Cryptic splicing adversely affects LINE-1 retrotransposition. P. A. Larson1, C. R. Beck1, J. V. Moran1,2,3 1) Department of Human Genetics; 2) Department of Internal Medicine; 3) Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, 48109 USA.

   Long INterspersed Element 1 (LINE-1 or L1) retrotransposons are a prolific family of mobile genetic elements that comprise approximately 17% of the human genome reference sequence (HGR). Most L1s have been rendered inactive by mutational processes and can be considered to be molecular fossils. However, the average human genome contains approximately 80-100 full-length, retrotransposition-competent L1s (RC-L1s). Ongoing RC-L1 retrotransposition contributes to both intra- and inter- individual genetic diversity, and on occasion causes sporadic cases of human disease. Previous studies from the Belancio and Deininger laboratories revealed that human L1 mRNAs contain cryptic splice sites, and that their utilization results in internally deleted L1 mRNAs. Given that the proteins (ORF1p and ORF2p) encoded by RC-L1s exhibit cis-preference (i.e., they preferentially bind to their respective encoding mRNA to enable retrotransposition), we sought to determine the effects of splicing on L1 retrotransposition. Here, we report the identification, frequency, and characterization of two separate classes of Spliced Integrated Retrotransposed L1 Elements (SpIREs) in the HGR. Strikingly, over the last 20 million years, SpIREs have been responsible for approximately 2% of presumed full-length L1s. The first class of SpIRE is generated by the retrotransposition of L1 mRNAs containing an intra-5 untranslated region (UTR) splicing event, which deletes cis-acting transcription factor binding sites critical for L1 transcription. Northern blot and luciferase-based assays reveal that the 5UTRs of these SpIREs have severely reduced promoter activity. The second class of SpIRE is generated from L1 mRNAs containing an intra-5 UTR/ORF1 splicing event. These L1s lack promoter activity and cannot produce a full-length ORF1 protein. Using a cultured cell retrotransposition assay, we determined that neither class of SpIRE could retrotranspose efficiently in cis. Our data demonstrate that SpIREs are essentially dead on arrival and likely cannot undergo subsequent retrotransposition. Thus, the use of cryptic splice sites within L1 mRNAs is detrimental to L1 retrotransposition and may be a mechanism to limit and/or regulate L1 retrotransposition in a developmental or cell-type specific manner.