Abstract
The U1 snRNP is essential for recognition of the
pre-mRNA 5′-splice site and the subsequent assembly
of the spliceosome. Yeast U1 snRNP is considerably more
complex than its metazoan counterpart, which suggests possible
differences between yeast and metazoa in early splicing
events. We have comprehensively analyzed the composition
of yeast U1 snRNPs using a combination of biochemical,
mass spectrometric, and genetic methods. We demonstrate
the specific association of four novel U1 snRNP proteins,
Snu71p, Snu65p, Nam8p, and Snu56p, that have no known metazoan
homologues. A fifth protein, Npl3p, is an abundant cellular
component that reproducibly co-purifies with the U1 snRNP,
but its association is salt-sensitive. Therefore, we are
unable to establish conclusively whether it binds specifically
to the U1 snRNP. Interestingly, Nam8p and Npl3p were previously
assigned functions in (pre-m)RNA-metabolism; however, so
far, no association with U1 snRNP has been demonstrated
or proposed. We also show that the yeast SmB protein is
a U1 snRNP component. Yeast U1 snRNP therefore contains
16 different proteins, including seven snRNP core proteins,
three homologues of the metazoan U1 snRNP-specific proteins,
and six yeast-specific U1 snRNP proteins. We have simultaneously
continued the characterization of additional mutants isolated
in a synthetic lethal (MUD) screen for genes that functionally
cooperate with U1 snRNA. Consistent with the biochemical
results, mud10, mud15, and mud16
are alleles of SNU56, NAM8, and SNU65,
respectively. mud10 and mud15 affect
the in vivo splicing efficiency of noncanonical introns.
Moreover, mud10p strongly affects the in vitro formation
of splicing complexes, and extracts from the mud15
strain contain a U1 snRNP that migrates aberrantly on native
gels. Finally, we show that Nam8p/Mud15p contributes to
the stability of U1 snRNP.