bims-micpro Biomed News
on Discovery and characterization of microproteins
Issue of 2022‒07‒24
five papers selected by
Thomas Farid Martínez
University of California, Irvine

  1. Nucleic Acids Res. 2022 Jul 18. pii: gkac611. [Epub ahead of print]
      Sustaining DNA damage response (DDR) signalling via retention of DDR factors at damaged sites is important for transmitting damage-sensing and repair signals. Herein, we found that DNA damage provoked the association of ribosomes with IRES region in lncRNA CTBP1-DT, which overcame the negative effect of upstream open reading frames (uORFs), and elicited the novel microprotein DNA damage-upregulated protein (DDUP) translation via a cap-independent translation mechanism. Activated ATR kinase-mediated phosphorylation of DDUP induced a drastic 'dense-to-loose' conformational change, which sustained the RAD18/RAD51C and RAD18/PCNA complex at damaged sites and initiated RAD51C-mediated homologous recombination and PCNA-mediated post-replication repair mechanisms. Importantly, treatment with ATR inhibitor abolished the effect of DDUP on chromatin retention of RAD51C and PCNA, thereby leading to hypersensitivity of cancer cells to DNA-damaging chemotherapeutics. Taken together, our results uncover a plausible mechanism underlying the DDR sustaining and might represent an attractive therapeutic strategy in improvement of DNA damage-based anticancer therapies.
  2. Mol Cell. 2022 Jul 07. pii: S1097-2765(22)00606-2. [Epub ahead of print]
      Translated small open reading frames (smORFs) can have important regulatory roles and encode microproteins, yet their genome-wide identification has been challenging. We determined the ribosome locations across six primary human cell types and five tissues and detected 7,767 smORFs with translational profiles matching those of known proteins. The human genome was found to contain highly cell-type- and tissue-specific smORFs and a subset that encodes highly conserved amino acid sequences. Changes in the translational efficiency of upstream-encoded smORFs (uORFs) and the corresponding main ORFs predominantly occur in the same direction. Integration with 456 mass-spectrometry datasets confirms the presence of 603 small peptides at the protein level in humans and provides insights into the subcellular localization of these small proteins. This study provides a comprehensive atlas of high-confidence translated smORFs derived from primary human cells and tissues in order to provide a more complete understanding of the translated human genome.
    Keywords:  Ribo-seq; Ribosome profiling; dORFs; lncRNAs; smORFs; translation; uORFs; untranslated regions
  3. mBio. 2022 Jul 19. e0124722
      Streptococcus pneumoniae, an opportunistic human pathogen, is the leading cause of community-acquired pneumonia and an agent of otitis media, septicemia, and meningitis. Although genomic and transcriptomic studies of S. pneumoniae have provided detailed perspectives on gene content and expression programs, they have lacked information pertaining to the translational landscape, particularly at a resolution that identifies commonly overlooked small open reading frames (sORFs), whose importance is increasingly realized in metabolism, regulation, and virulence. To identify protein-coding sORFs in S. pneumoniae, antibiotic-enhanced ribosome profiling was conducted. Using translation inhibitors, 114 novel sORFs were detected, and the expression of a subset of them was experimentally validated. Two loci associated with virulence and quorum sensing were examined in deeper detail. One such sORF, rio3, overlaps with the noncoding RNA srf-02 that was previously implicated in pathogenesis. Targeted mutagenesis parsing rio3 from srf-02 revealed that rio3 is responsible for the fitness defect seen in a murine nasopharyngeal colonization model. Additionally, two novel sORFs located adjacent to the quorum sensing receptor rgg1518 were found to impact regulatory activity. Our findings emphasize the importance of sORFs present in the genomes of pathogenic bacteria and underscore the utility of ribosome profiling for identifying the bacterial translatome. IMPORTANCE This work employed pleuromutilin-assisted ribosome profiling using retapamulin (Ribo-RET) to identify genome-wide translation start sites in the human pathogen Streptococcus pneumoniae. We identified 114 unannotated intergenic small open reading frames (sORFs). The described procedures and data sets provide a model for microbiologists seeking to explore the translational landscape of bacteria. The biological roles of four sORF examples are characterized: two control the regulation of a cell-cell communication (quorum sensing) system, one contributes to the ability of S. pneumoniae to colonize the upper respiratory tract of mice, and a fourth governs the translation of PrfB, a protein enabling ribosome release at stop codons. We propose that Ribo-RET is a valuable approach to identifying unstudied microproteins and difficult-to-find pheromone genes used by Gram-positive organisms, whose genomes are replete with pheromone receptors.
    Keywords:  Streptococcus pneumoniae D39; quorum sensing; ribosome profiling; small open reading frames; small proteins; translation inhibitors; translational control; virulence
  4. mBio. 2022 Jul 13. e0091222
      Copper is essential to most living beings but also highly toxic and as such is an important player at the host-pathogen interface. Bacteria have thus developed homeostatic mechanisms to tightly control its intracellular concentration. Known Cu export and import systems are under transcriptional control, whereas posttranscriptional regulatory mechanisms are yet to be characterized. We identified a three-gene operon, bp2923-bfrG-bp2921, downregulated by copper and notably encoding a TonB-dependent transporter in Bordetella pertussis. We show here that the protein encoded by the first gene, which is a member of the DUF2946 protein family, represents a new type of upstream Open Reading Frame (uORF) involved in posttranscriptional regulation of the downstream genes. In the absence of copper, the entire operon is transcribed and translated. Perception of copper by the nascent bp2923-coded protein via its conserved CXXC motif triggers Rho-dependent transcription termination between the first and second genes by relieving translation arrest on a conserved C-terminal RAPP motif. Homologs of bp2923 are widespread in bacterial genomes, where they head operons predicted to participate in copper homeostasis. This work has thus unveiled a new mode of genetic regulation by a transition metal and identified a regulatory function for a member of an uncharacterized family of bacterial proteins that we have named CruR, for copper-responsive upstream regulator. IMPORTANCE Copper is a transition metal necessary for living beings but also extremely toxic. Bacteria thus tightly control its homeostasis with transcriptional regulators. In this work, we have identified in the whooping cough agent Bordetella pertussis a new control mechanism mediated by a small protein called CruR, for copper-responsive upstream regulator. While being translated by the ribosome CruR is able to perceive intracellular copper, which shuts down the transcription of downstream genes of the same operon, coding for a copper uptake system. This mechanism limits the import of copper in conditions where it is abundant for the bacterium. This is the first report of "posttranscriptional regulation" in response to copper. Homologs of CruR genes head many operons harboring copper-related genes in various bacteria, and therefore the regulatory function unveiled here is likely a general property of this new protein family.
    Keywords:  Bordetella pertussis; DUF2946 family; copper homeostasis; posttranscriptional regulation; upstream ORF
  5. Peptides. 2022 Jul 13. pii: S0196-9781(22)00106-1. [Epub ahead of print] 170840
      MOTS-c (mitochondrial open reading frame of the 12S rRNA-c) is a newly discovered peptide that has been shown to have a protective role in whole-body metabolic homeostasis. This could be a consequence of the effect of MOTS-c on muscle tissue. Here, we investigated the role of MOTS-c in the differentiation of human (LHCN-M2) and murine (C2C12) muscle progenitor cells. Cells were treated with peptides at the onset of differentiation or after myotubes had been formed. We identified in silico a putative Src Homology 2 (SH2) binding motif in the YIFY region of the MOTS-c sequence, and created a Y8F mutant MOTS-c peptide to explore the role of this region. In both cellular models, treatment with wild-type MOTS-c peptide increased myotube formation whereas treatment with the Y8F peptide did not. MOTS-c wild-type, but not Y8F peptide, also protected against interleukin-6 (IL-6)-induced reduction of nuclear myogenin staining in myocytes. Thus, we investigated whether MOTS-c interacts with the IL-6/Janus kinase/ Signal transducer and activator of transcription 3 (STAT3) pathway, and found that MOTS-c, but not the Y8F peptide, blocked the transcriptional activity of STAT3 induced by IL-6. Altogether, our findings suggest that, in muscle cells, MOTS-c interacts with STAT3 via the putative SH2 binding motif in the YIFY region to reduce STAT3 transcriptional activity, which enhances myotube formation. This newly discovered mechanism of action highlights MOTS-c as a potential therapeutic target against muscle-wasting in several diseases.
    Keywords:  Atrophy; Mitochondria derived peptides; Muscle