The ribosome is a structurally and functionally conserved macromolecular machine universally responsible for catalyzing protein synthesis. Within eukaryotic cells, mitochondria contain their own ribosomes (mitoribosomes), which synthesize a handful of proteins, all essential for the biogenesis of the oxidative phosphorylation system. High-resolution cryo-EM structures of the yeast, porcine and human mitoribosomal subunits and of the entire human mitoribosome have uncovered a wealth of new information to illustrate their evolutionary divergence from their bacterial ancestors and their adaptation to synthesis of highly hydrophobic membrane proteins. With such structural data becoming available, one of the most important remaining questions is that of the mitoribosome assembly pathway and factors involved. The regulation of mitoribosome biogenesis is paramount to mitochondrial respiration, and thus to cell viability, growth and differentiation. Moreover, mutations affecting the rRNA and protein components produce severe human mitochondrial disorders. Despite its biological and biomedical significance, knowledge on mitoribosome biogenesis and its deviations from the much-studied bacterial ribosome assembly processes is scarce, especially the order of rRNA processing and assembly events and the regulatory factors required to achieve fully functional particles. This article focuses on summarizing the current available information on mitoribosome assembly pathway, factors that form the mitoribosome assembly machinery, and the effect of defective mitoribosome assembly on human health.

SUMMARY Proteins in a cell are universally synthesized by ribosomes. Mitochondria contain their own ribosomes, which specialize on the synthesis of a handful of proteins required for oxidative phosphorylation. The pathway of mitoribosomal biogenesis and factors involved are poorly characterized. A case in point are the DEAD-Box proteins, widely known to participate in the biogenesis of bacterial and cytoplasmic eukaryotic ribosomes as either RNA helicases or RNA chaperones, whose mitochondrial counterparts remain completely unknown. Here, we have identified the Saccharomyces cerevisiae mitochondrial DEAD-Box protein Mrh4 as essential for large mitoribosome subunit biogenesis. Mrh4 interacts with the 21S rRNA, mitoribosome subassemblies and fully assembled mitoribosomes. In the absence of Mrh4, the 21S rRNA is matured and forms part of a large on-pathway assembly intermediate missing proteins Mrpl16 and Mrpl39. We conclude that Mrh4 plays an essential role during the late stages of mitoribosome assembly by promoting remodeling of the 21S rRNA-protein interactions.

Members of the DEAD-box family are often multifunctional proteins involved in several RNA transactions. Among them, yeast Saccharomyces cerevisiae Mss116 participates in mitochondrial intron splicing and, under cold stress, also in mitochondrial transcription elongation. Here, we show that Mss116 interacts with the mitoribosome assembly factor Mrh4, is required for efficient mitoribosome biogenesis, and consequently, maintenance of the overall mitochondrial protein synthesis rate. Additionally, Mss116 is required for efficient COX1 mRNA translation initiation and elongation. Mss116 interacts with a COX1 mRNA-specific translational activator, the pentatricopeptide repeat protein Pet309. In the absence of Mss116, Pet309 is virtually absent, and although mitoribosome loading onto COX1 mRNA can occur, activation of COX1 mRNA translation is impaired. Mutations abolishing the helicase activity of Mss116 do not prevent the interaction of Mss116 with Pet309 but also do not allow COX1 mRNA translation. We propose that Pet309 acts as an adaptor protein for Mss116 action on the COX1 mRNA 5΄-UTR to promote efficient Cox1 synthesis. Overall, we conclude that the different functions of Mss116 in the biogenesis and functioning of the mitochondrial translation machinery depend on Mss116 interplay with its protein cofactors.

Activation of T cells requires a rapid surge in cellular protein synthesis. However, the role of translation initiation in the early induction of specific genes remains unclear. Here we show human translation initiation factor eIF3 interacts with select immune system related mRNAs including those encoding the T cell receptor (TCR) subunits TCRA and TCRB. Binding of eIF3 to the TCRA and TCRB mRNA 3'-untranslated regions (3'-UTRs) depends on CD28 coreceptor signaling and regulates a burst in TCR translation required for robust T cell activation. Use of the TCRA or TCRB 3'-UTRs to control expression of an anti-CD19 chimeric antigen receptor (CAR) improves the ability of CAR-T cells to kill tumor cells in vitro. These results identify a new mechanism of eIF3-mediated translation control that can aid T cell engineering for immunotherapy applications.

Chimeric antigen receptor T cell (CAR T) therapy has demonstrated unprecedented therapeutic activity in hematologic malignancies. However, generating potent clinical responses against solid tumors remains a challenge for CAR T therapy. As the field strives to improve the therapeutic efficacy of CAR T cells with novel target antigens and enhanced potency, the risks of on-target toxicity pose a major barrier to progress. To address these challenges, we have developed engineered CAR T cells to target solid tumors through AND logic gates, where CAR expression is conditionally induced by a transcription factor released from a priming receptor (PrimeRTM) upon binding to the PrimeR antigen. The AND gate limits off-tumor toxicity as it requires both CAR and PrimeR antigen expression in the tumor microenvironment. To ensure PrimeR expression and signal transduction upon antigen binding, while minimizing residual ‘‘leaky’’ CAR induction in the absence of PrimeR antigen, we screened hundreds of PrimeR binders using both arrayed and pooled strategies. In an arrayed strategy, we engineered T cells from four donors in multiwell plates using CRISPR-mediated, non-viral, site-specific integration of logic gates bearing a variable PrimeR binder and a fixed MSLN CAR. In addition, we employed a pooled screening strategy, where we engineered T cells from two independent donors with a pool containing a subset of >300 of the same logic gates. Engineered T cells from both strategies were co-cultured with cell lines bearing either both CAR and PrimeR antigens or a single antigen, in order to evaluate fidelity and on-target functionality. In the arrayed setting, on-target functionality was quantified based on the levels of CAR induction, cytokine secretion, T cell activation, and target cell killing in the presence of both antigens, while fidelity was assessed based on the absence of these activity signals in the presence of a single antigen. In the pooled setting, sorting based on functional markers was performed and sequencing was used to quantify the relative abundance of cells with each logic gate in different sorted populations. On-target activity and circuit fidelity were then quantified based on enrichments in different sorted populations. Results from the pooled and arrayed screens were highly concordant. We combined the screen readouts to nominate a small set of PrimeR binders that exhibited both high fidelity and on-target functionality. We confirmed the desired characteristics of these binders with targeted arrayed screens in additional conditions as well as in in-vivo models. We have applied both screen strategies to select a small set of leads from hundreds of candidate PrimeR binders in the context of a logic-gated MSLN CAR. As pooled and arrayed screens come with different sets of limitations and advantages, both serve as important tools for the effective selection of receptors in the development of novel cell therapies. Citation Format: Li Wang, Sofia Kyriazopoulou Panagiotopoulou, Rona Harari-Steinfeld, Dasmanthie De Silva, Michelle Tan, Laura Lim, Angela Boroughs, Cate Sue, Jon Chen, Jamie Thomas, Mary Chua, Ed Yashin, Christine Shieh, Ryan Fong, Sophie Xu, Grace Zheng, Brendan Galvin, Aaron Cooper, Tarjei Mikkelsen, Nicholas Haining. High throughput screening strategies in the development of logic gated cell therapies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5329.

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...

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