The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A)-translation
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PROVIDER: PRJNA506725 | ENA |
REPOSITORIES: ENA
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Project description:Human cells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and protein products. A common source of aberrant mRNAs is premature polyadenylation, which can result in non-functional protein products. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and rapid decay of the truncated nascent polypeptide via the ribosome-associated quality control (RQC). Here, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at continuous A-tracts during RQC. Using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), we show that MKRN1 is positioned upstream of A-rich stretches and poly(A) tails in mRNAs through an interaction with the cytoplasmic poly(A)-binding protein (PABP). Ubiquitin remnant profiling uncovered PABP and ribosomal protein RPS10 as well as additional translational regulators as main substrates of MRKN1. We propose that MKRN1 serves as a first line of poly(A) recognition at the mRNA level to prevent production of erroneous proteins, thus maintaining proteome integrity.
2019-08-19 | GSE122869 | GEO
Project description:Human cells have evolved multiple quality control mechanisms to ensure protein homeostasis by detecting aberrant mRNAs and protein products which are condemned for rapid decay. Translating ribosomes that run into poly(A) tails are terminally stalled, followed by ribosome recycling and rapid decay of the truncated nascent polypeptide via the ribosome-associated quality control (RQC). Here, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) acts as a novel sensor of poly(A) sequences to initiate ribosome stalling in RQC. Using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), we show that MKRN1 is positioned upstream of A-rich stretches and poly(A) tails in mRNAs via interaction with the cytoplasmic poly(A)-binding protein (PABP). Ubiquitin remnant profiling uncovers PABP and ribosomal protein RPS10 as well as multiple translational regulators as main targets of MRKN1-mediated ubiquitylation. The modified lysine in RPS10 is distinct from the main target sites of ZNF598, the second E3 ubiquitin ligase involved in initial ribosome stalling, indicating that multiple modifications need to converge to prevent promiscuous ubiquitylation of lysine-translating ribosomes. We propose that MKRN1 serves as a first line of poly(A) recognition at the RNA level to prevent erroneous protein products and maintain proteome integrity.
2019-08-19 | PXD011772 | Pride
Project description:Sarcopenia is a degenerative condition that consists in the age-induced atrophy and functional decline of skeletal muscle cells (myofibers). A common hypothesis is that inducing myofiber hypertrophy should also reinstate myofiber contractile function but such model has not been extensively tested. Here, we find that the levels of the ubiquitin ligase UBR4 increase in skeletal muscle with aging, and that muscle-specific UBR4 loss rescues age-associated myofiber atrophy in mice. However, UBR4 promotes proteolysis by the proteasome and consequently UBR4 loss reduces the muscle specific force and accelerates the decline in muscle protein quality that occurs with aging in mice. Similarly, hypertrophic signaling induced via muscle-specific loss of UBR4 and of several other ubiquitin ligases consistently compromises muscle function and shortens lifespan in Drosophila by reducing protein quality control. Altogether, these findings indicate that ubiquitin ligases regulate in opposite fashion myofiber size and muscle protein quality control during aging.
2021-01-07 | GSE149637 | GEO
Project description:Translation elongation rates are regulated to ensure proper conformation and biological function of proteins. Translation of either non-stop mRNA or transcripts coding for poly-basic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control system (RQC). During this process, the stalled ribosome is dissociated into subunits, and the polypeptide is ubiquitinated by the E3 ubiquitin ligase Listerin on the 60S large ribosomal subunit (LSU) leading to subsequent proteasomal degradation. However, it is largely unknown how stalled ribosomes are recognized and dissociated into subunits. Here we report that ubiquitination of the ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 is required for the production of the RQC substrate. RQC-trigger (RQT) factors, a RNA helicase-family protein Slh1/Rqt2, ubiquitin binding protein Cue3/Rqt3 and yKR023W/Rqt4, were also required for the primary steps of RQC, and associated with Hel2-ribosome complexes. Rqt2-4 factors were dispensable for the ubiquitination of uS10 by Hel2/Rqt1 and associated with ribosomes independent of the ubiquitination of uS10. However, the ubiquitin-binding activity of Rqt3 were crucial to trigger RQC. Cryo-electron microscopy (cryo-EM) analysis revealed that Hel2 bound ribosomes are in an rotated state containing hybrid state AP- and PE-tRNAs. Furthermore, ribosome profiling revealed that short footprints, hallmarks of hybrid state ribosomes18, were accumulated at tandem CGA rare codons at the beginning of the poly arginine stalling sequence and long footprints at subsequent codons, respectively. Short footprints at CGA codons were decreased in rqt1 mutant but drastically increased in uS10 mutants defective in the ubiquitination or rqt2 mutant. Collectively, our results demonstrate that Hel2 stabilizes ratcheted ribosomes leading to ubiquitination of uS10. Subsequently, Rqt2-4 factors target these hybrid state ribosomes specifically, allowing subsequent RQC reactions.
2017-07-31 | GSE79622 | GEO
Project description:Mutations in the human gene encoding the neuron-specific Eag1 voltage-gated K+ channel are associated with neurodevelopmental diseases, indicating an important role of Eag1 during brain development. A disease-causing Eag1 mutant channel is linked to defective protein stability that involves enhanced protein degradation by the E3 ubiquitin ligase cullin 7 (CUL7). The detailed mechanisms governing protein homeostasis of plasma membrane- and endoplasmic reticulum (ER)-localized Eag1 K+ channels, however, remains unclear. By using yeast two-hybrid screening, we identified another E3 ubiquitin ligase, makorin ring finger protein 1 (MKRN1), as a novel binding partner primarily interacting with the carboxyl-terminal region of Eag1. MKRN1 mainly interacts with ER-localized immature core-glycosylated, as well as nascent non-glycosylated, Eag1 proteins. MKRN1 promotes polyubiquitination and ER-associated proteasomal degradation of immature Eag1 proteins. Although both CUL7 and MKRN1 contribute to ER quality control of immature core-glycosylated Eag1 proteins, MKRN1, but not CUL7, associates with and promotes degradation of nascent, non-glycosylated Eag1 proteins at the ER. In direct contrast to the role of CUL7 in regulating both ER and peripheral quality controls of Eag1, MKRN1 is exclusively responsible for the early stage of Eag1 maturation at the ER. We further demonstrated that both CUL7 and MKRN1 contribute to protein quality control of additional disease-causing Eag1 mutants associated with defective protein homeostasis. Our data suggest that the presence of this dual ubiquitination system differentially maintains Eag1 protein homeostasis and may ensure efficient removal of disease-associated misfolded Eag1 mutant channels.
2021-09-10 | PXD023984 | Pride
Project description:Translation elongation stalling has the potential to produce toxic truncated protein fragments. Translation of either non-stop mRNA or transcripts coding for poly-basic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. During this process, the stalled ribosome is dissociated into subunits, and the polypeptide is ubiquitinated by the E3 ubiquitin ligase Listerin on the 60S large ribosomal subunit, leading to subsequent proteasomal degradation. However, it is largely unknown how the specific stalled ribosomes are recognized as aberrant to engage the RQC system. Here, we report that ubiquitination of the ribosomal protein uS10 of the 40S small ribosomal subunit, by the E3 ubiquitin ligase Hel2 (or RQC-trigger (Rqt) 1) initiates RQC. We identified a novel RQC-trigger (RQT) complex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlated with sensitivity to anisomycin, which stalls ribosome at the rotated form, suggesting that RQT factors rescue ribosomes stalled by this drug. Our un-biased survey by ribosome profiling revealed that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits. Our results provide mechanistic insight into the surveillance system for aberrant proteins induced by ribosome stalling and mediated by ribosome ubiquitination.
2017-07-31 | GSE90919 | GEO
Project description:Ubiquitin ligase COP1 suppresses microglia-mediated inflammation
| PRJNA607249 | ENA
Project description:We identified the ubiquitin ligase Huwe1 as a crucial regulator of hematopoietic stem cell (HSC) functions. We generated Huwe1 conditional knock-out mice and discovered that the loss of this ligase causes an increased proliferation and stem cell exhaustion, together with a decreased lymphoid specification in vivo. We observed that the ubiquitin ligase Huwe1 is controlling the expression of N-myc at the level of the most immature stem and progenitor hematopoietic populations, mediating the described effects. Hematopoietic stem cells (HSC) from the bone marrow of transgenic animals carrying a N-myc/mCherry fusion gene were sorted according to N-myc expression (defined by mCherry) into N-myc high and low subpopulations. Each of these 2 populations was subjected to microarray gene expression profiling.
2016-08-20 | E-GEOD-85832 | biostudies-arrayexpress
Project description:In the 1950s the drug thalidomide administered as a sedative to pregnant women led ot the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and ist IMiD derivatives recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4(CRBN)) ubiquitin ligase. Through an unbiased screen we identify the homeobox trranscription factor MEIS2 as an endogenous substrate of CRL4(CRBN).
2014-05-13 | GSE57554 | GEO
Project description:We identified the ubiquitin ligase Huwe1 as a crucial regulator of hematopoietic stem cell (HSC) functions. We generated Huwe1 conditional knock-out mice and discovered that the loss of this ligase causes an increased proliferation and stem cell exhaustion, together with a decreased lymphoid specification in vivo. We observed that the ubiquitin ligase Huwe1 is controlling the expression of N-myc at the level of the most immature stem and progenitor hematopoietic populations, mediating the described effects.
2016-08-20 | GSE85832 | GEO