Retroviral RNA Processing, Avian Leukosis Virus Tumorigenesis, TERT expression
My lab studies retroviral gene expression at the post-transcriptional level. An unusual feature of all retroviruses is that their primary RNA transcript is both a major viral mRNA, encoding capsid proteins, and a pre-mRNA. In addition, this unspliced RNA is packaged into viral particles as genomic RNA. We study control of retroviral RNA splicing, stability, and export. We have identified a Negative Regulator of Splicing (NRS) RNA element within the avian retroviral RNA intron, which helps maintain a portion of the primary transcripts as unspliced mRNA and genomic RNA. The NRS behaves like a defective 5' splice site, binding all of the splicing snRNPs, and interacting with 3' splice sites. It appears to compete with the authentic 5' splice site for interactions with 3' splice sites. While the NRS pseudo-spliceosome contains all of the splicing snRNPs, their arrangement is aberrant. We have observed the pRP8 splicing scaffold protein is not localized properly in the pseudo-spliceosome. We have also found that the NRS promotes polyadenylation in vitro.
Inactivating mutations in the NRS of an avian leukosis virus have been associated with rapid-onset chicken lymphomas, involving viral integration into the c-myb locus. We are also investigating possible additional integration sites. In several tumors, we have found viral integrations within 3 kb upstream of the telomerase reverse transcriptase (TERT) and associated with elevated expression of TERT and telomerase activity. These integrations are within a novel long non-coding RNA in the TERT promoter that we called TAPAS (TERT Antisense Promoter-Associated). Over-expression of truncated TAPAS affects the expression of cellular genes involving the cell cycle and cell mobility.
We have also studied TERT and TAPAS in human cells. We observed that TERT mRNA is alternatively spliced, frequently skipping the large exon 2 to generate an inactive mRNA. Further, we found that the bulk of TERT mRNA is nuclear and has some retained introns. We think this may be a regulatory mechanism. In addition, hTAPAS appears to negatively regulate TERT mRNA and has some regions of complementarity to TERT introns.
In addition, we are exploring the mechanism of nonsense-mediated RNA decay (NMD), induced by premature termination codons in the gag gene of unspliced retroviral RNAs. This is particularly interesting because NMD in higher organisms is thought to be coupled to splicing, and the deposition of exon-junction complexes. We have shown that NMD of unspliced viral RNAs is dependent on Upf1 and translation. Further, we have observed a stability element downstream of the normal gag termination codon. When these downstream sequences are removed, the RNA undergoes NMD. We think these stability sequences may be necessary to stabilize RNAs having a long 3' UTR. These stability sequences have been shown to bind PTBP1 and to suppress binding of UPF1. Finally, hundreds of cellular mRNAs have been found to similarly stabilize their mRNA.
Yang, F., Xian, R.R., Li, Y., Polony, T. and Beemon, K.L. 2007. Telomerase reverse transcriptase expression elevated by avian leukosis virus integration in B cell lymphomas. Proc. Natl. Acad. Sci. USA. 104:18952-7.
Bolisetty, M.T., Dy, G., Tam, W., and Beemon, K.L. 2009. Reticuloendotheliosis virus strain T induces microRNA-155, which targets JARID2 and promotes cell survival. J. Virol. 83:12009-17.
Bolisetty, M.T. and Beemon, K.L. 2012. Splicing of internal large exons is defined by novel cis-acting sequence elements. Nucleic Acids Res. 40:9244-9254.
Bolisetty, M.T., Blomberg, J., Benachenhou, F., Sperber, G., and Beemon, K.L. 2012. Unexpeced diversity and expression of avian endogenous retroviruses. MBio 3(5). doi:pii: e00344-12. 10.1128/mBio.00344-12.
Withers, J.B., Ashvetiya, T., and Beemon, K.L. 2012. Exclusion of exon 2 is a common mRNA splice variant of primate telomerase reverse transcriptases. PLoS One. 7(10):e48016. doi: 10.1371/journal.pone.0048016.
LeBlanc, J., Weil J. and Beemon, K. 2013. Post-transcriptional regulation of retroviral gene expression: primary RNA transcripts play three roles as pre-mRNA, mRNA and genomic RNA. WIREs RNA 4(5):567-80.
Justice, JF 4th, Morgan, RW, and Beemon, KL. 2015. Common viral integration sites identified in avian leukosis virus-induced B-cell lymphomas. mBio. 6(6) pii: e01863-15. doi: 10.1128/mBio.01863-15.
Ge, Z, Quek, BL, Beemon, KL, and Hogg, JR. 2016. Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway. eLIFE, Jan 8;5. pii: e11155. doi: 10.7554/eLife.11155.
Nehyba, J., Malhotra, S, Winans, S, O’Hare, TH, Justice, J IV and Beemon, K.L. 2016 Avian leukosis virus activation of an antisense RNA upstream of TERT in B-cell lymphomas. J. Virol. 90: 9509—17. PMCID: PMC5044821
Lam G, Xian RR, Li Y, Burns KH, and Beemon KL. 2016. Lack of TERT promoter mutations in human B-cell non-Hodgkin lymphoma. Genes (Basel). 7(11). pii: E93.
Winans S, Larue RC, Abraham CM, Shkriabai N, Skopp A, Winkler D, Kvaratskhelia M, Beemon KL. 2017. The FACT complex promotes avian leukosis virus DNA integration. J. Virol. 91(7) pii: JVI.00082-17. doi: 10.1128/JVI.00082-17. PMCID: PMC5355599
Balagopal V. and Beemon KL. 2017. Rous sarcoma virus RNA stability element inhibits deadenylation of mRNAs with long 3’ UTRs. Viruses 9(8). pii: E204. Doi: 10.3390/v9080204. PMCID: PMC5580461
Winans S, Flynn A, Malhotra S, Balagopal V, and Beemon KL. 2017. Integration of ALV into CTDSPL and CTDSPL2 genes in B-cell lymphomas promotes cell immortalization, migration and survival. Oncotarget 8(34):57302-57315. doi: 10.1863/oncotarget.19328. PMCID: PMC5593642
Malhotra S, Winans S, Lam G, Justice J, Morgan R, and Beemon K. 2017. Selection for avian leukosis virus integration sites determines the clonal progression of B-cell lymphomas. PLoSPathog. 13(11):e1006708. doi: 10.1371/journal.ppat. 1006708. PMCID: PMC5687753.
Malhotra S, Freeberg MA, Winans SJ, Taylor J, and Beemon KL. 2017. A novel long non-coding RNA in the hTERT promoter region regulates hTERT expression Non-coding RNA 4, 1; doi:10.3390/ncrna401001.
Lam G. and Beemon K.L. 2018. ALV integration-associated hypomethylation at the TERT promoter locus. Viruses 10, 74. Doi 10.3390/v10020074. PMCID: PMC5850381