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.