HIV-1 alternative splicing
During a processing step of precursor eukaryotic messenger RNAs (pre-mRNA) called splicing, so-called intronic sequences are mostly co-transcriptionally removed followed by the fusion of the remaining exonic sequence segments. Alternative splicing enables a variable set of mature mRNA transcripts originating from the same pre-mRNA sequence through ligation of different exon pairs or varying exon borders. Looseness in the exon usage pattern facilitates production of various protein isoforms in metazoans and higher eukaryotes. Whereas 95% of all human genes are affected by alternative splicing, constitutively spliced exons are present in most mature mRNAs. Mutations within regulatory sequence segments like splice site sequences and splicing regulatory elements, which among others mediate splicing, constitute around 25% of inherited diseases.
Replication of the human immunodeficiency virus type 1 (HIV-1) is essentially dependent on pre-mRNA splicing. Alternative splicing of the HIV-1 primary transcript leads to over 50 different mRNAs. Disruption of this process might be a promising target for future antiviral therapies. One focus of our research is to uncover the principle signals of splicing and regulation of alternative splicing. During this work we previously developed an algorithm to estimate the intrinsic strength of human and viral splice donor sequences (https://www2.hhu.de/rna/html/hbond_score.php), based on mutation analysis of the HIV-1 splice donor site 4 (Freund et al. 2003). Since binding of splicing regulatory proteins in proximity to splice sites critically influences splice site usage, we additionally developed an algorithm called HEXplorer (https://www2.hhu.de/rna/html/hexplorer_score.php), which assesses the potential of a genomic sequence to enhance or repress splice site usage (Erkelenz et al. 2014, Brillen et al. 2017a).
Regulation of splice site choice
We are also interested in RNA binding proteins (RBPs) which play important roles in numerous post-transcriptional processes including the splicing reaction (Dassi 2017). They interact with cis-located binding sites, so called splicing regulatory elements (SREs) on the RNA. Together with the splice sites themselves, they highly contribute to the generation of the ‘splicing code’. Two distinct protein families, serine and arginine rich proteins (SR proteins) and heterogeneous nuclear ribonucleoproteins (hnRNP) proteins are main interaction partners of SREs (Wang and Burge 2008). We study the tight regulation of splice donor and splice acceptor choice that is influenced by RBPs binding to splicing regulatory elements in the vicinity of splice sites. Additionally, we employ the HEXplorer algorithm to understand the impact of mutations on the binding landscape of RBPs and the subsequent splicing reaction.
Inhibition of HIV-1 replication by locked nucleic acid mixmer-modified antisense oligonucleotides
Our aim is to inhibit HIV-1 replication. Following this purpose, we masked viral splicing regulatory elements (SREs) by transfecting cells with locked nucleic acid mixmer-modified antisense oligonucleotides (LNA mixmers). This approach successfully interfered with viral particle production (Brillen et al. 2017b, Erkelenz et al. 2015, Widera et al. 2014). However, transfection reagents are not suitable for in vivo administration. Therefore, to better understand possible effects in vivo we simply added our LNA mixmers into the cell culture medium of HIV-1 infected cells (gymnosis), thereby circumventing transfection reagent-caused bias. Surprisingly, reduced levels of viral RNA species containing the respective LNA mixmer target sequence indicated specific degradation of LNA mixmer-bound RNA (Hillebrand et al. 2019). Thus, since we could reveal a novel productive cellular pathway for LNA mixmers for inhibition of HIV-1 replication, we now aim to understand the cellular mechanism underlying LNA mixmer-induced degradation of HIV-1 target RNA.
Brillen, A. L., K. Schoneweis, L. Walotka, L. Hartmann, L. Muller, J. Ptok, W. Kaisers, G. Poschmann, K. Stuhler, E. Buratti, S. Theiss & H. Schaal (2017a) Succession of splicing regulatory elements determines cryptic 5ss functionality. Nucleic Acids Res, 45, 4202-4216.
Brillen, A. L., L. Walotka, F. Hillebrand, L. Muller, M. Widera, S. Theiss & H. Schaal (2017b) Analysis of Competing HIV-1 Splice Donor Sites Uncovers a Tight Cluster of Splicing Regulatory Elements within Exon 2/2b. J Virol, 91.
Dassi, E. (2017) Handshakes and Fights: The Regulatory Interplay of RNA-Binding Proteins. Front Mol Biosci, 4, 67.
Erkelenz, S., F. Hillebrand, M. Widera, S. Theiss, A. Fayyaz, D. Degrandi, K. Pfeffer & H. Schaal (2015) Balanced splicing at the Tat-specific HIV-1 3'ss A3 is critical for HIV-1 replication. Retrovirology, 12, 29.
Erkelenz, S., S. Theiss, M. Otte, M. Widera, J. O. Peter & H. Schaal (2014) Genomic HEXploring allows landscaping of novel potential splicing regulatory elements. Nucleic Acids Res, 42, 10681-97.
Freund, M., C. Asang, S. Kammler, C. Konermann, J. Krummheuer, M. Hipp, I. Meyer, W. Gierling, S. Theiss, T. Preuss, D. Schindler, J. Kjems & H. Schaal (2003) A novel approach to describe a U1 snRNA binding site. Nucleic Acids Res, 31, 6963-75.
Hillebrand, F., P. N. Ostermann, L. Muller, D. Degrandi, S. Erkelenz, M. Widera, K. Pfeffer & H. Schaal (2019) Gymnotic Delivery of LNA Mixmers Targeting Viral SREs Induces HIV-1 mRNA Degradation. Int J Mol Sci, 20.
Wang, Z. & C. B. Burge (2008) Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA, 14, 802-13.
Widera, M., F. Hillebrand, S. Erkelenz, A. A. Vasudevan, C. Münk & H. Schaal (2014) A functional conserved intronic G run in HIV-1 intron 3 is critical to counteract APOBEC3G-mediated host restriction. Retrovirology, 11, 72.