bims-micpro 2024-03-24 papers

Issue of 2024‒03‒24
three papers selected by
Thomas Farid Martínez, University of California, Irvine

mBio. 2024 Mar 21. e0033324

Proteins à la carte: riboproteogenomic exploration of bacterial N-terminal proteoform expression.

Igor Fijalkowski, Valdes Snauwaert, Petra Van Damme.

In recent years, it has become evident that the true complexity of bacterial proteomes remains underestimated. Gene annotation tools are known to propagate biases and overlook certain classes of truly expressed proteins, particularly proteoforms-protein isoforms arising from a single gene. Recent (re-)annotation efforts heavily rely on ribosome profiling by providing a direct readout of translation to fully describe bacterial proteomes. In this study, we employ a robust riboproteogenomic pipeline to conduct a systematic census of expressed N-terminal proteoform pairs, representing two isoforms encoded by a single gene raised by annotated and alternative translation initiation, in Salmonella. Intriguingly, conditional-dependent changes in relative utilization of annotated and alternative translation initiation sites (TIS) were observed in several cases. This suggests that TIS selection is subject to regulatory control, adding yet another layer of complexity to our understanding of bacterial proteomes.IMPORTANCE: With the emerging theme of genes within genes comprising the existence of alternative open reading frames (ORFs) generated by translation initiation at in-frame start codons, mechanisms that control the relative utilization of annotated and alternative TIS need to be unraveled and our molecular understanding of resulting proteoforms broadened. Utilizing complementary ribosome profiling strategies to map ORF boundaries, we uncovered dual-encoding ORFs generated by in-frame TIS usage in Salmonella. Besides demonstrating that alternative TIS usage may generate proteoforms with different characteristics, such as differential localization and specialized function, quantitative aspects of conditional retapamulin-assisted ribosome profiling (Ribo-RET) translation initiation maps offer unprecedented insights into the relative utilization of annotated and alternative TIS, enabling the exploration of gene regulatory mechanisms that control TIS usage and, consequently, the translation of N-terminal proteoform pairs.

Keywords: (retapamulin-assisted) ribosome profiling (Ribo-RET); N-terminal proteoforms; N-terminal proteomics; alternative translation initiation; riboproteogenomics

DOI: https://doi.org/10.1128/mbio.00333-24

Cell Rep. 2024 Mar 19. pii: S2211-1247(24)00304-8. [Epub ahead of print]43(4): 113976

Stem-loop-induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control.

Anna M Smirnova, Vladislava Hronová, Mahabub Pasha Mohammad, Anna Herrmannová, Stanislava Gunišová, Denisa Petráčková, Petr Halada, Štěpán Coufal, Michał Świrski, Justin Rendleman, Kristína Jendruchová, Maria Hatzoglou, Petra Beznosková, Christine Vogel, Leoš Shivaya Valášek.

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response, leading cells toward adaptation or death. ATF4's induction under stress was thought to be due to delayed translation reinitiation, where the reinitiation-permissive upstream open reading frame 1 (uORF1) plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrated that the canonical ATF4 translation start site is substantially leaky scanned. Thus, ATF4's translational control is more complex than originally described, underpinning its key role in diverse biological processes.

Keywords: ATF4; CP: Molecular biology; integrated stress response; ribosome; ribosome queuing; translation reinitiation; translational control; unfolded protein response

DOI: https://doi.org/10.1016/j.celrep.2024.113976

J Immunother Cancer. 2024 Mar 19. pii: e008402. [Epub ahead of print]12(3):

Circular RNA as a source of neoantigens for cancer vaccines.

Yi Ren, Thamizhanban Manoharan, Beijia Liu, Cyrus Zai Ming Cheng, Bei En Siew, Wai-Kit Cheong, Kai Yin Lee, Ian Jse-Wei Tan, Bettina Lieske, Ker-Kan Tan, Gloryn Chia.

BACKGROUND: The effectiveness of somatic neoantigen-based immunotherapy is often hindered by the limited number of mutations in tumors with low to moderate mutation burden. Focusing on microsatellite-stable colorectal cancer (CRC), this study investigates the potential of tumor-associated circular RNAs (circRNAs) as an alternative source of neoepitopes in CRC.METHODS: Tumor-associated circRNAs in CRC were identified using the MiOncoCirc database and ribo-depletion RNA sequencing of paired clinical normal and tumor samples. Candidate circRNA expression was validated by quantitative real-time PCR (RT-qPCR) using divergent primers. TransCirc database was used for translation prediction. Human leukocyte antigen binding affinity of open reading frames from potentially translatable circRNA was predicted using pVACtools. Strong binders from messenger RNA-encoded proteins were excluded using BlastP. The immunogenicity of the candidate antigens was functionally validated through stimulation of naïve CD8+ T cells against the predicted neoepitopes and subsequent analysis of the T cells through enzyme-linked immunospot (ELISpot) assay, intracellular cytokine staining (ICS) and granzyme B (GZMB) reporter. The cytotoxicity of T cells trained with antigen peptides was further tested using patient-derived organoids.
RESULTS: We identified a neoepitope from circRAPGEF5 that is upregulated in CRC tumor samples from MiOncoCirc database, and two neoepitopes from circMYH9, which is upregulated across various tumor samples from our matched clinical samples. The translation potential of candidate peptides was supported by Clinical Proteomic Tumor Analysis Consortium database using PepQuery. The candidate peptides elicited antigen-specific T cells response and expansion, evidenced by various assays including ELISpot, ICS and GZMB reporter. Furthermore, T cells trained with circMYH9 peptides were able to specifically target and eliminate tumor-derived organoids but not match normal organoids. This observation underscores the potential of circRNAs as a source of immunogenic neoantigens. Lastly, circMYH9 was enriched in the liquid biopsies of patients with CRC, thus enabling a detection-to-vaccination treatment strategy for patients with CRC.
CONCLUSIONS: Our findings underscore the feasibility of tumor-associated circRNAs as an alternative source of neoantigens for cancer vaccines targeting tumors with moderate mutation levels.

Keywords: circular RNA; colorectal cancer; neoantigen

DOI: https://doi.org/10.1136/jitc-2023-008402