A U6 snRNA:pre-mRNA interaction can be rate-limiting for U1-independent splicing.

The full set of consensus sequences at the 5' splice site is recognized during splicing of pre-mRNA in extracts depleted of U1 snRNP. High concentrations of HeLa SR proteins or purified SC35 alone promote the splicing of specific RNA substrates, bypassing the requirement for U1 snRNP in formation of the U2 snRNP-pre-mRNA complex. Under these conditions, mutations in the substrate that increase the sequence complementarity between U6 snRNA and the 5' splice site region can facilitate splicing. This provides additional strong evidence that U1 snRNP is not essential for splicing. Thus, the consensus sequence at the 5' splice site is probably recognized twice during splicing of most introns; however, some pre-mRNAs could potentially be processed in the absence of interactions with U1 snRNP in regions of the nucleus containing high concentrations of SR protein.