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 KD values) that leads to oligomerization of the RBD on its RNA ligands.

24The morphogenesis of the vertebrate eye consists of a complex choreography of cell 25 movements, tightly coupled to axial regionalization and cell type specification processes. Any 26 disturbance in these events can lead to developmental defects and blindness. Here we have 27 deciphered the sequence of defective events leading to coloboma phenotype in the 28 embryonic eye of the blind cavefish of the species Astyanax mexicanus. Using comparative 29 live imaging on targeted enhancer-trap Zic1:hsp70:GFP reporter lines of both the normal, 30 river-dwelling morph and the cave morph of the species, we identified major defects in initial 31 optic vesicle size and optic cup invagination in cavefish. Combining these results with gene 32 expression analyses, we also discovered defects in axial patterning affecting mainly the 33 temporal retina, in optic stalk tissue specification, and in the spreading processes involving 34 the retinal pigmented epithelium cells. Based on these results, we propose a developmental 35 scenario to explain the cavefish phenotype and discuss developmental constraints to 36 morphological evolution. The cavefish eye appears as an outstanding natural mutant model 37 to study molecular and cellular processes involved in optic region morphogenesis. 38 39 65 pigmented epithelium (RPE), expand and flatten to cover the back of the retina (Cechmanek 66 and McFarlane, 2017; Heermann et al., 2015). Together with the basal constriction of lens-67 facing epithelial cells (Martinez-Morales et al., 2009; Nicolas-Perez et al., 2016), these 68 movements lead to optic cup invagination. The invagination process leads to the formation of 69 the optic fissure at the level of the connection of the eye with the optic stalk. This fissure 70allows bloods vessels to invade the eye and leads the way of retino-fugal axons, but needs to 71 close to have a functional and round eye (Gestri et al., 2018). Finally, the entire eye, together 72 with the forebrain, rotates anteriorly, bringing the optic fissure in its final ventral position. 73 Hence, cells that are initially located in the dorsal or ventral part of the optic vesicles 74 contribute to the nasal or temporal quadrant of the retina, respectively (Picker et al., 2009). 75 Failure to correctly complete any of these steps can lead to vision defects; for example, failure 76 to properly close the optic fissure is termed coloboma and can lead to congenital blindness. 77 During the eye morphogenetic process, three types of tissues emerge: (1) the neural retina, 78 facing the lens and composed of various neuronal types, (2) the RPE at the back of the neural 79 retina, with multiple functions including nurturing of photoreceptors (Strauss, 2018), and (3) 80 the optic stalk, transiently connecting the retina to the neural tube. This thin ventral structure 81 is invaded by the ganglionic cells axons and guides them on their way to the tectum. Optic 82 stalk cells then differentiate into reticular astrocytes surrounding the optic nerve (Macdonald 83 et al.,...

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 behaviors during evagination which 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.