Abstract
MicroRNAs (miRNAs) play important roles in plant growth, development, and response to infection by microbes. Double-stranded RNA binding protein 1 (DRB1) facilitates the processing of primary miRNA transcripts into mature miRNAs. Recently, we found that NS3 protein encoded by rice stripe virus (RSV) associates with DRB1 and promotes miRNA biogenesis during RSV infection (Zheng et al., 2017). RNA co-immunoprecipitation (RIP) method was applied to identity association patterns among DRB1, NS3, and miRNA transcript.
Keywords: Rice stripe virus, NS3, Double-stranded RNA binding protein, Primary-miRNA, miRNA, Plant-microbe interaction
Background
MicroRNAs (miRNAs) are processed from their primary transcripts (pri-miRNAs) by the RNase III enzyme DICER-LIKE 1 (DCL1) with the help of the double-stranded RNA (dsRNA) binding protein HYPONASTIC LEAVES1 (DRB1/HYL1) and the zinc finger protein SERRATE (SE). Rice stripe virus (RSV) infection broadly perturbs miRNA accumulation. We found that RSV-encoding nonstructural protein 3 (NS3) promotes miRNA accumulation by downregulating pri-miRNAs through interaction with DRB1 in rice (Zheng et al., 2017). To reveal how NS3 enhances pri-miRNA processing, we used co-immunoprecipitation (Co-IP) to illustrate the relationship of NS3, DRB1 and pri-miRNA in vivo. This protocol contributes to understand association patterns between two proteins and one RNA transcript.
Materials and Reagents
Equipment
Procedure
Data analysis
Co-IP products were reversely transcribed using gene specific primer. Reverse transcripts were separated by electrophoresis on a 2% agarose gel. As shown in Figure 1, both OsDRB1a and mOsDRB1a interacted with apri-miR528, but neither of them recognized mapri-miR528; and NS3 and mNS3 were associated with OsDRB1a instead of mOsDRB1a. With the expression of NS3, both OsDRB1a and mOsDRB1a interacted with apri-miR528. However, with the expression of mNS3, only mOsDRB1a was associated with apri-miR528. These results indicate that NS3 may act as a scaffold to mediate the interaction between OsDRB1 and pri-miRNA. Figure 1. NS3 acts as a scaffold between DRB1 and pri-miRNA. RT-PCR detection of coimmunoprecipitated and input products of transiently co-expressed protein (DRB1a or mDRB1a), pri-miRNA (aprimiR528 or mapri-miR528), and protein (empty vector, NS3, or mNS3) in N. benthamiana. For more information, see Zheng et al., 2017.
Notes
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Acknowledgments
This work was supported by grants to JG. W. and L. Y. from the National Natural Science Foundation of China (Nos. 31722045; 31772128; 31701757 and 31201491); the National Basic Research Program 973 (2014CB138400); and the Natural Science Foundation of Fujian Province of China, Outstanding Young Scientific Research Plan and Excellent Talent Plan in the New Century of Fujian Province (JA3091 and 2014J06011). This protocol was modified from Saleh et al., 2008 and Terzi et al., 2009. We declare no conflicts of interest or competing interests.
References
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