crosslink mapped to the branch site. The kinetics of 
crosslink formation and disappearance delineates a 
temporal pathway for the action of small RNAs in the 
spliceosome. Potential base-pairing between con- 
served sequences in both the snRNAs and pre-mRNA 
at the sites of crosslinking argue that the 5' splice 
site is inspected by the U5 and U6 as well as the Ul 
snRNP prior to the first cleavage event. The data 
further suggest functional analogies between U6 
snRNA and domain 5 of group II self-splicing 
introns. 
Multiple functions for the Ul snRNPF There are 
many indications that the Ul snRNP, which is 
slightly more abundant than the other splicing 
snRNPs, may play several roles in splicing as well as 
in related RNA-processing events. 
1. Two functions for the 5' end of Ul in pre- 
mRNA splicing. To probe functions of the Ul snRNP 
during in vitro splicing, Dr. Steitz and her col- 
leagues examined unusual splicing substrates that 
replace the 5' splice site region of an adenovirus 
substrate with SL RNA sequences from Leptomonas 
collosoma or Caenorhabditis elegans. Such chi- 
meric transcripts were previously shown not to re- 
quire 5' splice site/Ul snRNA base-pairing and thus 
uncouple this function from other roles the Ul 
snRNP may perform in splicing. When antisense 2'- 
O-methyl oligoribonucleotides were used to se- 
quester stably positions 1-10 of Ul snRNA involved 
in 5' splice site recognition, splicing of the SL con- 
structs continued; however, longer oligoribonu- 
cleotides that also disrupt the first stem of Ul snRNA 
inhibit the splicing of the SL as well as the adenovi- 
rus substrates. Native gel analysis indicated that the 
longest oligoribonucleotide inhibits splicing by 
blocking spliceosome formation subsequent to U2 
snRNP association but before joining of the U4/U5/ 
U6 tri-snRNP. Thus, although the extreme 5' end of 
Ul snRNA base-pairs with the 5' splice site, the se- 
quence or structural integrity of stem I is essential 
for some later function in spliceosome assembly. 
2. Ul snRNP association with terminal exons. 
Psoralen crosslinking experiments in HeLa cell nu- 
clear extracts have revealed the binding of Ul 
snRNA to substrates containing the simian virus 40 
(SV40) late or adenovirus L3 polyadenylation sig- 
nals. The sites of Ul crosslinking to the substrates 
mapped different distances upstream of the AAUAAA 
sequence to regions with limited complementarity 
to the 5' end of Ul snRNA. Ul crosslinking to the 
same site in the SV40 late pre-mRNA was enhanced 
by addition of an upstream 3' splice site, which also 
enhances polyadenylation. Examination of different 
nuclear extracts revealed a correlation between Ul 
crosslinking and the coupling of splicing and poly- 
adenylation, suggesting that the Ul snRNP par- 
ticipates in the coordination of these two RNA- 
processing events. The mechanism of communica- 
tion between the splicing and polyadenylation 
machineries, as well as how interaction of the Ul 
snRNP with 3'-terminal exons might contribute to 
mRNA export, is under further study. 
Minor snRNPs Related to Splicing snRNPs 
In addition to the splicing snRNPs, mammalian 
cells contain a number of less abundant snRNPs 
(10^-10^ copies/cell), which also have 5'-trimeth- 
ylguanosine caps on their RNAs and contain proteins 
reactive with anti-Sm autoantibodies. Ul 1 and U12 
are two low-abundance snRNPs that coexist in a 
two-snRNP complex. Their functions are under 
study in part using serum from a scleroderma pa- 
tient that contains novel antibodies that recognize 
protein component (s) of the Ul 1 particle as well as 
antibodies against the trimethylguanosine cap of U 
RNAs. Psoralen crosslinking and genetic suppres- 
sion approaches are being used to analyze the low- 
abundance U7 snRNP, which participates in the 3'- 
end maturation of histone pre-mRNAs. 
Mammalian Nucleolar snRNPs 
Mammalian cell nucleoli contain a family of re- 
lated small RNP particles, whose RNAs are called 
U3, U8, U13, U14, U15, and Ul6. Human U8 and 
U13 are present at about 7? and ^/2o the amount of 
U3 (lO^cell), respectively, and, like U3, contain 
5'-trimethylguanosine cap structures. All these par- 
ticles can be immunoprecipitated by autoantibodies 
to fibrillarin, an abundant protein of the fibrillar 
subcompartment of the nucleolus. To date, only a 
function for U3 in the earliest processing step of 
preribosomal RNA maturation to yield 45S pre-rRNA 
has been established, but the other nucleolar 
snRNPs are likewise believed to participate in ribo- 
some biogenesis. 
Nucleolar targeting of snRNPs. The require- 
ments for import of the human U3 snRNA into the 
nucleus have been analyzed in the Xenopus oocyte. 
Neither cap trimethylation nor binding of the com- 
mon protein fibrillarin were found to be essential. 
Rather, a structural element that includes the 3' end 
of the RNA was identified as a novel determinant for 
nuclear import. Currently the protein composition 
of the U3 snRNP is being scrutinized, with the goal 
of finding proteins (and their binding sites on U3) 
that are required for nuclear and eventual nucleolar 
localization of this snRNP. 
Finding a function for U8. U8 RNA from Xeno- 
pus has been sequenced to identify regions con- 
served with mammalian US. Some of the highly ho- 
264 
