ferent populations of amino acids, the levels of indi- 
vidual charged tRNAs in the translation extract can 
now be manipulated. These studies should lead to 
an elucidation of the role that rare tRNAs play in 
ribosome pausing during translation. 
A tertiary structure in mRNA, known as an RNA 
pseudoknot, has also been postulated to force ribo- 
somes to pause during translation. This RNA struc- 
ture is required for ribosome frameshifting during 
translation of certain retroviral mRNAs. The pseu- 
doknot is thought to cause ribosomes to pause over 
certain "slippery" sequences in the mRNA, result- 
ing in frameshifting. Two retroviral mRNAs are 
currently being examined to determine if ribosomes 
actually pause at the site of the frameshift and if this 
pausing is due to the presence of a pseudoknot. 
The Ro Small Ribonucleoprotein Particles 
All eukaryotic cells contain an array of small RNA- 
protein complexes that play fundamental roles in 
cell metabolism. These ribonucleoprotein particles 
(RNPs) are often classified based on their subcellu- 
lar location, i.e., small nuclear ribonucleoproteins 
(snRNPs), small cytoplasmic ribonucleoproteins 
(scRNPs), and small nucleolar ribonucleoproteins 
(snoRNPs). Some of the best-characterized small 
RNPs are the small nuclear U RNPs, which are in- 
volved in mRNA processing, and the cytoplasmic 
signal recognition particle, which is important for 
targeting nascent secretory proteins to the endoplas- 
mic reticulum membrane. 
In addition to these relatively well-characterized 
small RNPs, there are additional small RNPs in cells 
whose functions remain obscure. One class of these 
RNPs, the Ro RNPs, was discovered because these 
particles are frequent targets of the autoimmune re- 
sponse in patients with two rheumatologic dis- 
orders, systemic lupus erythematosus and Sjogren's 
syndrome. The Ro RNPs (named because they are 
immunoprecipitated by anti-Ro lupus antibodies) 
consist of several small RNA molecules of ~ 1 00 nu- 
cleotides in length, each of which is complexed 
with a 60-kDa polypeptide. Each Ro RNP is present 
in ~10^ copies per cell, or ~1% the number of 
ribosomes. 
To define possible functions of the Ro RNPs, it is 
important to know their distribution within cells. 
Immunofluorescence experiments with patient au- 
toantibodies have long given conflicting results, pre- 
sumably because of the multiple specificities pres- 
ent in patient sera. Thus, to determine definitively 
the subcellular location of Ro RNPs, the distribution 
of these particles was determined in enucleated 
mammalian cells and karyoplasts. In these cell enu- 
cleation experiments, the Ro RNPs appear to be pri- 
marily cytoplasmic. As a further test, nucleic acid 
probes specific for the RNA components of these 
particles are being used to determine the distribu- 
tion of Ro RNPs within the cytoplasm, in relation to 
known organelles. This information will be valuable 
in making predictions for Ro RNP function. 
The Ro RNPs must function in a basic cellular pro- 
cess, because they have been found in every verte- 
brate cell type examined. Cloning and sequencing 
studies of the Xenopus protein and RNA compo- 
nents have revealed that the human and Xenopus 
components are nearly 80% identical, indicating 
that these particles are extremely conserved cellular 
components. Xenopus eggs have been demon- 
strated to contain stores of the 60-kDa Ro protein, 
which can be used to assemble heterologous Ro 
RNPs by mixing egg extracts with RNAs from other 
species. Future work will focus on identifying mole- 
cules that interact with Ro RNPs and on identifying 
Ro RNPs in species amenable to genetic analysis. 
Dr. Wolin is also Assistant Professor of Cell Biol- 
ogy at the Yale University School of Medicine. 
ENDOTHELINS AND THEIR RECEPTORS 
Masashi Yanagisawa, M.D., Ph.D., Associate Investigator 
The endothelins are a family of three small pep- 
tides with a variety of potent biological activities 
that are mediated by their specific G protein- 
coupled receptors. Dr. Yanagisawa and his col- 
leagues earlier identified the original member of the 
family, endothelin- 1 , a strong and extremely long- 
lasting vasopressor molecule secreted by vascular 
endothelial cells. This year the laboratory has initi- 
ated a number of projects aimed at further character- 
ization of the physiological role and regulation of 
the endothelins and their receptors. 
The Endothelin/Endothelin-Receptor System 
Despite the relative wealth of information regard- 
ing molecular components of the endothelin/en- 
dothelin-receptor system, its regulation, and its 
CELL BIOLOGY AND REGULATION 
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