TLR-mediated signaling pathways play critical roles in host defense against microbials. However, dysregulation of innate immune and inflammatory responses triggered by TLRs would result in harmful ...damage to the host. Using a
gene-knockout mouse model, we show that tripartite motif (TRIM) 8 negatively regulates TLR3- and TLR4-mediated innate immune and inflammatory responses. TRIM8 deficiency leads to increased polyinosinic-polycytidylic acid- and LPS-triggered induction of downstream anti-microbial genes including
,
,
, and
, evaluated serum cytokine levels, and increased susceptibility of mice to polyinosinic-polycytidylic acid- and LPS-induced inflammatory death as well as
infection-induced loss of body weight and septic shock. TRIM8 interacted with Toll/IL-1 receptor domain-containing adapter-inducing IFN-β and mediated its K6- and K33-linked polyubiquitination, leading to disruption of the Toll/IL-1 receptor domain-containing adapter-inducing IFN-β-TANK-binding kinase-1 association. Our findings uncover an additional mechanism on the termination of TLR3/4-mediated inflammatory and innate immune responses.
Recognition of viral nucleic acids by pattern recognition receptors initiates type I IFN induction and innate antiviral immune response. Here we show that LSm14A, a member of the LSm family involved ...in RNA processing in the processing bodies, binds to synthetic or viral RNA and DNA and mediates IRF3 activation and IFN-β induction. Knockdown of LSm14A inhibits cytosolic RNA- and DNA-trigger type I IFN production and cellular antiviral response. Moreover, LSm14A is essential for early-phase induction of IFN-β after either RNA or DNA virus infection. We further found that LSm14A-mediated IFN-β induction requires RIG-I–VISA or MITA after RNA or DNA virus infection, respectively, and viral infection causes translocation of LSm14A to peroxisomes, where RIG-I, VISA, and MITA are located. These findings suggest that LSm14A is a sensor for both viral RNA and DNA and plays an important role in initiating IFN-β induction in the early phase of viral infection.
The MHC class I antigen presentation system enables T cell immunosurveillance of cancers and viruses. A substantial fraction of the immunopeptidome derives from rapidly degraded nascent polypeptides ...(DRiPs). By knocking down each of the 80 ribosomal proteins, we identified proteins that modulate peptide generation without altering source protein expression. We show that 60S ribosomal proteins L6 (RPL6) and RPL28, which are adjacent on the ribosome, play opposite roles in generating an influenza A virus-encoded peptide. Depleting RPL6 decreases ubiquitin-dependent peptide presentation, whereas depleting RPL28 increases ubiquitin-dependent and -independent peptide presentation. 40S ribosomal protein S28 (RPS28) knockdown increases total peptide supply in uninfected cells by increasing DRiP synthesis from non-canonical translation of “untranslated” regions and non-AUG start codons and sensitizes tumor cells for T cell targeting. Our findings raise the possibility of modulating immunosurveillance by pharmaceutical targeting ribosomes.
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•Ribosome heterogeneity controls MHC class I peptide ligand presentation•RPL6 and RPL28 play opposing roles in viral peptide generation•RPS28 controls MHC class I peptide generation by modulating non-canonical translation•Ribosomal proteins influence CD8+ T cell cancer immunosurveillance
Wei et al. show that cells with ribosomes lacking any one of three ribosomal protein subunits have an altered capacity to generate MHC class I peptides for immunosurveillance and that tumor cells can potentially use this mechanism to avoid CD8 T cell immunosurveillance.
Abstract
Dynamic mRNA modification in the form of
N
6
-methyladenosine (m
6
A) adds considerable richness and sophistication to gene regulation. The m
6
A mark is asymmetrically distributed along ...mature mRNAs, with approximately 35% of m
6
A residues located within the coding region (CDS). It has been suggested that methylation in CDS slows down translation elongation. However, neither the decoding feature of endogenous mRNAs nor the physiological significance of CDS m
6
A has been clearly defined. Here, we found that CDS m
6
A leads to ribosome pausing in a codon-specific manner. Unexpectedly, removing CDS m
6
A from these transcripts results in a further decrease of translation. A systemic analysis of RNA structural datasets revealed that CDS m
6
A positively regulates translation by resolving mRNA secondary structures. We further demonstrate that the elongation-promoting effect of CDS methylation requires the RNA helicase-containing m
6
A reader YTHDC2. Our findings established the physiological significance of CDS methylation and uncovered non-overlapping function of m
6
A reader proteins.
The differentiation of B cells into immunoglobulin-secreting plasma cells is controlled by two transcription factors, Blimp-1 and XBP1. By gene expression profiling, we defined a set of genes whose ...induction during mouse plasmacytic differentiation is dependent on Blimp-1 and/or XBP1. Blimp-1-deficient B cells failed to upregulate most plasma cell-specific genes, including xbp1. Differentiating xbp1-deficient B cells induced Blimp-1 normally but failed to upregulate genes encoding many secretory pathway components. Conversely, ectopic expression of XBP1 induced a wide spectrum of secretory pathway genes and physically expanded the endoplasmic reticulum. In addition, XBP1 increased cell size, lysosome content, mitochondrial mass and function, ribosome numbers, and total protein synthesis. Thus, XBP1 coordinates diverse changes in cellular structure and function resulting in the characteristic phenotype of professional secretory cells.
The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither ...straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation.
In the human genome, translation initiation from non-AUG codons plays an important role in various gene regulation programs. However, mechanisms regulating the non-AUG initiation rate remain poorly ...understood. Here, we show that the non-AUG initiation rate is nearly consistent under a fixed nucleotide context in various human and insect cells. Yet, it ranges from <1% to nearly 100% compared to AUG translation, depending on surrounding sequences, including Kozak, and possibly additional nucleotide contexts. Mechanistically, this range of non-AUG initiation is controlled in part, by the eIF5-mimic protein (5MP). 5MP represses non-AUG translation by competing with eIF5 for the Met-tRNAi-binding factor eIF2. Consistently, eIF5 increases, whereas 5MP decreases translation of NAT1/EIF4G2/DAP5, whose sole start codon is GUG. By modulating eIF5 and 5MP1 expression in combination with ribosome profiling we identified a handful of previously unknown non-AUG initiation sites, some of which serve as the exclusive start codons. If the initiation rate for these codons is low, then an AUG-initiated downstream ORF prevents the generation of shorter, AUG-initiated isoforms. We propose that the homeostasis of the non-AUG translatome is maintained through balanced expression of eIF5 and 5MP.
To survive, mammalian cells must adapt to environmental challenges. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show ...here that under severe hyperosmotic stress, cells enter a transient hibernation-like state in anticipation of recovery. We demonstrate this adaptive pausing response (APR) is a coordinated cellular response that limits ATP supply and consumption through mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery. We further show that recovery from severe stress involves ISR (integrated stress response) signaling that permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress via a hibernation-like mechanism that preserves vital elements of cellular function under harsh environmental conditions.
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•Cells exposed to extremely harsh environments preserve the ability to recover•Severe stress induces dramatic mitochondrial fragmentation•Severe stress induces a hibernation-like state entailing ribosome pausing•Exit from the hibernation-like state requires induction of ISR
Jobava et al. show that near-lethal environmental stress induces a hibernation-like state of severe mitochondrial fragmentation with 80S ribosomes stalled at the start codons of a select group of mRNAs. Induction of ISR during removal of stress reverses the hibernation-like state and promotes a return to homeostasis.
Regulation of translation initiation is a central control point in protein synthesis. Variations of start codon selection contribute to protein diversity and complexity. Systemic mapping of start ...codon positions and precise measurement of the corresponding initiation rate would transform our understanding of translational control. Here we describe a ribosome profiling approach that enables identification of translation initiation sites on a genome-wide scale. By capturing initiating ribosomes using lactimidomycin, this approach permits qualitative and quantitative analysis of alternative translation initiation.
Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To ...maintain a content of 2.6 × 10
9 proteins, each cell's 6 × 10
6 ribosomes produce 4 × 10
6 proteins min
−1. Each of the cell's 8 × 10
5 proteasomes degrades 2.5 substrates min
−1, creating one MHC class I-peptide complex for each 500–3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate.
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