RESUMO
Influenzavirus non-structural protein NS1 is involved in several steps of the virus replication cycle. It counteracts the interferon response, and also exhibits other activities towards viral and cellular RNAs. NS1 is known to bind non-specifically to double-stranded RNA (dsRNA) as well as to viral and cellular RNAs. We set out to search whether NS1 could preferentially bind sequence-specific RNA patterns, and performed an in vitro selection (SELEX) to isolate NS1-specific aptamers from a pool of 80-nucleotide(nt)-long RNAs. Among the 63 aptamers characterized, two families were found to harbour a sequence that is strictly conserved at the 5' terminus of all positive-strand RNAs of influenzaviruses A. We found a second virus-specific motif, a 9 nucleotide sequence located 15 nucleotides downstream from NS1's stop codon. In addition, a majority of aptamers had one or two symmetrically positioned copies of the 5'-GUAAC / 3'-CUUAG double-stranded motif, which closely resembles the canonical 5'-splice site. Through an in-depth analysis of the interaction combining fluorimetry and gel-shift assays, we showed that NS1's RNA-binding domain (RBD) specifically recognizes sequence patterns in a structure-dependent manner, resulting in an intimate interaction with high affinity (low nanomolar to subnanomolar K(D) values) that leads to oligomerization of the RBD on its RNA ligands.
Assuntos
Vírus da Influenza A/genética , RNA Viral/química , Proteínas de Ligação a RNA/química , Proteínas não Estruturais Virais/química , Substituição de Aminoácidos , Aptâmeros de Nucleotídeos/química , Motivos de Nucleotídeos , Ligação Proteica , Estrutura Terciária de Proteína , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
The morphogenesis of the vertebrate eye consists of a complex choreography of cell movements, tightly coupled to axial regionalization and cell type specification processes. Disturbances in these events can lead to developmental defects and blindness. Here, we have deciphered the sequence of defective events leading to coloboma in the embryonic eye of the blind cavefish of the species Astyanax mexicanus. Using comparative live imaging on targeted enhancer-trap Zic1:hsp70:GFP reporter lines of both the normal, river-dwelling morph and the cave morph of the species, we identified defects in migratory cell behaviours during evagination that participate in the reduced optic vesicle size in cavefish, without proliferation defect. Further, impaired optic cup invagination shifts the relative position of the lens and contributes to coloboma in cavefish. Based on these results, we propose a developmental scenario to explain the cavefish phenotype and discuss developmental constraints to morphological evolution. The cavefish eye appears as an outstanding natural mutant model to study molecular and cellular processes involved in optic region morphogenesis.