Martynova NY et al. (2021), Using RNA-binding proteins for immunoprecipitat...

XB-ART-58251

STAR Protoc 2021 Apr 21;22:100552. doi: 10.1016/j.xpro.2021.100552.

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Using RNA-binding proteins for immunoprecipitation of mRNAs from Xenopus laevis embryos.

Martynova NY , Parshina EA , Zaraisky AG .

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This protocol is developed for identifying mRNAs that form complexes with mRNA-binding proteins (mRBPs) in Xenopus laevis embryos at different developmental stages. Here, we describe the use of the Ybx1 mRBP for immunoprecipitation-based mRNA isolation. This protocol features the translation of the mRBP of interest directly in living embryos following injection of synthetic mRNA templates encoding a hybrid of this protein with a specific tag. This approach allows precipitation of mRNA-protein complexes from embryonic lysates using commercially available anti-tag antibodies. For complete details on the use and execution of this protocol, please refer to Parshina et al. (2020).

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Species referenced: Xenopus laevis

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References [+] :

Guo, Nanog RNA-binding proteins YBX1 and ILF3 affect pluripotency of embryonic stem cells. 2016, Pubmed


	Figure 1. Overall schematic of workflow in this protocolFor each stage (dotted box), the expected finish time and the corresponding steps are annotated.
	Figure 2. The main steps of embryo preparation (steps 15–17, before you begin and step 4, step-by-step method details)(A) Fertilized eggs before cleavage begins.(B) Two-cell stage embryo.(C) Embryos in Terasaki plates before manipulation.(D) Embryo at the gastrula stage (stage 12), top (animal) view.(E) Embryo at the gastrula stage (stage 12), bottom (vegetal) view.(F) Alive and dead (indicated by a red dashed line) embryos.Scale bars are 500 μm.
	Figure 3. The main steps of capillary preparation (step 2, step-by-step method details)(A) Initial capillary(B) The capillary is broken off with a light touch.(C). Prepared capillary after tip break
	Figure 4. Immunoprecipitation of 6Myc-Ybx1 and RT-qPCR determination of the mRNA of interest precipitated in a complex with 6Myc-Ybx1 (expected outcomes)(A) An equal amount of 6Myc-Ybx1 protein precipitated on Myc resin from a lysate of embryos microinjected with mRNA encoding 6myc-ybx1 and with a mixture of mRNA encoding 6myc-ybx1 and zyxin mRNA. Loading control is shown by the immunoglobulin light chain.(B) RIP experiments demonstrate the ability of Ybx1 to bind mRNA of pluripotency genes. The addition of zyxin mRNA has different effects on the stability of these complexes.The transcript levels of the housekeeping genes odc and efα are used for normalization. The error bars indicate the standard deviations derived from three independent experiments. ∗p<0.01; Student’s t-test.