Dr. Soriano is also Assistant Professor of Molec- 
ular Genetics and of Cell Biology at Baylor Col- 
lege of Medicine. 
Articles 
Friedrich, G. , and Soriano, P. 1 99 1 . Promoter traps 
in embryonic stem cells: a genetic screen to iden- 
tify and mutate developmental genes in mice. 
Genes Dev 5:1513-1523. 
Thomas, J.E., Soriano, P., and Brugge, J.S. 1991. 
Phosphorylation of c-Src on tyrosine 527 by an- 
other protein tyrosine kinase. Science 254:568- 
571. 
STRUCTURE AND FUNCTION OF SMALL RIBONUCLEOPROTEINS IN MAMMALIAN CELLS 
Joan Argetsinger Steitz, Ph.D., Investigator 
A variety of small ribonucleoproteins (RNPs, tight 
complexes between one or more proteins and a 
short RNA molecule) inhabit all higher eukaryotic 
cells. Many are highly abundant (> 10^ copies/cell) 
and highly conserved across species. Different types 
are localized specifically in the cell nucleus, nu- 
cleolus, or cytoplasm and are often the targets of 
autoantibodies found in the sera of patients with 
rheumatic disease. Dr. Steitz's laboratory would like 
to evolve a full description of both the variety of 
basic cellular processes that require input from 
small RNPs and the various ways in which the RNA 
moieties contribute to the action of individual parti- 
cles. So far, all small RNPs appear to participate in 
gene expression or genome maintenance. 
Small RNPs Involved in Splicing 
The most abundant (~10Vcell) of all small nu- 
clear RNPs (snRNPs) contain Ul, U2, U5, or U4 
+ U6 RNAs and ~6-12 proteins, some unique and 
some common. These snRNPs belong to the Sm class 
and are precipitable by anti-Sm patient antibodies. 
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-mRNAs of eu- 
karyotic cells. 
New snRNA • snRNA and snRNA • pre-mRNA con- 
tacts in the spliceosome. Although mechanistic sim- 
ilarities between group II self-splicing and nuclear 
pre-mRNA splicing have been apparent for some 
time, only a few parallels in RNA - RNA interactions 
between the two systems are known. Two different 
crosslinking strategies have been used to character- 
ize specific contacts between snRNAs, snRNAs and 
the pre-mRNA, and proteins and the pre-mRNA dur- 
ing the course of splicing in HeLa cell extracts. In 
addition to previously suspected or documented in- 
teractions, novel contacts between the pre-mRNA 
and the U5 and U6 snRNPs have been mapped, and 
kinetic analyses that allow these interactions to be 
sequentially ordered have been performed. 
1. Site-specific crosslinking of mammalian U5 
snRNP to the 5' splice site prior to the first step of 
pre-mRNA splicing. The standard adenovirus sub- 
strate and a spliced leader (SL) RNA-containing sub- 
strate were synthesized with a single '^P-labeled 
photoactivatable 4-thiouridine residue two nucleo- 
tides upstream of the 5' splice site. Selective pho- 
toactivation of the 4-thiouridine after incubation of 
either substrate under splicing conditions in HeLa 
nuclear extract resulted in crosslinks to the U5 
snRNA and the U5 snRNP protein p220. These ATP- 
dependent interactions occur prior to the first step 
of splicing. The U5 snRNA crosslinks map to a phy- 
logenetically invariant nine-nucleotide loop se- 
quence but do not require Watson-Crick comple- 
mentarity to the 5' exon. The results therefore 
provide biochemical evidence for a U5 snRNP- 5' 
exon interaction, previously suggested based on ge- 
netic studies in yeast. In addition, crosslinks of this 
position in the 5' exon to Ul — but not to U2, U4, or 
U6 snRNAs — are observed and are now being 
characterized. 
2. Psoralen crosslinking detects specific inter- 
actions of Ul, U2, U5, and U6 snRNAs with pre- 
mRNA during in vitro splicing. The crosslinking re- 
agent psoralen has been used to analyze the 
interactions of snRNAs with an adenovirus pre- 
mRNA in HeLa cell nuclear extract. An endogenous 
U2-U4-U6 crosslinkable complex dissociated upon 
incubation with the splicing substrate. During splic- 
ing, Ul , U2, U5, and U6 became crosslinked to pre- 
mRNA, and U2, U5, and U6 became crosslinked to 
the excised lariat intron. U2 also formed a doubly 
crosslinked complex with U6 and pre-mRNA. The 
Ul, U5, and U6 crosslinks mapped to intron se- 
quences near the 5' splice site, whereas the U2 
GENETICS 263 
