RNA-binding protein immunoprecipitation as a tool to investigate plant miRNA processing interference by regulatory proteins of diverse origin

GARCÍA, M. L.; GARCÍA, M. L.; REYES, C. A.; REYES, C. A.; MARMISOLLE, F. E.; MARMISOLLE, F. E.

PLANT METHODS

BIOMED CENTRAL LTD

Año: 2018 vol. 14

1746-4811

Background: Due to the nature of viral RNA genomes, RNA viruses depend on many RNA-binding proteins (RBP) ofviral and host origin for replication, dissemination and evasion of host RNA degradation pathways. Some viruses interferewith the microRNA (miRNA) pathway to generate better ftness. The development of an adjusted, reliable andsensitive ribonucleoprotein immunoprecipitation (RIP) assay is needed to study the interaction between RBP of diferentorigin (including viral origin) and miRNA precursors. The method could be further applied to transiently expressedheterologous proteins in diferent plant species.Results: Here we describe a modifed RIP assay applied to nuclear epitope-tagged proteins of heterologous originand transiently expressed in Nicotiana benthamiana. The assay includes a combination of optimized steps as wellas the careful selection of control samples and rigorous data analysis. It has proven efcient to detect and quantifymiRNA processing intermediates associated with regulatory proteins.Conclusions: The RIP method described here provides a reliable tool to study the interaction of RBPs, such as transientlyexpressed regulatory proteins with lowly represented host RNA, as is the case of miRNA precursors. This modifedmethod was efciently adjusted to recover nuclear proteins and reduce unspecifc background. The purifcationscheme optimized here for GFP-tagged proteins can be applied to a wide array of RBPs. The subsequent applicationof next-generation sequencing technologies will permit to sequence and characterize all RNA species bound in vivoby a given RBP.