Genomic HEXploring allows landscaping of novel potential splicing regulatory elements.

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.

Differential hnRNP D isoform incorporation may confer plasticity to the ESSV-mediated repressive state across HIV-1 exon 3.

Even though splicing repression by hnRNP complexes bound to exonic sequences is well-documented, the responsible effector domains of hnRNP proteins have been described for only a select number of hnRNP constituents. Thus, there is only limited information available for possible varying silencer activities amongst different hnRNP proteins and composition changes within possible hnRNP complex assemblies. In this study, we identified the glycine-rich domain (GRD) of hnRNP proteins as a unifying feature in splice site repression. We also show that all four hnRNP D isoforms can act as genuine splicing repressors when bound to exonic positions. The presence of an extended GRD, however, seemed to potentiate the hnRNP D silencer activity of isoforms p42 and p45. Moreover, we demonstrate that hnRNP D proteins associate with the HIV-1 ESSV silencer complex, probably through direct recognition of "UUAG" sequences overlapping with the previously described "UAGG" motifs bound by hnRNP A1. Consequently, this spatial proximity seems to cause mutual interference between hnRNP A1 and hnRNP D. This interplay between hnRNP A1 and D facilitates a dynamic regulation of the repressive state of HIV-1 exon 3 which manifests as fluctuating relative levels of spliced vpr- and unspliced gag/pol-mRNAs.