of LINE-1 Retrotransposition Inhibition

[摘要] Long INterspersed Element-1 (LINE-1 or L1) retrotransposons are an ancient family of repeated DNA sequences present in all inspected mammalian genomes. L1s are the only active autonomous mobile element in the human genome and are present at over 500,000 copies, representing ~17% of genomic DNA. A full-length human L1 is ~6 kb in length and contains an internal RNA polymerase II promoter within its 5’ untranslated region (UTR). Following the 5’UTR are two open reading frames (ORF1 and ORF2) that encode two functional proteins (ORF1p and ORF2p) that are required for mobilization (i.e., retrotransposition). L1s end with a 3’UTR and a poly(A) tail. The evolutionary success of L1 relies on the reiterative retrotransposition of full-length L1 RNAs. The vast majority (>99.9%) of genomic L1s are inactive; however, on average, ~80-100 L1s per diploid genome are capable of retrotransposition. Since, L1 retrotransposition by its nature is mutagenic, it is likely that cellular host-factors have evolved to inhibit or restrict unabated L1 retrotransposition. Previous studies identified functional splice donor, splice acceptor, and polyadenylation sequences in full-length L1 RNA. Here, I demonstrate that retrotransposition of intra-5’UTR or 5’UTR/ORF1 spliced L1 RNAs leads to the generation of Spliced Integrated Retrotransposed Elements (SpIREs). Additionally, I uncovered a new intra-5’UTR SpIRE that is approximately ten times more abundant than previously identified SpIREs. Using biochemical and genetic approaches, I definitively demonstrate that intra-5’UTR SpIREs lack cis-acting transcription factor binding sites, resulting in reduced 5’UTR promoter activity compared to a full-length 5’UTR. Moreover, I demonstrate that 5’UTR/ORF1 SpIREs lack cis-acting sequences required for L1 transcription and produce non-functional ORF1p variants. These results establish that SpIREs are evolutionary ;;dead ends,” which are unlikely to contribute to additional rounds of L1 retrotransposition. Finally, in agreement with previous publications, I demonstrate that a subset of splicing factors may repress L1 expression and/or retrotransposition. Previous experiments in embryonic human carcinoma-derived cells (hECs) revealed that an engineered L1 tagged with a retrotransposition indicator cassette (L1-reporter) successfully retrotransposed into hEC genomic DNA. However, either during or immediately after genomic integration, expression of the L1-reporter is silenced in hECs. Previous experiments demonstrated that hEC cells treated with histone deacetylase (HDAC) inhibitors swiftly reverse L1-reporter silencing. I sought to identify host proteins that may be involved in L1-reporter silencing by developing a forward genetic screen using CRISPR/Cas9-based genome editing technology. Using the PA-1 hEC cell line that permits L1-reporter retrotransposition, but subsequently silences L1-reporter expression, I identified potential candidate genes that may play a role in L1-reporter mediated gene silencing. Future work will test whether the candidate genes identified in this screen directly or indirectly inhibit L1-reporter mediated gene silencing. A continued understanding of the interplay between L1 and host-factors is critical to understanding human genomic variation. Additionally, as the L1-host interaction largely recapitulates a traditional host-parasite ;;arms-race,” new insights gained from these studies may contribute to our understanding of how L1 retrotransposition continues to influence human health and disease.

[发布机构] University of Michigan

[效力级别] Genetics

[关键词] LINE-1 Retrotransposition;Genetics;Science;Human Genetics