Conserved loop I of U5 small nuclear RNA is dispensable for both catalyticsteps of pre-mRNA splicing in HeLa nuclear extracts

The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) fr om purified snRNP proteins and HeLa or Xenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts. Substit ution of conserved nucleotides comprising internal loop 2 or deletion of in ternal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing. However, deletion of internal loop 2 abolish ed U5 activity in splicing and spliceosome formation. Surprisingly, substit ution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effe ct on U5 activity. Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5' and 3' halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing. Thus, in contrast to yeas t, loop 1 of the human U5 snRNA is dispensable for both steps of splicing i n HeLa nuclear extracts. This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins as sociated with the U5 snRNP, Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution.