STRUCTURE AND FUNCTION OF SMALL RIBONUCLEOPROTEINS IN MAMMALLVN CELLS 
Joan Argetsinger Steitz, Ph.D., Investigator 
A variety of small ribonucleoproteins (tight com- 
plexes between one or more proteins and a short 
RNA molecule) inhabit all higher eukaryotic cells. 
Many are highly abundant (more than 10^ cop- 
ies/cell) and highly conserved across species, sug- 
gesting important roles in cellular metabolism. Dif- 
ferent types are localized specifically in the cell 
nucleus or cytoplasm. Small ribonucleoproteins 
(RNPs) are often the targets of autoantibodies 
found in the sera of patients with rheumatic dis- 
ease. Dr. Steitz's laboratory has been using autoan- 
tibody probes to decipher the structure and func- 
tion of small RNPs in mammalian cells. So far, they 
all appear to participate in gene expression or ge- 
nome maintenance. Progress in the past year is 
summarized according to the category of small RNP 
I. Small RNPs Involved in Splicing. 
The most abundant (~10^/cell) of all small nu- 
clear RNPs (snRNPs) contain Ul, U2, U5, or U4 -I- 
U6 RNAs and —6-9 proteins, some unique and 
some common. These snRNPs belong to the Sm 
class, as they are precipitable by anti-Sm patient an- 
tibodies. They assemble on the pre-mRNA, together 
with many additional protein factors, to form a 
large body called the spliceosome, which carries 
out the two-step excision of introns from the pre- 
messenger RNAs of eukaryotic cells. 
A. Use of antibodies directed against yeast splicing 
proteins to characterize mammalian analogues. 
Genetic dissection of the splicing reaction in the 
yeast Saccharomyces cerevisiae has provided in- 
sights into spliceosome composition and function. 
However, such analyses have been largely lacking in 
the mammalian system. Using antibodies against 
prp8 (courtesy of Dr. Jean Beggs, University of Ed- 
inburgh), a yeast protein essential for splicing and a 
unique component of the yeast U5 snRNP, Dr. 
Steitz's laboratory identified an analogue in HeLa 
cell extracts. Like many other snRNP proteins, this 
novel 200 kDa protein is also reactive with anti-Sm 
antibodies. Immunoprecipitation and gradient frac- 
tionation analyses have characterized it as a compo- 
nent of the U5 snRNP and of the U4AJ5/U6 snRNP 
complex, which is a precursor to spliceosome 
assembly. The 200 kDa protein is also present in 
affinity-purified spliceosomes. Antibodies directed 
against other yeast splicing proteins (courtesy of 
Dr. John Abelson, California Institute of Technol- 
ogy) are being exploited to identify additional HeLa 
proteins implicated in splicing. 
B. Dissecting the mechanism of trans-splicing. The 
trans-splicing reaction occurring in trypanosomes 
and related species {Trypanosoma brucei, T. cruzi, 
T. vivax, Leishmania enriettii, Leptomonas col- 
losoma, Crithidia fasciculata), as well as in the 
nematode Caenorhabditis elegans, involves the 
transfer of a 5' exon from a spliced leader transcript 
(SL RNA) onto a pre-mRNA transcript possessing a 
3' splice acceptor. Dr. Steitz's laboratory previously 
discovered that SL RNAs assemble together with 
proteins precipitable by anti-Sm antibodies. Thus 
SL RNAs seem to fulfill a dual function in the trans- 
splicing process— that of both the 5' exon and an 
snRNP RNA. To test the prediction that the SL 
snRNP might autonomously activate its own 5' 
splice site and thereby eliminate the need for a Ul- 
like snRNP in the trans-splicing machinery, splicing 
substrates that contain an SL RNA sequence (from 
L. collosoma) connected to a 3' splice site (from 
the adenovirus major late-transcription unit) have 
been constructed. Such constructs splice well in 
HeLa cell nuclear extracts. Next, Ul snRNPs were 
inactivated in the extract by extensive oligonucleo- 
tide-directed RNase H degradation of the 5' end of 
Ul. Splicing of a control transcript containing in- 
tron 1 of adenovirus was completely abolished; 
however, the SL RNA- 3' splice-site construct re- 
tained substantial activity. Thus the SL RNA se- 
quence does appear to substitute for Ul during 
splicing in HeLa cell nuclear extracts. Studies em- 
ploying mutant SL RNA- 3' splice-site constructs are 
currently in progress to define the precise se- 
quence requirements for Ul snRNP-independent 
splicing. These results underscore the close rela- 
tionship of the cis- and trans-splicing machineries 
and suggest that trans-splicing could exist in mam- 
malian cells. 
11. 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) that also have 5' trimethyl- 
guanosine on their RNAs and contain proteins reac- 
tive with anti-Sm autoantibodies. Two of the RNAs 
in such low abundance snRNPs (Ull and U12) 
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