A burst in T cell receptor translation mediated by eIF3 interactions with T cell receptor mRNAs

Abstract

39Activation of T cells requires a global surge in cellular protein synthesis, which is 40 accompanied by a large increase in translation initiation [1][2][3][4] . A central component of the 41 translation initiation machinery-the multi-subunit eukaryotic initiation factor 3 (eIF3)-is 42 rapidly turned on when quiescent T cells are stimulated 3 . However, the precise role eIF3 43 plays in activated T cells is not known. Using a global transcriptome cross-linking 44 approach, we show that human eIF3 interacts with a distinct set of mRNAs in activated 45 Jurkat cells. A subset of these mRNAs, including those encoding the T cell receptor (TCR) 46 subunits TCRA and TCRB, crosslink to eIF3 across the entire length of the mRNA. 47Main 55 Several lines of evidence indicate eIF3 serves specialized roles in cellular translation, by 56 recognizing specific RNA structures in the 5'-untranslated regions (5'-UTRs) of target 57 mRNAs 5 , binding the 7-methyl-guanosine (m 7 G) cap 6 or through interactions with N-6-58 methyl-adenosine (m 6 A) post-transcriptional modifications in mRNAs 7 . Binding to these 59 cis-regulatory elements in mRNA can lead to translation activation or repression, 60 depending on the RNA sequence and structural context 5,7,8 . These functions for eIF3 can 61 aid cell proliferation 5 , or allow cells to rapidly adapt to stress such as heat shock 7 . 62 Additionally, eIF3 plays an important role in the development of specific tissues 9-11 . 63 64 In the immune system, T cell activation involves a burst in translation 1-4 and correlates 65 with assembly of subunit EIF3J with the main eIF3 complex 3 . However, whether eIF3 66 serves a general or more specific role in T cell activation is not known. To delineate how 67 eIF3 contributes to T cell activation, we first identified mRNAs that directly interact with 68 eIF3 in Jurkat cells activated for 5 hours with ionomycin and phorbol 12-myristate 13-69 acetate (I+PMA), or in non-activated Jurkat cells as a control, using photoactivatable 70 ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) 5,12,13 (Fig. 71 1a). We used Jurkat cells as a model for T cells, as PAR-CLIP experiments require a 72 large number of cells labeled with 4-thiouridine at a non-toxic concentration 14 . Jurkat cells 73 also have a defined T cell receptor and transcriptome, avoiding the donor-to-donor 74 variability of primary T cells. In the Jurkat PAR-CLIP experiments, RNA crosslinked to 75 eight of the thirteen eIF3 subunits, as identified by mass spectrometry: subunits EIF3A, 76 EIF3B, EIF3D and EIF3G as seen in HEK293T cells 5 , as well as subunits EIF3C, EIF3E, 77 De Silva et al. 4EIF3F, and EIF3L (Fig. 1b, Extended Data Fig. 1, Supplementary Table 1). In activated 78 Jurkat cells, eIF3 crosslinked to a substantially larger number of mRNAs compared to the 79 non-activated cells (Extended Data Figs. 2 and 3, Supplementary Tables 2 and 3). 80 Notably, eIF3 interacted with a completely new suite of mRNAs in activated Jurkat cells, 81 co...