Genes within Genes: Multiple LAGLIDADG Homing Endonucleases Target the Ribosomal Protein S3 Gene Encoded within an rnl Group I Intron of Ophiostoma and Related Taxa

In some ascomycete fungi, ribosomal protein S3 (Rps3) is encoded within a group I intron (mL2449) that is inserted in the U11 region of the mitochondrial large Subunit rDNA (rnl) gene. Previous characterization of the mL2449 intron in strains of Ophiostoma novo-ulmi subspecies americana (Dutch Elm Disease) revealed a complex genes-within-genes arrangement whereby a LAGLIDADG homing endonuclease gene (HEG) is inserted into the RPS3 gene near the 3' terminus, creating a hybrid Rps3-LAGLIDADG fusion protein. Here. we examined 119 additional strains of Ophiostoma and related taxa representing 85 different species by a polymerase chain reaction- based survey and detected both short (similar to 1.6 kb) and long (>2.2 kb) versions of the mL2449 intron in 88 and 31 strains, respectively. Among the long versions encountered, 21 were sequenced, revealing the presence of either intact or degenerated HEG-coding regions inserted within the RPS3 gene. Surprisingly, we identified two new HEG insertion sites in RPS3; one near the original C-terminal insertion site and one near the N-terminus of RPS3. In all instances, the HEGs are fused in-frame with the RPS3-coding sequences to create fusion proteins. However, comparative sequence analysis showed that upon insertion, the HEGs displaced a portion of the RPS3-coding region. Remarkably, the displaced RPS3-coding segments are duplicated and fused in-frame to the 3' end of RPS3, restoring a full-length RPS3 gene. We cloned and expressed the LAGLIDADG portion of two Rps3-HEG fusions, and showed that I-OnuI and I-LirI generate 4 nucleotide (nt), 3' overhangs, and cleave at or I nt upstream of the HEG insertion site, respectively. Collectively, Our data indicate that RPS3 genes are a refuge for distinct types of LAGLIDADG HEGs that are defined by the presence of duplicated segments of the host gene that restore the RPS3 gene, thus minimizing the impact of the HEG insertion on Rps3 function.