Human Endogenous Retrovirus Expression in Fetal Tumors and Analysis of Factors Involved in the Post-Transcriptional Regulation of mRNA with Retained Introns

Human Endogenous Retroviruses (HERVs) are a class of genomic elements that resulted from repeated retroviral infection and integration in the human germline. HERV-K (HML-2) is the most recent proviral integration and has been shown to be biologically active in certain cancers and during fetal development. In my investigation for this thesis, I developed a transcriptional annotation of the approximately 90 Human Endogenous Retrovirus-K (HML-2) proviruses in the human genome. I used this transcriptional annotation to characterize HERV-K expression in fetal tumors and found that mRNA from specific integrated HERV-K proviruses was increased in both Hepatoblastoma and Wilms’ tumor. These findings raise the possibility that such expression could be used as targets for immune therapy or biomarkers for each respective disease.

HERV-K, like all retroviruses, expresses mRNA with retained introns. Nucleocytoplasmic export of mRNAs with retained introns requires special mechanisms, since the cell normally restricts the export of incompletely spliced mRNA. Previous investigations from our lab demonstrated that retroviral RNAs contain cis-acting RNA regulatory elements which pair with specific nuclear export proteins to allow efficient export and translation of retroviral mRNA that contain retained introns. In some cases, the export protein is encoded by the retrovirus (HERV-K, HIV, MMTV), while in other cases (MPMV, MLV) the cellular protein Nxf1 is utilized.

Our lab discovered that the RNA element present in MPMV RNA, which facilitates nuclear export, was exapted and duplicated from the cellular Nxf1 gene. This element has been named the Constitutive Transport Element (CTE). In the Nxf1 gene, the function of the RNA element is also to meditate the nucleocytoplasmic export of an Nxf1 isoform with a retained intron. This discovery raised the possibility that other mammalian genes might similarly contain CTE-like elements which facilitate the export of cellular mRNA isoforms with retained introns.

In this thesis, I identified several hundred novel CTEs in mammalian genes, by analyzing sequences that were derived from a retroviral vector trap system, which selected for elements that would allow the export of an mRNA with a retained intron. I then experimentally confirmed that many of these elements function together with Nxf1 to mediate nucleocytoplasmic export and translation of mRNA with a retained intron.

I additionally analyzed how the expression of Nxf1 and the post-transcriptional isoform of WT1(+KTS) alter the expression of cytoplasmic mRNA in a model cell line, using short and long read RNA Seq methods. I found that both proteins increase cytoplasmic mRNAs that contain retained introns from genes that have a direct role in post-transcriptional regulation. Additionally, there was significant overlap between genes with increased intron retention in the cytoplasm following expression of Nxf1 and WT1+KTS and the cellular CTEs discovered in the vector trap.