Genomic HEXploring allows landscaping of novel potential splicing regulatory elements

Abstract

Effective splice site selection is critically controlled by flanking splicing regulatory elements (SREs) that can enhance or repress splice site use. Although several computational algorithms currently identify a multitude of potential SRE motifs, their predictive power with respect to mutation effects is limited. Following a RESCUE-type approach, we defined a hexamer-based ‘HEXplorer score’ as average Z-score of all six hexamers overlapping with a given nucleotide in an arbitrary genomic sequence. Plotted along genomic regions, HEXplorer score profiles varied slowly in the vicinity of splice sites. They reflected the respective splice enhancing and silencing properties of splice site neighborhoods beyond the identification of single dedicated SRE motifs. In particular, HEXplorer score differences between mutant and reference sequences faithfully represented exonic mutation effects on splice site usage. Using the HIV-1 pre-mRNA as a model system highly dependent on SREs, we found an excellent correlation in 29 mutations between splicing activity and HEXplorer score. We successfully predicted and confirmed five novel SREs and optimized mutations inactivating a known silencer. The HEXplorer score allowed landscaping of splicing regulatory regions, provided a quantitative measure of mutation effects on splice enhancing and silencing properties and permitted calculation of the mutationally most effective nucleotide.

Analysis of Competing HIV-1 Splice Donor Sites Uncovers a Tight Cluster of Splicing Regulatory Elements within Exon 2/2b

“…Upregulation of D2b use following G I2 -1 inactivation indicated that an SR protein binding site could be located within exon 2b. We had previously found an enhancing element located downstream of D2 in a HEXplorer-based screen of total HIV-1 mRNA (13). Continuing analysis of this element here showed that the enhancer ESE2b strongly activates D2b and simultaneously inhibits D2, which is facilitated by binding of SRSF10 and Tra2.…”

“…We used the sequence CCAAACAA (23) as a splicing-neutral reference and the very strongly enhancing purine-rich SRE HIV-1 GAR E42 fragment as a positive control (GAR contains GAA or GAG repeats [R is A or G]) (20,22). As expected, fragment IV, covering G I2 -1, did not support downstream splice donor use, while ESE2b (ESE 5005-5032 [13]), contained in fragment III, enhanced D2b use. Neither fragment I nor fragment II led to increased eGFP expression (Fig.…”

“…1A). Only recently, five novel SREs could be identified using the HEXplorer algorithm (13). This algorithm reflects potentially enhancing and silencing properties of hexamers in the neighborhood of a splice donor by calculating the frequency of hexamer occurrence within introns versus exons.…”

“…Within exon 2, serine/arginine-rich splicing factor 1 (SRSF1)-dependent exonic splicing enhancers (ESEs) M1 and M2 (15), as well as the SRSF4-dependent ESE-Vif (16), have been shown to activate D2, whereas two G runs suppress exon 2/2b inclusion (16,17). Furthermore, a novel HEXplorer-identified SRE within exon 2b, ESE2b (previously called ESE ), was shown to activate downstream splice donor usage in minigene analysis (13).…”

“…In a RESCUE-type approach, the HZ EI is based on different hexamer occurrences in exonic and intronic sequences in the neighborhood of splice donors, and it has been successfully used as a basis for the identification of exonic splicing regulatory elements (13,24).…”