bims-micpro Biomed News
on Discovery and characterization of microproteins
Issue of 2023‒03‒26
four papers selected by
Thomas Farid Martínez
University of California, Irvine

  1. bioRxiv. 2023 Mar 12. pii: 2023.03.09.531963. [Epub ahead of print]
      Ribosome profiling experiments indicate pervasive translation of short open reading frames (ORFs) outside of annotated protein-coding genes. However, shotgun mass spectrometry experiments typically detect only a small fraction of the predicted protein products of this noncanonical translation. The rarity of detection could indicate that most predicted noncanonical proteins are rapidly degraded and not present in the cell; alternatively, it could reflect technical limitations. Here we leveraged recent advances in ribosome profiling and mass spectrometry to investigate the factors limiting detection of noncanonical proteins in yeast. We show that the low detection rate of noncanonical ORF products can be explained by small size and low translation levels and does not indicate that they are unstable or biologically insignificant. In particular, no proteins encoded by evolutionarily young genes were detected, not even those with well-characterized biological roles. Additionally, we find that decoy biases can give misleading estimates of noncanonical protein false discovery rates, potentially leading to false detections. After accounting for these issues, we found strong evidence for four noncanonical proteins in mass spectrometry data, which were also supported by evolution and translation data. These results illustrate the power of mass spectrometry to validate unannotated genes predicted by ribosome profiling, but also its substantial limitations in finding many biologically relevant lowly-expressed proteins.
  2. J Proteome Res. 2023 Mar 24.
      Proteomic diversity in biological samples can be characterized by mass spectrometry (MS)-based proteomics using customized protein databases generated from sets of transcripts previously detected by RNA-seq. This diversity has only been increased by the recent discovery that many translated alternative open reading frames rest unannotated at unsuspected locations of mRNAs and ncRNAs. These novel protein products, termed alternative proteins, have been left out of all previous custom database generation tools. Consequently, genetic variations that impact alternative open reading frames and variant peptides from their translated proteins are not detectable with current computational workflows. To fill this gap, we present OpenCustomDB, a bioinformatics tool that uses sample-specific RNaseq data to identify genomic variants in canonical and alternative open reading frames, allowing for more than one coding region per transcript. In a test reanalysis of a cohort of 16 patients with acute myeloid leukemia, 5666 peptides from alternative proteins were detected, including 201 variant peptides. We also observed that a significant fraction of peptide-spectrum matches previously assigned to peptides from canonical proteins got better scores when reassigned to peptides from alternative proteins. Custom protein libraries that include sample-specific sequence variations of all possible open reading frames are promising contributions to the development of proteomics and precision medicine. The raw and processed proteomics data presented in this study can be found in PRIDE repository with accession number PXD029240.
    Keywords:  alternative ORFs; alternative proteins; database; multicoding; precision medicine; proteogenomics; variants
  3. Proteomics. 2023 Mar 22. e2200473
      Nostoc flagelliforme, a terrestrial cyanobacterium spread throughout arid and semi-arid areas, has been long known for its outstanding adaptability to extremely dry conditions. This microorganism is able to recover biological activities within hours after months of anhydrobiosis state, attracting investigation through proteomic analysis. Except for canonical proteome, microproteins encoded by small ORFs (smORFs) have recently been regarded as indispensable participants in metabolic processes. However, the involvement of smORFs in Nostoc flagelliforme remains unknown. Here we first constructed a smORF database in Nostoc flagelliforme using bioinformatic prediction, resulting in 6072 novel smORFs. Then LS-MS/MS analysis was applied to identify expression patterns of microproteins and seek smORFs and their encoded microprotein playing a role during rehydration. In total, 18 novel microproteins were mined based on a smORF searching strategy combined with three proteomic assays, of which five were annotated as ribosomal proteins, one as RNA polymerase subunit, and one as acetohydroxy acid isomeroreductase. We also suggested the possible functions of smORFs according to their expression pattern and discovered two neighboring and homologous smORFs. All these results will expand our knowledge of smORFs-encoded microproteins and their relation to the stress response of extremophilic microorganisms. This article is protected by copyright. All rights reserved.
    Keywords:  LC-MS/MS; SEP; cyanobacteria; drought stress ; smORF
  4. Cardiovasc Res. 2023 Mar 21. pii: cvad044. [Epub ahead of print]
      AIMS: The plasticity of vascular smooth muscle cells (VSMCs) enables them to alter phenotypes under various physiological and pathological stimuli. The alteration of VSMC phenotype is a key step in vascular diseases, including atherosclerosis. Although the transcriptome shift during VSMC phenotype alteration has been intensively investigated, uncovering multiple key regulatory signaling pathways, the translatome dynamics in this cellular process remain largely unknown. Here, we explored the genome-wide regulation at the translational level of human VSMCs during phenotype alteration.METHODS AND RESULTS: We generated nucleotide-resolution translatome and transcriptome data from human VSMCs undergoing phenotype alteration. Deep sequencing of ribosome-protected fragments (Ribo-seq) revealed alterations in protein synthesis independent of changes in mRNA levels. Increased translational efficiency of many translational machinery components, including ribosomal proteins, eukaryotic translation elongation factors and initiation factors, were observed during the phenotype alteration of VSMCs. In addition, hundreds of candidates for short ORF-encoded polypeptides (SEPs), a class of peptides containing 200 aa or less, were identified in a combined analysis of translatome and transcriptome data with a high positive rate in validating their coding capability. Three evolutionarily conserved SEPs were further detected endogenously by customized antibodies and suggested to participate in the pathogenesis of atherosclerosis by analyzing the transcriptome and single cell RNA-seq data from patient atherosclerotic artery samples. Gain- and loss-of-function studies in human VSMCs and genetically engineered mice showed that these SEPs modulate the alteration of VSMC phenotype through different signaling pathways, including the mitogen-activated protein kinase (MAPK) pathway and p53 pathway.
    CONCLUSION: Our study indicates that an increase in the capacity of translation, which is attributable to an increased quantity of translational machinery components, mainly controls alterations of VSMC phenotype at the level of translational regulation. In addition, SEPs could function as important regulators in the phenotype alteration of human VSMCs.
    TRANSLATIONAL PERSPECTIVE: Alterations of VSMC status are tightly associated with vascular diseases, including atherosclerosis. By incorporating Ribo-seq and RNA-seq of human VSMCs, we revealed that the increased translational capacity dominated the translational regulation of gene expression during alterations of VSMC phenotype. This finding of regulation in the translational level could provide new strategies of treating vascular diseases in the future. In addition, novel short ORF-encoded polypeptide (SEP) regulators for phenotype alteration were identified in the incorporated analyses. With the uncovered mechanisms, these SEPs may represent a new type of potential therapeutic target for clinical intervention of vascular diseases.
    Keywords:  Phenotype alteration; Ribo-seq; Translational landscape; Vascular smooth muscle cell; short ORF-encoded polypeptide