Figure 2. eIF3 remains bound to the coding sequences (CDS) of pan-mRNAs independent of their 5’-UTR and 3’-UTR elements in actively translating ribosomes.

(A) Schematic outlining the RNase H-based assay of eIF3 interactions with mRNAs in polysomes. DSP refers to the dithiobis (succinimidyl propionate) crosslinking agent. Oligos, DNA oligos designed for RNase H-mediated targeting and cleavage of specific mRNAs. (B) Strategy for detecting mRNA fragments released by RNase H digestion. Red arrows denote DNA oligos for RNase H-mediated targeting of mRNAs. RT-qPCR primers (black) were used to detect the CDS regions of the mRNAs. (C) Amount of eIF3-bound mRNA co-immunoprecipitated by an anti-EIF3B antibody (Lee et al., 2015), from polysome fractions of Jurkat cells treated with RNase H and oligos targeting the CDS-UTR junctions (red arrows diagrammed in panel B). (D) Amount of eIF3-bound mRNA co-immunoprecipitated with IgG beads, from polysome fractions of Jurkat cell lysate treated with RNase H and oligos targeting the CDS-UTR junctions. (E) Amount of eIF3-bound mRNA co-immunoprecipitated by the anti-EIF3B antibody, from polysome fractions of Jurkat cell lysate treated only with RNase H. (F) Amount of eIF3-bound mRNA co-immunoprecipitated by an anti-EIF3B antibody, from polysome fractions of primary human T cells treated with RNase H and oligos targeting the CDS-UTR junctions (red arrows diagrammed in panel B). (G) Amount of eIF3-bound mRNA co-immunoprecipitated with IgG beads, from polysome fractions of primary human T cell lysate treated with RNase H and oligos targeting the CDS-UTR junctions. (H) Amount of eIF3-bound mRNA co-immunoprecipitated by the anti-EIF3B antibody, from polysome fractions of primary human T cell lysate treated only with RNase H. In panels C–H, the percentage is relative to the amount of total mRNA present in the polysome fraction prior to immunoprecipitation. All the immunoprecipitation experiments in panels C–H were carried out in biological duplicate with one technical triplicate shown (n = 3, with mean and standard deviations shown). The primary human T cell experiment was done using two donors.

Figure 2—figure supplement 1. The TCRA and TCRB mRNAs remain bound to elongating ribosomes via eIF3 in activated T cells.

(A) Sucrose gradient fractionation of polysomes from crosslinked Jurkat cells. Cell lysates treated with RNase H, as indicated, were fractionated on 10–50% sucrose gradients. Shown is the absorbance at 254 nm, for one of two biological replicates. (B) Strategy for detecting mRNA cleavage by RNase H digestion. RT-qPCR primers were designed to span the mRNA digestion sites. (C) Strategy for detecting 5’-UTR or 3’-UTR mRNA fragments released by RNase H digestion. RT-qPCR primers were designed within the 5’-UTR or 3’-UTR regions. (D) Fraction of intact mRNA segments remaining after RNase H treatment of DSP-crosslinked cell lysates from Jurkat cells, in the presence or absence of mRNA-specific DNA oligos for the indicated mRNAs. The mRNAs were detected using RT-qPCR oligos as illustrated in panel B. (E) Amount of eIF3-bound 5’-UTR and 3’-UTR regions of the mRNA co-immunoprecipitated by anti-EIF3B antibody, from polysome fractions of lysate from Jurkat cells treated with RNase H, either in the absence (left) or presence (right) of mRNA-targeting DNA oligos. Percentage is relative to the amount of total mRNA present in the polysome fraction prior to immunoprecipitation. Primers to the mRNA 5’-UTR and 3’-UTR regions, as indicated in panel C, were used for quantification. (F) Sucrose gradient fractionation of polysomes from crosslinked primary human T cells. (G) Fraction of intact mRNA segments remaining after RNase H treatment of DSP-crosslinked cell lysates from primary T cells, in the presence or absence of mRNA-specific DNA oligos for the indicated mRNAs. The mRNAs were detected using RT-qPCR oligos as illustrated in panel B. (H) Amount of eIF3-bound 5’-UTR and 3’-UTR regions of the mRNA co-immunoprecipitated by anti-EIF3B antibody, from polysome fractions of lysate from primary human T cells treated with RNase H, either in the absence (left) or presence (right) of mRNA-targeting DNA oligos. Percentage is relative to mRNA present in the polysome fraction prior to immunoprecipitation. Primers to the mRNA 5’-UTR and 3’-UTR regions, as indicated in panel C, were used for quantification. All experiments were carried out in biological duplicate with one technical triplicate shown (n = 3, with mean and standard deviations shown). All the primary human T cell experiments were performed using samples from two donors and data from one representative donor are shown.