RiboTag: Ribosomal Tagging Strategy to Analyze Cell-Type-Specific mRNA Expression In Vivo

Abstract

Ribosome tagging has become a very useful in vivo approach for analyzing gene expression and mRNA translation in specific cell types that are difficult and time consuming to isolate by conventional methods. The approach is based on selectively expressing a hemagglutinin A (HA)-tagged ribosomal protein in a target cell type and then using antibodies against HA to purify the polysomes and associated mRNAs from the target cell. The original approach makes use of a mouse line (RiboTag) harboring a modified allele of Rpl22 (Rpl22-HA) that is induced by the action of Cre recombinase. The Rpl22-HA gene can also be introduced into the animal by stereotaxic injection of an AAV-DIO-Rpl22-HA that is then activated in Cre-expressing cells. Both methods for tagging ribosomes facilitate the immunoprecipitation of ribosome-bound mRNAs and their analysis by qRT-PCR or RNA-Seq. This protocol will discuss the technical procedures and describe important considerations relevant to the analysis of the data. © 2019 by John Wiley & Sons, Inc.

Keywords: RNA-Seq; gene expression; neuron-specific mRNA; polyribosome immunoprecipitation; ribosome tagging.

Figure 1.. Cartoon comparing the RiboTag mouse and AAV-DIO-RiboTag approaches

On the left side, the Rpl22 locus was modified by homologous recombination to insert LoxP sites on either end of the last exon (Ex4) of the Rpl22 gene and a duplicated Ex4 with an HA tag was inserted downstream. The CRE-Driver mouse removes the WT Ex4 sequence in specific cells and the transcript now splices in Ex4-HA (shown in green). Polyribosomes from cells expressing the Ex4-HA are recovered from a lysate by immunoprecipitation (IP) with an antibody directed against HA and magnetic beads coated with an artificial Protein A/G peptide. RNA is isolated from the IP polyribosomes and RNA is also isolated from the lysate. Gene expression analysis is done by RNA-Seq. On the right side the DIO-RiboTag viral vector is shown schematically. In the presence of Cre-recombinase the Rpl22-HA-IRES-YFP cassette is flipped and locked in the ON configuration so that the eIF1a promoter now drives expression of Rpl22-HA in specific cells. RNA is isolated from the IP and compared with the input.

Figure 2.. Example plots of hypothetical RNA Seq data.

A, Volcano plot of data with log base 2 transformed fold change (condition A vs condition B) plotted on the x axis and negative natural log transformed corrected p-value (q-value) plotted on the y axis. The solid horizontal line denotes where q-value is equal to 0.05. The two solid vertical lines denote where fold change is equal to 2 (or −2). Data points are colored red if they are both 2-fold different and have a q-value less than 0.05; otherwise they are black. B, Scatter plot of data with log2 transformed fragments per kilobase million (FPKM) plotted on the x axis for condition A and the y axis for condition B. As in A data points are colored red if they are both 2-fold different and have a q-value less than 0.05; otherwise they are black. C, Box plots for Gene A for condition A and B. FPKM are plotted on the y axis with the two conditions represented side by side on the x axis. D, Bar graphs denoting mean plus or minus standard error of the mean (SEM) for Gene A for condition A and B. FPKM are plotted on the y axis with the two conditions represented side by side on the x axis. Individual data points are superimposed on top of the bars for greater data reporting transparency. DE, differentially expressed.