Abstract
BackgroundDue to the nature of viral RNA genomes, RNA viruses depend on many RNA-binding proteins (RBP) of viral and host origin for replication, dissemination and evasion of host RNA degradation pathways. Some viruses interfere with the microRNA (miRNA) pathway to generate better fitness. The development of an adjusted, reliable and sensitive ribonucleoprotein immunoprecipitation (RIP) assay is needed to study the interaction between RBP of different origin (including viral origin) and miRNA precursors. The method could be further applied to transiently expressed heterologous proteins in different plant species.ResultsHere we describe a modified RIP assay applied to nuclear epitope-tagged proteins of heterologous origin and transiently expressed in Nicotiana benthamiana. The assay includes a combination of optimized steps as well as the careful selection of control samples and rigorous data analysis. It has proven efficient to detect and quantify miRNA processing intermediates associated with regulatory proteins.ConclusionsThe RIP method described here provides a reliable tool to study the interaction of RBPs, such as transiently expressed regulatory proteins with lowly represented host RNA, as is the case of miRNA precursors. This modified method was efficiently adjusted to recover nuclear proteins and reduce unspecific background. The purification scheme optimized here for GFP-tagged proteins can be applied to a wide array of RBPs. The subsequent application of next-generation sequencing technologies will permit to sequence and characterize all RNA species bound in vivo by a given RBP.
Highlights
Due to the nature of viral RNA genomes, RNA viruses depend on many RNA-binding proteins (RBP) of viral and host origin for replication, dissemination and evasion of host RNA degradation pathways
RNA-binding proteins (RBP)–RNA associations can be identified in RNP complexes involved in well-conserved core RNA processes, and in specialized processes regardless of whether the interaction occurs in a specific subcellular location like the nucleus
In the case of miRNA precursors, it is well known that they interact with nuclear RBPs like DICER-like 1 (DCL1) in order to be processed, and that the structural features of these precursors are recognized by processing proteins [22]
Summary
Due to the nature of viral RNA genomes, RNA viruses depend on many RNA-binding proteins (RBP) of viral and host origin for replication, dissemination and evasion of host RNA degradation pathways. RNA immunoprecipitation (RIP) is a powerful technique used to detect the association of individual proteins with specific RNA molecules in vivo. This assay has been successfully employed to purify ribonucleoprotein (RNP) complexes from plant tissue extracts, mostly from Arabidopsis [17,18,19,20]. In the case of miRNA precursors, it is well known that they interact with nuclear RBPs like DCL1 in order to be processed, and that the structural features of these precursors are recognized by processing proteins [22]. Methodologies aimed at detecting specific interactions between RBP of heterologous origin and lowly represented miRNAprocessing intermediates need to be optimized
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