A novel approach to describe a U1 snRNA binding site

• Published in: Nucleic acids research 2003-12, Vol.31 (23), p.6963-6975
• Main Authors: Freund, Marcel, Asang, Corinna, Kammler, Susanne, Konermann, Carolin, Krummheuer, Jörg, Hipp, Marianne, Meyer, Imke, Gierling, Wolfram, Theiss, Stephan, Preuss, Thorsten, Schindler, Detlev, Kjems, Jørgen, Schaal, Heiner
• Format: Article
• Language: English
• Subjects: Algorithms; Ataxia Telangiectasia Mutated Proteins; ATM gene; Base Sequence; Binding Sites; Cell Cycle Proteins; Cell Extracts; DNA-Binding Proteins; Exons - genetics; Gene Products, env - genetics; HeLa Cells; HIV-1 - genetics; Human immunodeficiency virus; Humans; Hydrogen Bonding; Mutation - genetics; Protein-Serine-Threonine Kinases - genetics; Ribonucleoprotein, U1 Small Nuclear - metabolism; Ribonucleoprotein, U2 Small Nuclear - metabolism; RNA Splice Sites - genetics; RNA, Small Nuclear - chemistry; RNA, Small Nuclear - genetics; RNA, Small Nuclear - metabolism; splice donor; Tumor Suppressor Proteins
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkg901

RNA duplex formation between U1 snRNA and a splice donor (SD) site can protect pre‐mRNA from degradation prior to splicing and initiates formation of the spliceosome. This process was monitored, using sub‐genomic HIV‐1 expression vectors, by expression analysis of the glycoprotein env, whose formation critically depends on functional SD4. We systematically derived a hydrogen bond model for the complementarity between the free 5′ end of U1 snRNA and 5′ splice sites and numerous mutations following transient transfection of HeLa‐T4+ cells with 5′ splice site mutated vectors. The resulting model takes into account number, interdependence and neighborhood relationships of predicted hydrogen bond formation in a region spanning the three most 3′ base pairs of the exon (–3 to –1) and the eight most 5′ base pairs of the intron (+1 to +8). The model is represented by an algorithm classifying U1 snRNA binding sites which can or cannot functionally substitute SD4 with respect to Rev‐mediated env expression. In a data set of 5′ splice site mutations of the human ATM gene we found a significant correlation between the algorithmic classification and exon skipping (P = 0.018, χ2‐test), showing that the applicability of the proposed model reaches far beyond HIV‐1 splicing. However, the algorithmic classification must not be taken as an absolute measure of SD usage as it may be modified by upstream sequence elements. Upstream to SD4 we identified a fragment supporting ASF/SF2 binding. Mutating GAR nucleotide repeats within this site decreased the SD4‐dependent Rev‐mediated env expression, which could be balanced simply by artificially increasing the complementarity of SD4.