Position-dependent inhibition of the cleavage step of pre-mRNA 3 '-end processing by U1 snRNP

The 3' ends of most eukaryotic pre-mRNAs are generated by 3' endonucleolyti c cleavage and subsequent polyadenylation. 3'-end formation can be influenc ed positively or negatively by various factors. In particular, U1 snRNP act s as an inhibitor when bound to a 5' splice site located either upstream of the 3'-end formation signals of bovine papilloma virus (BPV) late transcri pts or downstream of the 3'-end processing signals in the 5' LTR of the HIV -1 provirus. Previous work showed that in BPV it is not the first step, 3' cleavage, that is affected by U1 snRNP, but rather the second step, polyade nylation, that is inhibited, Since in HIV-1 the biological requirement is t o produce transcripts that read through the 5' LTR cleavage site rather tha n being cleaved there, this mechanism seemed unlikely to apply, The obvious difference between the two examples was the relative orientation of the 3' -end formation signals and the U1 snRNP-binding site, In vitro assays were therefore used to assess the effect of U1 snRNP bound at various locations relative to a cleavage/polyadenylation site on the 3' cleavage reaction, U1 snRNP was found to inhibit cleavage when bound to a 5' splice site downstr eam of the cleavage/polyadenylation site, as in the HIV-1 LTR, U1 snRNP bin ding at this location was shown not to affect the recruitment of multiple c leavage/polyadenylation factors to the cleavage substrate, indicating that inhibition is unlikely to be due to steric hindrance. Interactions between U1A, U1 70K, and poly(A) polymerase, which mediate the effect of U1 snRNP o n polyadenylation of other pre-mRNAs, were shown not to be required for cle avage inhibition. Therefore, U1 snRNP bound to a 5' splice site can inhibit cleavage and polyadenylation in two mechanistically different ways dependi ng on whether the 5' splice site is located upstream or downstream of the c leavage site.