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RNA Folding and Misfolding Within Cells
Summary: Sandra Wolin is interested in how RNA molecules fold into complex structures within cells, how misfolded RNA molecules are handled, and the consequences of RNA misfolding for cell function and disease.
All cells contain large numbers of noncoding RNAs that function in diverse cellular processes. In the cytoplasm, ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) are critical components of the protein synthesis machinery. In the nucleus, noncoding RNAs function in processes ranging from chromosome replication to pre-mRNA splicing. For these RNAs, it is often the three-dimensional folded structure of the RNA, rather than the primary sequence, that is important for function. Also, most RNAs do not function as naked RNAs. Instead, they assemble with proteins, and sometimes with other RNAs, to form ribonucleoprotein complexes.
We are interested in understanding how RNA molecules fold into intricate structures and assemble with proteins inside cells. Most studies of RNA folding have involved allowing RNA to fold in a test tube. These studies have revealed that while RNA can fold by itself, the buffer conditions needed are often different from the conditions inside cells. For this reason, it has been proposed that RNA-binding proteins assist RNA folding in cells. Few proteins with a bona fide role in RNA folding have been identified, however. In addition, many studies have revealed that RNAs have a tendency to misfold into incorrect structures. Although cells contain well-described mechanisms for recognizing misfolded proteins, little is known of the mechanisms by which cells recognize and handle misfolded RNAs.
Our entry point into this problem has been the study of two RNA-binding proteins, the Ro and La autoantigens. Both proteins are major targets of the immune response in patients suffering from the rheumatic disease systemic lupus erythematosus. Our work has revealed that La assists folding of certain newly synthesized RNAs, while Ro is part of a system by which cells recognize and degrade misfolded and defective RNA molecules. We also discovered that Ro is important for cell survival following exposure to ultraviolet irradiation. Most interestingly, mice lacking Ro develop an autoimmune syndrome resembling systemic lupus erythematosus in humans. Thus, Ro may be important for the prevention of autoimmune disease.
The La Autoantigen Is a Molecular Chaperone for Newly Transcribed Small RNAs
The La autoantigen is the first protein that binds many newly synthesized noncoding RNAs. These RNAs include precursors to tRNAs, the ribosomal 5S rRNA, small spliceosomal RNAs, and the signal recognition particle RNA. La binds many different RNAs because it recognizes a uridylate-rich sequence found at the end of all these nascent RNAs. Using genetics and biochemistry in the yeast Saccharomyces cerevisiae, we discovered that La stabilizes newly synthesized RNAs from degradation. This La-mediated stabilization has different consequences for the various RNAs. For pre-tRNAs, La binding influences the pathway by which the precursors are processed to mature tRNAs. For the spliceosomal U4 RNA, La stabilizes the form of the RNA that is preferentially bound by proteins, thus facilitating assembly of this RNA into a functional ribonucleoprotein particle.
Recently, we obtained evidence that La is required for efficient folding of certain pre-tRNAs. These pre-tRNAs have an intrinsic tendency to misfold into incorrect structures inside cells. For these RNAs, La binding stabilizes the correctly folded structure. Thus, as has been appreciated for protein folding, a protein that binds transiently to a precursor form of an RNA can influence the final folded structure of the mature RNA. Our current studies are directed toward identifying other proteins involved in RNA folding and examining the role of La in a quality control pathway in which cells recognize and degrade aberrant noncoding RNAs.
To understand how La recognizes nascent RNAs, we are collaborating with Karin Reinisch (Yale University) to obtain a crystal structure of La. Recently, we obtained the structure of the most conserved portion of La, a domain called the La motif. The La motif adopts a winged helix-turn-helix fold, similar to that of many DNA-binding proteins. By combining structural studies with biochemistry, we demonstrated that La uses a different surface of the motif to interact with RNA than that used by winged helix proteins for DNA binding. Specifically, a conserved aromatic pocket on the surface of the motif is crucial for recognition of 3' ends. (A grant from the National Institutes of Health provided support for these projects.)
The Ro Autoantigen Functions in a Quality Control Pathway for Noncoding RNAs
The Ro autoantigen is a 60-kDa protein that is normally bound to small RNAs called Y RNAs. Although Ro/Y RNA complexes are abundant components of all vertebrate cells, their function was mysterious for many years. One of the few clues was our discovery that Ro associates with variant 5S rRNA precursors in frog oocyte nuclei. The 5S rRNA variants bound by Ro have mutations that disrupt the conserved 5S rRNA structure, causing the RNA to misfold. This finding suggested that Ro might function in a quality control pathway in which incorrectly folded pre-5S rRNAs are recognized and targeted for degradation.
By studying Ro function in mouse cells, we discovered that Ro also associates with variant spliceosomal small RNAs. Like the pre-5S rRNA variants, association of Ro with these RNAs occurs in nuclei and requires that the RNAs misfold. It is known that cells possess mechanisms to recognize and degrade abnormal mRNAs. As targeting of defective mRNAs for degradation requires that the mRNAs undergo translation, this pathway is not accessible to noncoding RNAs. Our results suggest that Ro may be part of a novel pathway by which cells recognize abnormal RNA molecules that do not undergo translation.
To understand how Ro recognizes several classes of RNAs, we collaborated with the Reinisch laboratory to obtain a crystal structure of Ro complexed with RNA. We found that Ro is shaped like a doughnut with a central hole. Y RNAs bind on the outside of the ring, while single-stranded RNA binds in the hole. Mutagenesis experiments suggest that helical portions of misfolded RNAs bind on the surface of the doughnut, while the 3' ends of these RNAs insert into the cavity. Moreover, our studies suggest that one role of Y RNAs may be to regulate access of Ro to misfolded RNAs. We are pursuing the possibility that Ro is part of a cellular machine involved in refolding and/or degrading misfolded RNAs.
Interestingly, Ro is important for cell survival after ultraviolet irradiation. Although Ro is not present in yeast, a radiation-resistant bacterium, Deinococcus radiodurans, contains orthologs of both Ro and a Y RNA. By constructing a strain lacking Ro, we found that Ro is important for survival of the bacterium after ultraviolet irradiation. Consistent with a role in handling radiation damage, both Ro and the Y RNA increase during recovery of the bacterium from irradiation. By examining mouse cells lacking Ro, we showed that Ro is also important for mammalian cell survival after ultraviolet irradiation. Experiments in progress suggest that Ro may be involved in the degradation of damaged RNA after ultraviolet irradiation.
To examine the role of Ro in a mammalian organism, we collaborated with Richard Flavell (HHMI, Yale University) to generate mice lacking Ro. Surprisingly, these mice develop a syndrome that resembles systemic lupus erythematosus in humans. Similar to patients, the mice produce antibodies against their own chromatin and ribosomes, and develop kidney lesions due to autoantibody deposition. In one genetic background, the mice are also sensitive to sunlight, a common manifestation of human lupus. Although the mechanism by which loss of Ro results in autoimmunity is being explored, several mutations that cause autoimmunity in mice affect components of the machinery involved in clearing extracellular debris. One possibility is that in mice lacking Ro, a small population of incorrectly assembled ribosomes containing misfolded 5S rRNAs causes a breach of tolerance by exposing normally cryptic determinants to the immune system.
Last updated February 24, 2005
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