La antigen recognizes and binds to the 3''-oligouridylate tail of a small RNA.

The La antigen is a cellular protein which interacts with many RNA species that are products of RNA polymerase III, including the adenovirus virus-associated (VA) RNAs. We demonstrate that the efficiency of antigen binding in vitro is determined by the number of U residues at the RNA 3' terminus. Forms of VA RNAI with more than two terminal U residues are fully bound, forms with two U residues are partially bound, and forms with fewer than two U residues are not bound at all. The antigen can be covalently linked to VA RNA by UV irradiation, and the site of cross-linking is shown to contain the 3' terminus of the RNA. We conclude that the antigen recognizes the U-rich 3' tail of VA RNA, and presumably that of other polymerase III products, and that it binds at or close to this site.     

Binding sites of rat liver 5S RNA to ribosomal protein L5

The ribonucleoprotein complex consisting of 5S RNA and the protein L5 was prepared from the large subunit of rat liver ribosomes. The RNA in the complex was digested in situ with RNase A or RNase T1. The RNase-resistant RNA fragments bound to the protein were recovered and purified by 2D-PAGE, and their nucleotide sequences were determined in order to elucidate the binding sites of the RNA to the protein. The results showed that the fragments had arisen from the 5'-end region (residues 1-21), from the second hairpin loop (residues 77-102) and from the 3'-end region (residues 106-120). Harsher digestion trimmed these fragments to shorter fragments. It was concluded that the minimal interactive sequences of 5S RNA to the protein L5 were residues 13-21, residues 85-102, and residues 106-114. A part of the first hairpin loop, residues 41-52, was also suspected to interact with the protein. These protein-binding sites of rat liver 5S RNA were compared with those of Escherichia coli, Halobacterium cutirubrum and yeast, and their probable conservation from eubacteria to eukaryotes is discussed.     

Eukaryotic small ribonucleoproteins. Anti-La human autoantibodies react with U1 RNA-protein complexes

Anti-La sera from patients with autoimmune disorders precipitate a set of nuclear and cytoplasmic small RNA-protein complexes. Up to now, it has been thought that the La antigen is associated only with RNAs transcribed by RNA polymerase III, including precursors of tRNA and 5 S ribosomal RNA. Here we report that anti-La sera also react with ribonucleoprotein particles containing small nuclear RNA U1, which is transcribed by RNA polymerase II. Anti-La sera from 12 out of 12 patients tested were found to precipitate U1 RNA-protein complexes from HeLa cell nuclear extracts, under conditions where nonimmune sera do not. Ribonucleoprotein particles containing a second small nuclear RNA, U2, do not react appreciably with anti-La sera although they are present in HeLa cell nuclei at the same concentration as U1 RNA. Anti-La sera also react with U1 RNA-protein complexes in mouse and frog cells, but not in Drosophila or Chironomus, two organisms which lack the La antigen. Hybridization of cloned U1 DNA with anti-La-reactive RNA from HeLa cell nuclear extracts reveals mature U1 RNA, whereas anti-La-reactive cytoplasmic RNA contains a series of hybridizing bands that represent molecules 1-7 nucleotides longer than U1 and which may include precursors of nuclear U1 RNA (Madore, S. J., Wieben, E. D., and Pederson, T. (1984) J. Cell Biol., 188-192). Pulse-chase experiments suggest that the association of La antigenicity with these cytoplasmic U1 RNA molecules is transient. These results are discussed in relation to the presence of uridylate-rich sequences in the 3' termini of U1 RNA precursors and mature U1 RNA, which are similar to La antigen binding sites in several RNAs transcribed by RNA polymerase III.     

Structure and assembly of turnip crinkle virus. VI. Identification of coat protein binding sites on the RNA

Structural studies of turnip crinkle virus have been extended to include the identification of high-affinity coat protein binding sites on the RNA genome. Virus was dissociated at elevated pH and ionic strength, and a ribonucleoprotein complex (rp-complex) was isolated by chromatography on Sephacryl S-200. Genomic RNA fragments in the rp-complex, resistant to RNase A and RNase T1 digestion and associated with tightly bound coat protein subunits, were isolated using coat-protein-specific antibodies. The identity of the protected fragments was determined by direct RNA sequencing. These approaches allowed us to study the specific RNA-protein interactions in the rp-complex obtained from dissociated virus particles. The location of one protected fragment downstream from the amber terminator codon in the first and largest of the three viral open reading frames suggests that the coat protein may play a role in the regulation of the expression of the polymerase gene. We have also identified an additional cluster of T1-protected fragments in the region of the coat protein gene that may represent further high-affinity sites involved in assembly recognition.