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 (approximately 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-LtrI generate 4 nucleotide (nt), 3' overhangs, and cleave at or 1 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.

The mitochondrial ribosomal protein S3 (rps3) gene within the fungi is extremely diverse in location and organization, some versions of this gene have been incorporated into a group I intron, others appear to have gained large insertions, microsatellite expansions, or have been invaded by homing endonucleases. Among the ascomycetes fungi the group I intron encoded version of rps3 appears to have a rather complex evolutionary history including first the acquisition of rps3 by a group I intron (mL2449), the loss of the mL2499 intron and the establishment of rps3 as a free-standing gene, and the eventual loss of the intron derived version of rps3.

In many cancers, combination therapy regimens are successfully improving response and survival rates, but the challenges of toxicity remain. GRP78, the master regulator of the unfolded protein response, is emerging as a target that is upregulated in tumors, specifically following treatment, and one that impacts tumor cell survival and disease recurrence. Here, we show IT-139, an antitumor small molecule inhibitor, suppresses induction of GRP78 from different types of endoplasmic reticulum (ER) stress in a variety of cancer cell lines, including those that have acquired therapeutic resistance, but not in the non-cancer cells being tested. We further determined that IT-139 treatment exacerbates ER stress while at the same time suppresses GRP78 induction at the transcriptional level. Our studies revealed a differential effect of IT-139 on chaperone protein family expression at multiple levels in different cancer cell lines. In xenograft studies, IT-139 decreased BRAF inhibitor upregulation of GRP78 expression in the tumor, while having minimal effect on GRP78 expression in the adjacent normal cells. The preferential decrease in GRP78 levels in tumor cells over normal cells, supported by the manageable safety profile seen in the Phase 1 clinical trial, reinforce the value IT-139 brings to combination therapies as it continues its clinical development.

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