Genetic analysis of the role of human U1 snRNA in mRNA splicing: I. Effect of mutations in the highly conserved stem-loop I of U1.

The 5' splice site mutation known as hr440 can be suppressed efficiently in vivo by a compensatory base change in U1 small nuclear RNA (snRNA). We have now begun a second-site reversion analysis of this suppressor U1-4u snRNA (which has a C----U change at position 4) to identify U1 nucleotides that are essential for mRNA splicing. Point mutations in U1-4u that disrupt the structure of stem-loop I or alter phylogenetically conserved nucleotides within the loop cause loss of suppression. The level of suppressor activity observed for most mutants correlated with the abundance of the corresponding suppressor RNA, suggesting that mutations in stem-loop I cause loss of suppression by destabilizing U1 snRNA or the U1 snRNP (small nuclear ribonucleoprotein particle). We favor the interpretation that incompletely or improperly assembled U1 snRNPs are unstable, because two severe point mutations in stem-loop I were found to decrease the binding of U1 snRNP-specific proteins in vitro. In a separate set of experiments, we found that increasing the distance between stem-loop I and the 5' end of U1 snRNA also inhibited suppression but did not affect assembly or stability of the U1 snRNP. This suggests that the relationship between the 5' splice site and the body of the U1 snRNP is important for mRNA splicing.