HBond Score
HEXplorer score
ModCon
HBond Score background
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://rna.hhu.de/HBond/), 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://rna.hhu.de/HEXplorer/), 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.
Figure 1: The splicing regulatory effect of SREs is position dependent. SR proteins enhance recognition of downstream splice donors and repress usage of upstream splice donors. In contrast, hnRNPs show the opposite effect on splice site selection. hnRNP: heterogeneous nuclear ribonucleoproteins, SD: splice donor, SR: serine/arginine-rich proteins, SRE: splicing regulatory element
Codon degeneracy of amino acid sequences permits an additional "mRNP code" layer underlying the genetic code that is related to RNA processing. Usage of splice donor sites within the coding sequence of exons or reporter constructs is influenced by the splicing regulatory properties of the sequence neighborhood around a potential splice site, which can be approximated by its Splice Site HEXplorer Weight (SSHW) based on the HEXplorer score algorithm. To systematically modify splice donor neighborhoods, either minimizing or maximizing their SSHW, we designed the novel stochastic optimization algorithm ModCon (https://rna.hhu.de/ModCon/) that applies a genetic algorithm with stochastic crossover, insertion and random mutation elements supplemented by a heuristic sliding window approach. ModCon optimization can be applied, to induce or repress usage of splice donor sites within the coding sequence of splice donor competition reporters, while retaining the amino acid coding information. The ModCon algorithm and its R package implementation (available on Bioconductor) can assist in reporter design by either introducing novel splice sites, silencing accidental, undesired splice sites, and by generally modifying the entire mRNP code while maintaining the genetic code (Ptok et al. 2021).
References
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., 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.
Wang, Z. & C. B. Burge (2008) Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA, 14, 802-13.
Ptok J, Müller L, Ostermann PN, Ritchie A, Dilthey AT, Theiss S, Schaal H. Modifying splice site usage with ModCon: Maintaining the genetic code while changing the underlying mRNP code. Comput Struct Biotechnol J. 2021 May 21;19:3069-3076.