bims-micpro 2023-05-28 papers

iScience. 2023 Jun 16. 26(6): 106781

Microproteins: Overlooked regulators of physiology and disease.

Keira R Hassel, Omar Brito-Estrada, Catherine A Makarewich.

Ongoing efforts to generate a complete and accurate annotation of the genome have revealed a significant blind spot for small proteins (<100 amino acids) originating from short open reading frames (sORFs). The recent discovery of numerous sORF-encoded proteins, termed microproteins, that play diverse roles in critical cellular processes has ignited the field of microprotein biology. Large-scale efforts are currently underway to identify sORF-encoded microproteins in diverse cell-types and tissues and specialized methods and tools have been developed to aid in their discovery, validation, and functional characterization. Microproteins that have been identified thus far play important roles in fundamental processes including ion transport, oxidative phosphorylation, and stress signaling. In this review, we discuss the optimized tools available for microprotein discovery and validation, summarize the biological functions of numerous microproteins, outline the promise for developing microproteins as therapeutic targets, and look forward to the future of the field of microprotein biology.

Keywords: Biochemistry; Biological sciences; Cell biology; Molecular biology

DOI: https://doi.org/10.1016/j.isci.2023.106781

Microlife. 2022 ;3 uqac005

Hidden in plain sight: challenges in proteomics detection of small ORF-encoded polypeptides.

Igor Fijalkowski, Patrick Willems, Veronique Jonckheere, Laure Simoens, Petra Van Damme.

Genomic studies of bacteria have long pointed toward widespread prevalence of small open reading frames (sORFs) encoding for short proteins, <100 amino acids in length. Despite the mounting genomic evidence of their robust expression, relatively little progress has been made in their mass spectrometry-based detection and various blanket statements have been used to explain this observed discrepancy. In this study, we provide a large-scale riboproteogenomics investigation of the challenging nature of proteomic detection of such small proteins as informed by conditional translation data. A panel of physiochemical properties alongside recently developed mass spectrometry detectability metrics was interrogated to provide a comprehensive evidence-based assessment of sORF-encoded polypeptide (SEP) detectability. Moreover, a large-scale proteomics and translatomics compendium of proteins produced by Salmonella Typhimurium (S. Typhimurium), a model human pathogen, across a panel of growth conditions is presented and used in support of our in silico SEP detectability analysis. This integrative approach is used to provide a data-driven census of small proteins expressed by S. Typhimurium across growth phases and infection-relevant conditions. Taken together, our study pinpoints current limitations in proteomics-based detection of novel small proteins currently missing from bacterial genome annotations.

Keywords: Salmonella Typhimurium; in silico proteomics; proteomics; riboproteogenomics; sORF; sORF-encoded polypeptides (SEPs)

DOI: https://doi.org/10.1093/femsml/uqac005

Microlife. 2023 ;4 uqad012

Unraveling the small proteome of the plant symbiont Sinorhizobium meliloti by ribosome profiling and proteogenomics.

Lydia Hadjeras, Benjamin Heiniger, Sandra Maaß, Robina Scheuer, Rick Gelhausen, Saina Azarderakhsh, Susanne Barth-Weber, Rolf Backofen, Dörte Becher, Christian H Ahrens, Cynthia M Sharma, Elena Evguenieva-Hackenberg.

The soil-dwelling plant symbiont Sinorhizobium meliloti is a major model organism of Alphaproteobacteria. Despite numerous detailed OMICS studies, information about small open reading frame (sORF)-encoded proteins (SEPs) is largely missing, because sORFs are poorly annotated and SEPs are hard to detect experimentally. However, given that SEPs can fulfill important functions, identification of translated sORFs is critical for analyzing their roles in bacterial physiology. Ribosome profiling (Ribo-seq) can detect translated sORFs with high sensitivity, but is not yet routinely applied to bacteria because it must be adapted for each species. Here, we established a Ribo-seq procedure for S. meliloti 2011 based on RNase I digestion and detected translation for 60% of the annotated coding sequences during growth in minimal medium. Using ORF prediction tools based on Ribo-seq data, subsequent filtering, and manual curation, the translation of 37 non-annotated sORFs with ≤ 70 amino acids was predicted with confidence. The Ribo-seq data were supplemented by mass spectrometry (MS) analyses from three sample preparation approaches and two integrated proteogenomic search database (iPtgxDB) types. Searches against standard and 20-fold smaller Ribo-seq data-informed custom iPtgxDBs confirmed 47 annotated SEPs and identified 11 additional novel SEPs. Epitope tagging and Western blot analysis confirmed the translation of 15 out of 20 SEPs selected from the translatome map. Overall, by combining MS and Ribo-seq approaches, the small proteome of S. meliloti was substantially expanded by 48 novel SEPs. Several of them are part of predicted operons and/or are conserved from Rhizobiaceae to Bacteria, suggesting important physiological functions.

Keywords: Alphaproteobacteria; Ribosome profiling; Sinorhizobium meliloti; proteogenomics; proteomics; small open reading frame; small proteins

DOI: https://doi.org/10.1093/femsml/uqad012

Microlife. 2023 ;4 uqad001

Revealing the small proteome of Haloferax volcanii by combining ribosome profiling and small-protein optimized mass spectrometry.

Lydia Hadjeras, Jürgen Bartel, Lisa-Katharina Maier, Sandra Maaß, Verena Vogel, Sarah L Svensson, Florian Eggenhofer, Rick Gelhausen, Teresa Müller, Omer S Alkhnbashi, Rolf Backofen, Dörte Becher, Cynthia M Sharma, Anita Marchfelder.

In contrast to extensively studied prokaryotic 'small' transcriptomes (encompassing all small noncoding RNAs), small proteomes (here defined as including proteins ≤70 aa) are only now entering the limelight. The absence of a complete small protein catalogue in most prokaryotes precludes our understanding of how these molecules affect physiology. So far, archaeal genomes have not yet been analyzed broadly with a dedicated focus on small proteins. Here, we present a combinatorial approach, integrating experimental data from small protein-optimized mass spectrometry (MS) and ribosome profiling (Ribo-seq), to generate a high confidence inventory of small proteins in the model archaeon Haloferax volcanii. We demonstrate by MS and Ribo-seq that 67% of the 317 annotated small open reading frames (sORFs) are translated under standard growth conditions. Furthermore, annotation-independent analysis of Ribo-seq data showed ribosomal engagement for 47 novel sORFs in intergenic regions. A total of seven of these were also detected by proteomics, in addition to an eighth novel small protein solely identified by MS. We also provide independent experimental evidence in vivo for the translation of 12 sORFs (annotated and novel) using epitope tagging and western blotting, underlining the validity of our identification scheme. Several novel sORFs are conserved in Haloferax species and might have important functions. Based on our findings, we conclude that the small proteome of H. volcanii is larger than previously appreciated, and that combining MS with Ribo-seq is a powerful approach for the discovery of novel small protein coding genes in archaea.

Keywords: Haloferax volcanii; Ribo-seq; archaea; mass spectrometry; proteomics; ribosome profiling; sORF; small protein; small proteome; sprotein

DOI: https://doi.org/10.1093/femsml/uqad001

Elife. 2023 May 25. pii: e69611. [Epub ahead of print]12

Unraveling the influences of sequence and position on yeast uORF activity using massively parallel reporter systems and machine learning.

Gemma E May, Christina Akirtava, Matthew Agar-Johnson, Jelena Micic, John Woolford, Joel McManus.

Upstream open reading frames (uORFs) are potent cis-acting regulators of mRNA translation and nonsense-mediated decay (NMD). While both AUG- and non-AUG initiated uORFs are ubiquitous in ribosome profiling studies, few uORFs have been experimentally tested. Consequently, the relative influences of sequence, structural, and positional features on uORF activity have not been determined. We quantified thousands of yeast uORFs using massively parallel reporter assays in wildtype and ∆upf1 yeast. While nearly all AUG uORFs were robust repressors, most non-AUG uORFs had relatively weak impacts on expression. Machine learning regression modeling revealed that both uORF sequences and locations within transcript leaders predict their effect on gene expression. Indeed, alternative transcription start sites highly influenced uORF activity. These results define the scope of natural uORF activity, identify features associated with translational repression and NMD, and suggest that the locations of uORFs in transcript leaders are nearly as predictive as uORF sequences.

Keywords: S. cerevisiae; genetics; genomics

DOI: https://doi.org/10.7554/eLife.69611

iScience. 2023 May 19. 26(5): 106757

TP53BP1, a dual-coding gene, uses promoter switching and translational reinitiation to express a smORF protein.

Marta A Inchingolo, Aurélie Diman, Maxime Adamczewski, Tom Humphreys, Pascale Jaquier-Gubler, Joseph A Curran.

The complexity of the metazoan proteome is significantly increased by the expression of small proteins (<100 aa) derived from smORFs within lncRNAs, uORFs, 3' UTRs and, reading frames overlapping the CDS. These smORF encoded proteins (SEPs) have diverse roles, ranging from the regulation of cellular physiological to essential developmental functions. We report the characterization of a new member of this protein family, SEP53BP1, derived from a small internal ORF that overlaps the CDS encoding 53BP1. Its expression is coupled to the utilization of an alternative, cell-type specific promoter coupled to translational reinitiation events mediated by a uORF in the alternative 5' TL of the mRNA. This uORF-mediated reinitiation at an internal ORF is also observed in zebrafish. Interactome studies indicate that the human SEP53BP1 associates with components of the protein turnover pathway including the proteasome, and the TRiC/CCT chaperonin complex, suggesting that it may play a role in cellular proteostasis.

Keywords: Cell biology; Molecular biology; Protein

DOI: https://doi.org/10.1016/j.isci.2023.106757