A human U2 RNA mutant stalled in 3 ' end processing is impaired in nuclearimport

The biosynthesis of U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRN As) involves the nuclear export of precursor molecules extended at their 3' ends, followed by a cytoplasmic phase during which the pre-snRNAs assemble into ribonucleoprotein particles and undergo hypermethylation of their 5' caps and 3' end processing prior to nuclear import, Previous studies have d emonstrated that the assembly of pre-snRNAs into ribonucleoprotein particle s containing the Sm core proteins is essential for nuclear import in mammal ian cells but that 5' cap hypermethylation is not. In the present investiga tion we have asked whether or not 3' end processing is required for nuclear import of U2 RNA. We designed human pre-U2 RNAs that carried modified 3' t ails, and identified one that was stalled (or greatly slowed) in 3' end pro cessing, leading to its accumulation in the cytoplasm of human cells. Nonet heless, this 3' processing arrested pre-U2 RNA molecule was found to underg o cytoplasmic assembly into Sm protein-containing complexes to the same ext ent as normal pre-U2 RNA, The Sm protein-associated, unprocessed mutant pre -U2 RNA was not observed in the nuclear fraction. Using an assay based on s uppression of a genetically blocked SV40 pre-mRNA splicing pathway, we foun d that the 3' processing deficient U2 RNA was significantly reduced in its ability to rescue splicing, consistent with its impaired nuclear import.