RESUMO
The Let-7:LIN28 regulatory loop is a paradigm in miRNA regulation. LIN28 harbors two RNA binding domains, which interact with well-conserved sequences in pre-let-7 RNAs, the GNGAY and the GGAG motifs. Here, the differential binding between LIN28B and pre-let-7 members was associated with the structural characteristics of the pre-let-7 family mapped by SHAPE, uncovering diverse structural patterns within pre-let-7 members. Pre-let-7 mutants supported a relevant role of the GGAG motif location and the preE-stem stability for the interaction with LIN28B. Based on these results, we propose a core RNA structure for LIN28B interaction.
Assuntos
MicroRNAs/química , MicroRNAs/metabolismo , Precursores de RNA/química , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Humanos , MicroRNAs/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Precursores de RNA/genéticaRESUMO
A reverse genetics approach was used to identify viral genetic determinants of the differential virulence displayed by two field foot-and-mouth disease virus (FMDV) strains (A/Arg/00 and A/Arg/01) isolated in Argentina during the 2000-2001 epidemics. A molecular clone of A/Arg/01 strain and viral chimeras containing the S-fragment or the internal ribosome entry site (IRES) of A/Arg/00 in the A/Arg/01 backbone were constructed and characterized. The IRES appeared as a determining factor of the lower level of A/Arg/00 replication in cell culture. High-throughput RNA probing revealed structural differences between both IRESs. Translation experiments using either synthetic viral RNAs (in vitro) or bicistronic plasmids (in vivo) showed that these IRESs' activities differ when the viral 3' untranslated region (UTR) is present, suggesting that their function is differentially modulated by this region. This work provides experimental evidence supporting the role of the IRES-3'UTR modulation in determining the level of FMDV replication in field strains.