Towards reconstructing the dipteran demise of an ancient essential gene: E3 ubiquitin ligase Murine double minute.

Genome studies have uncovered many examples of essential gene loss, raising the question of how ancient genes transition from essentiality to dispensability. We explored this process for the deeply conserved E3 ubiquitin ligase Murine double minute (Mdm), which is lacking in Drosophila despite the conservation of its main regulatory target, the cellular stress response gene p53. Conducting gene expression and knockdown experiments in the red flour beetle Tribolium castaneum, we found evidence that Mdm has remained essential in insects where it is present. Using bioinformatics approaches, we confirm the absence of the Mdm gene family in Drosophila, mapping its loss to the stem lineage of schizophoran Diptera and Pipunculidae (big-headed flies), about 95-85 million years ago. Intriguingly, this gene loss event was preceded by the de novo origin of the gene Companion of reaper (Corp), a novel p53 regulatory factor that is characterized by functional similarities to vertebrate Mdm2 despite lacking E3 ubiquitin ligase protein domains. Speaking against a 1:1 compensatory gene gain/loss scenario, however, we found that hoverflies (Syrphidae) and pointed-wing flies (Lonchopteridae) possess both Mdm and Corp. This implies that the two p53 regulators have been coexisting for ~ 150 million years in select dipteran clades and for at least 50 million years in the lineage to Schizophora and Pipunculidae. Given these extensive time spans of Mdm/Corp coexistence, we speculate that the loss of Mdm in the lineage to Drosophila involved further acquisitions of compensatory gene activities besides the emergence of Corp. Combined with the previously noted reduction of an ancestral P53 contact domain in the Mdm homologs of crustaceans and insects, we conclude that the loss of the ancient Mdm gene family in flies was the outcome of incremental functional regression over long macroevolutionary time scales.

Spliced integrated retrotransposed element (SpIRE) formation in the human genome.

Human Long interspersed element-1 (L1) retrotransposons contain an internal RNA polymerase II promoter within their 5' untranslated region (UTR) and encode two proteins, (ORF1p and ORF2p) required for their mobilization (i.e., retrotransposition). The evolutionary success of L1 relies on the continuous retrotransposition of full-length L1 mRNAs. Previous studies identified functional splice donor (SD), splice acceptor (SA), and polyadenylation sequences in L1 mRNA and provided evidence that a small number of spliced L1 mRNAs retrotransposed in the human genome. Here, we demonstrate that the retrotransposition of intra-5'UTR or 5'UTR/ORF1 spliced L1 mRNAs leads to the generation of spliced integrated retrotransposed elements (SpIREs). We identified a new intra-5'UTR SpIRE that is ten times more abundant than previously identified SpIREs. Functional analyses demonstrated that both intra-5'UTR and 5'UTR/ORF1 SpIREs lack Cis-acting transcription factor binding sites and exhibit reduced promoter activity. The 5'UTR/ORF1 SpIREs also produce nonfunctional ORF1p variants. Finally, we demonstrate that sequence changes within the L1 5'UTR over evolutionary time, which permitted L1 to evade the repressive effects of a host protein, can lead to the generation of new L1 splicing events, which, upon retrotransposition, generates a new SpIRE subfamily. We conclude that splicing inhibits L1 retrotransposition, SpIREs generally represent evolutionary "dead-ends" in the L1 retrotransposition process, mutations within the L1 5'UTR alter L1 splicing dynamics, and that retrotransposition of the resultant spliced transcripts can generate interindividual genomic variation.