Pre-mRNA splicing is regulated through combinatorial activity of RNA motifs including splice sites and splicing regulatory elements (SREs). Here, we show that the activity of a major class of mammalian SREs is highly sensitive to the strength of the adjacent 5 splice site (5ss) sequence, and that this has important functional and evolutionary implications. Activity of G-run SREs was higher for intermediate strength 5ss by ~4-fold relative to weak 5ss, and by ~1.3-fold relative to strong 5ss. The dependence on 5ss strength was supported both by comparative genomics and by microarray and Illumina mRNA-Seq analyses of splicing changes following RNAi against the splicing factor heterogeneous nuclear ribonucleoprotein (hnRNP) H, which binds G-runs. This dependence implies that the responsiveness of exons to changes in hnRNP H levels is a bivariate function of both SRE abundance and 5ss strength; this relationship may hold also for other splicing factors. This pattern of activity enables G-runs and hnRNP H to buffer the effects of 5ss mutations, augmenting both the frequency of 5ss polymorphism and the evolution of new splicing patterns. Overall design: Examine mRNA expression in 293T cells following hnRNP H or control siRNA knockdown