The 3′ ends of most eukaryotic pre-mRNAs
are generated by 3′ endonucleolytic cleavage and
subsequent polyadenylation. 3′-end formation can
be influenced positively or negatively by various factors.
In particular, U1 snRNP acts 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 transcripts 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, polyadenylation, that is inhibited. Since
in HIV-1 the biological requirement is to produce transcripts
that read through the 5′ LTR cleavage site rather
than 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
downstream of the cleavage/polyadenylation site, as in
the HIV-1 LTR. U1 snRNP binding at this location was shown
not to affect the recruitment of multiple cleavage/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 on polyadenylation of other pre-mRNAs,
were shown not to be required for cleavage inhibition.
Therefore, U1 snRNP bound to a 5′ splice site can
inhibit cleavage and polyadenylation in two mechanistically
different ways depending on whether the 5′ splice
site is located upstream or downstream of the cleavage
site.