RESUMEN
In this study, phylogenetically conserved structural features of the Ro RNP associated Y RNAs were investigated. The human, iguana, and frog Y3 and Y4 RNA sequences have been determined previously and the respective RNAs were subjected to enzymatic and chemical probing to obtain structural information. For all of the analyzed RNAs, the probing data were used to compose secondary structures, which partly deviate from previously predicted structures. Our results confirm the existence of two stem structures, which are also found at similar positions in hY1 and hY5 RNA. For the remaining parts of hY3 and hY4 RNA the secondary structures differ from those previously proposed based upon computer predictions. What might be more important is that certain parts of the RNAs appear to be flexible, i.e., to adopt several conformations. Another striking feature is that a characteristic pyrimidine-rich region, present in every Y RNA known, is single-stranded in all secondary structures. This may suggest that this region is readily available for base pairing inter-actions with other cellular nucleic acids, which might be important for the as yet unknown function of the RNAs.
Asunto(s)
Conformación de Ácido Nucleico , ARN/genética , Animales , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Filogenia , ARN/química , Sondas ARN , Homología de Secuencia de Ácido NucleicoRESUMEN
This is the first study in which the complex of a monoclonal autoantibody fragment and its target, stem loop II of U1 snRNA, was investigated with enzymatic and chemical probing. A phage display antibody library derived from bone marrow cells of an SLE patient was used for selection of scFvs specific for stem loop II. The scFv specificity was tested by RNA immunoprecipitation and nitrocellulose filter binding competition experiments. Immunofluorescence data and immunoprecipitation of U1 snRNPs containing U1A protein, pointed to an scFv binding site different from the U1A binding site. The scFv binding site on stem loop II was determined by footprinting experiments using RNase A, RNase V1, and hydroxyl radicals. The results show that the binding site covers three sequence elements on the RNA, one on the 5' strand of the stem and two on the 3' strand. Hypersensitivity of three loop nucleotides suggests a conformational change of the RNA upon antibody binding. A three-dimensional representation of stem loop II reveals a juxtapositioning of the three protected regions on one side of the helix, spanning approximately one helical turn. The location of the scFv binding site on stem loop II is in full agreement with the finding that both the U1A protein and the scFv are able to bind stem loop II simultaneously. As a consequence, this recombinant monoclonal anti-U1 snRNA scFv might be very useful in studies on U1 snRNPs and its involvement in cellular processes like splicing.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Autoanticuerpos/química , Autoanticuerpos/genética , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/genética , ARN Nuclear Pequeño/inmunología , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Autoanticuerpos/metabolismo , Bacteriófagos/genética , Secuencia de Bases , Sitios de Unión/genética , Biblioteca de Genes , Humanos , Fragmentos de Inmunoglobulinas/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Conformación Proteica , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The structure of the conserved region of the U1A pre-mRNA (AgRNA) and its complex with U1A protein was investigated. The previously proposed secondary structure of Ag RNA, derived from enzymatic probing and analysis of the structure and function of mutant mRNAs, is now confirmed by chemical probing data and further refined in the regions where the enzymatic data were not conclusive. The two unpaired nucleotides in the internal loops opposite of the Box sequences as well as the tetraloop could not be cleaved by ribonucleases, but are accessible to chemical probes. Concerning the RNA-protein complex, the protection experiments showed that the Box regions are largely protected when the U1A protein is present. All stem regions in the 5' part of the structure seem protected against ribonucleases. Unexpectedly, the nucleotides of the tetraloop become accessible to ribonucleases in the RNA-protein complex. This result indicates that the tetraloop undergoes a conformational change upon U1A protein binding. The 3' part of the Ag RNA sequence, containing the polyadenylation signal in a hairpin structure, showed hardly any protection, a finding that agrees with the fact that U1A does not interfere with the binding of the cleavage polyadenylation specificity factor (CPSF) to the polyadenylation signal.