Research Article
Defective Human SRP Induces Protein Quality Control and Triggers Stress Response

https://doi.org/10.1016/j.jmb.2022.167832Get rights and content

Highlights

  • Human SRP targets secretory proteins to ER and protects their mRNAs.

  • SRP54 depletion globally downregulates secretory and membrane protein mRNAs.

  • SRP54 knockdown upregulates specific chaperone network and ubiquitination.

  • Loss of SRP54 leads to ribosomal stress and change in expression of RPS27 and RPS27L.

  • Data demonstrate complex nature of SRP function and dramatic consequences of defects.

Abstract

Regulation of Aberrant Protein Production (RAPP) is a protein quality control in mammalian cells. RAPP degrades mRNAs of nascent proteins not able to associate with their natural interacting partners during synthesis at the ribosome. However, little is known about the molecular mechanism of the pathway, its substrates, or its specificity. The Signal Recognition Particle (SRP) is the first interacting partner for secretory proteins. It recognizes signal sequences of the nascent polypeptides when they are exposed from the ribosomal exit tunnel. Here, we reveal the generality of the RAPP pathway on the whole transcriptome level through depletion of human SRP54, an SRP subunit. This depletion triggers RAPP and leads to decreased expression of the mRNAs encoding a number of secretory and membrane proteins. The loss of SRP54 also leads to the dramatic upregulation of a specific network of HSP70/40/90 chaperones (HSPA1A, DNAJB1, HSP90AA1, and others), increased ribosome associated ubiquitination, and change in expression of RPS27 and RPS27L suggesting ribosome rearrangement. These results demonstrate the complex nature of defects in protein trafficking, mRNA and protein quality control, and provide better understanding of their mechanisms at the ribosome.

Introduction

Cells synthesize thousands of proteins that need to be transported to different organelles, secreted outside of the cell, integrated into different membranes, or remain in the cytoplasm. Cells evolved several mechanisms for proper and efficient protein transport and folding. These processes are tightly regulated to eliminate the appearance of incorrect proteins in the wrong subcellular localization that may be harmful for a cell. However, sometimes defects in the transported proteins can occur due to mutations, premature stop codons in mRNA templates, mRNA truncations, or defects in the components of the transport machinery. These proteins are not able to reach their correct subcellular localizations or do not fold properly. Aberrant proteins are potentially dangerous, and in many cases lead to human diseases.1, 2, 3, 4, 5 Therefore, there are several mRNA and protein quality control mechanisms that were evolved to prevent synthesis or remove these undesired defective proteins.6, 7, 8, 9, 10, 11, 12, 13, 14 Many of these processes happen co-translationally when polypeptide nascent chains are being synthesized by the ribosome. Defective mRNAs with premature stop codons, truncations, or with missing stop codons are detected and eliminated by nonsense mediated decay, no-go decay, or non-stop decay, respectively. Truncated polypeptides resulting from incomplete synthesis or because of stress are removed by the ribosome quality control complex (RQC).8, 14 The Regulation of Aberrant Protein Production (RAPP) pathway senses protein interactions during their translation and degrades their mRNAs if these interactions are disrupted by mutations in the synthesized proteins or in the interacting factors (see recent reviews for details.1, 6) Several protein quality control pathways in the cytosol and the endoplasmic reticulum (ER) remove misfolded proteins missed by the quality control mechanisms at the ribosome. However, the precise details of these processes are still poorly understood.

More than a third of all cellular proteins are secretory and membrane proteins.15 One of the major protein targeting pathways is Signal Recognition Particle (SRP) dependent protein targeting.16, 17, 18, 19, 20 SRP recognizes special N-terminal targeting signals termed signal sequences or signal peptides.21, 22, 23, 24, 25 Although signal sequences do not have strong amino acid homology, they share a similar organization and physicochemical properties. A typical signal sequence contains an N-terminal positively charged n-domain, a hydrophobic core (h-domain), and a C-terminal c-domain.26, 27 While the h-domain is crucial for protein translocation and the n-domain for its targeting efficiency, the c-domain is important for processing and cleavage of the signal sequence.25, 28, 29, 30

SRP is a ribonucleoprotein complex with multiple subunits. It targets proteins to the endoplasmic reticulum (ER) membrane for translocation, processing, modifications, and maturation in secretory protein biogenesis. In mammals, SRP consists of six proteins (SRP9, SRP14, SRP68, SRP72, SRP19 and SRP54) and one noncoding 7SL RNA.18 Defects in SRP subunits are associated with multiple human diseases.31 The SRP54 subunit of the targeting complex recognizes and directly binds the signal sequence of a secretory protein during its synthesis.22, 24, 25, 32 The integrity of the hydrophobic core of the signal sequence is crucial for its interaction with SRP54.25 The failure of SRP54 to bind to a polypeptide nascent chain due to a mutation in the hydrophobic core of the signal sequence triggers the RAPP protein quality control pathway and consequently leads to the mRNA degradation of the aberrant secretory protein.33, 34, 35, 36 RAPP is unique among other protein quality controls – it senses interactions of the polypeptide nascent chains during their synthesis on the ribosome and destroys their mRNA templates if normal interactions are disrupted in order to prevent synthesis of potentially harmful products. We found that the loss of the SRP54 subunit also triggers the RAPP response.33, 34, 36 However, it is still unknown if the RAPP pathway is a general mechanism controlling the synthesis and targeting of secretory and membrane proteins, or if it is involved in the regulation of a small number of specific proteins.

We hypothesize that the RAPP pathway surveys synthesis of many secretory and membrane proteins, and that its activation leads to specific changes in expression of proteins associated with a cellular stress response. In this study, we activated RAPP by depleting SRP54 and evaluated gene expression at the whole transcriptome level. Our data demonstrate that the loss of the SRP54 subunit results in mRNA depletion of many secretory and membrane proteins. It also leads to a complex stress response that occurs at the ribosome and includes specific chaperones upregulation, ribosome rearrangement, and ubiquitination. Our results support the idea that RAPP is a general mechanism of protein quality control, demonstrate involvement of distinct chaperone networks in the process, and uncover the complex nature of the co-translational events at the ribosome during translation.

Section snippets

Effect of SRP54 depletion on the whole transcriptome in human cells

To evaluate SRP function in the RAPP pathway on the whole transcriptome level in mammalian cells, we depleted SRP54 using RNAi technology, performed Deep RNA sequencing, and compared the results with the transcriptome of the control cells. About 90% of the reads were successfully mapped to the human genome GRCh38.p13, and the sample distance analysis showed excellent reproducibility between three biological replicates (Figure 1(A), Supplementary Table S1).

We were able to generate an efficient

Discussion

Regulation of Aberrant Protein Production (RAPP) is a protein quality control pathway in mammalian cells. It surveys proteins during their synthesis and degrades their mRNAs if the nascent proteins are not able to associate with their natural partners at the ribosome polypeptide exit site.33, 34, 36 This pathway is unique because it senses aberrant proteins and eliminates not only defective proteins but also their mRNAs. The specificity of the pathway and its place among other protein quality

Cells, growth conditions, siRNA transfections

HeLa Tet-On cells (Clontech) were grown in 6-well plates for Deep RNA-seq and in 12-well plates for other experiments at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium–high glucose (DMEM, Sigma Aldrich) supplied with 10% fetal bovine serum (FBS, Sigma Aldrich) and penicillin and streptomycin mixture (100 units/ml and 100 µg/ml correspondingly) (Sigma Aldrich). Where indicated, siSRP54-transfected HeLa Tet-On cells were treated with proteasome inhibitor MG132 (Sigma Aldrich) or

Data availability

Deep RNA-seq data have been submitted to the GEO database https://www.ncbi.nlm.nih.gov/geo/ and available under accession number GSE182922.

CRediT authorship contribution statement

Elena B. Tikhonova: Methodology, Validation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Sneider Alexander Gutierrez Guarnizo: Methodology, Formal analysis, Data curation, Investigation, Writing – review & editing. Morgana K. Kellogg: Validation, Formal analysis, Investigation, Writing – review & editing. Alexander Karamyshev: Validation, Investigation, Writing – review & editing. Igor M. Dozmorov: Formal analysis, Data curation, Writing

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors thank Sarah C. Miller for critical reading of the manuscript and help in its editing. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM135167. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

References (86)

  • A. Jomaa et al.

    Molecular mechanism of cargo recognition and handover by the mammalian signal recognition particle

    Cell Rep.

    (2021)
  • A.L. Karamyshev et al.

    Inefficient SRP interaction with a nascent chain triggers a mRNA quality control pathway

    Cell

    (2014)
  • E.S. Pinarbasi et al.

    Pathogenic Signal Sequence Mutations in Progranulin Disrupt SRP Interactions Required for mRNA Stability

    Cell Rep.

    (2018)
  • E.B. Tikhonova et al.

    Silencing of Aberrant Secretory Protein Expression by Disease-Associated Mutations

    J. Mol. Biol.

    (2019)
  • J.N. Rauch et al.

    Binding of human nucleotide exchange factors to heat shock protein 70 (Hsp70) generates functionally distinct complexes in vitro

    J. Biol. Chem.

    (2014)
  • H.H. Kampinga et al.

    Guidelines for the nomenclature of the human heat shock proteins

    Cell Stress Chaperones.

    (2009)
  • J.C. Hsieh et al.

    Mesd encodes an LRP5/6 chaperone essential for specification of mouse embryonic polarity

    Cell

    (2003)
  • S. Munro et al.

    An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein

    Cell

    (1986)
  • M.J. Gething

    Role and regulation of the ER chaperone BiP

    Semin. Cell Dev. Biol.

    (1999)
  • B. Liu et al.

    Cotranslational response to proteotoxic stress by elongation pausing of ribosomes

    Mol. Cell

    (2013)
  • R. Hjerpe et al.

    Alternative UPS drug targets upstream the 26S proteasome

    Int J Biochem Cell Biol.

    (2008)
  • N. Kondrashov et al.

    Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning

    Cell

    (2011)
  • N. Slavov et al.

    Differential Stoichiometry among Core Ribosomal Proteins

    Cell Rep.

    (2015)
  • J.E. Gerst

    Pimp My Ribosome: Ribosomal Protein Paralogs Specify Translational Control

    Trends Genet. : TIG.

    (2018)
  • R. Higgins et al.

    The Unfolded Protein Response Triggers Site-Specific Regulatory Ubiquitylation of 40S Ribosomal Proteins

    Mol. Cell

    (2015)
  • Y. Matsuo et al.

    The ribosome collision sensor Hel2 functions as preventive quality control in the secretory pathway

    Cell Rep.

    (2021)
  • A.L. Karamyshev et al.

    Translational Control of Secretory Proteins in Health and Disease

    Int. J. Mol. Sci.

    (2020)
  • M. Stefani et al.

    Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution

    J. Mol. Med. (Berl).

    (2003)
  • D.N. Hebert et al.

    In and out of the ER: protein folding, quality control, degradation, and related human diseases

    Physiol. Rev.

    (2007)
  • A.L. Karamyshev et al.

    Lost in Translation: Ribosome-Associated mRNA and Protein Quality Controls

    Front. Genet.

    (2018)
  • O. Brandman et al.

    Ribosome-associated protein quality control

    Nat. Struct. Mol. Biol.

    (2016)
  • E.M. Sontag et al.

    Mechanisms and Functions of Spatial Protein Quality Control

    Annu. Rev. Biochem.

    (2017)
  • C.A.P. Joazeiro

    Mechanisms and functions of ribosome-associated protein quality control

    Nat. Rev. Mol. Cell Biol.

    (2019)
  • T. Inada

    Quality controls induced by aberrant translation

    Nucleic Acids Res.

    (2020)
  • C.S. Sitron et al.

    Detection and Degradation of Stalled Nascent Chains via Ribosome-Associated Quality Control

    Annu. Rev. Biochem.

    (2020)
  • M. Uhlen et al.

    Proteomics. Tissue-based map of the human proteome

    Science.

    (2015)
  • H.G. Koch et al.

    Signal recognition particle-dependent protein targeting, universal to all kingdoms of life

    Rev. Physiol. Biochem. Pharmacol.

    (2003)
  • D. Akopian et al.

    Signal recognition particle: an essential protein-targeting machine

    Annu. Rev. Biochem.

    (2013)
  • K. Wild et al.

    SRP meets the ribosome

    Nat. Struct. Mol. Biol.

    (2004)
  • M.K. Kellogg et al.

    SRPassing Co-translational Targeting: The Role of the Signal Recognition Particle in Protein Targeting and mRNA Protection

    Int. J. Mol. Sci.

    (2021)
  • H.H. Hsieh et al.

    Fidelity of Cotranslational Protein Targeting to the Endoplasmic Reticulum

    Int. J. Mol. Sci.

    (2021)
  • P. Walter et al.

    Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein

    J. Cell Biol.

    (1981)
  • U.C. Krieg et al.

    Photocrosslinking of the signal sequence of nascent preprolactin to the 54-kilodalton polypeptide of the signal recognition particle

    Proc. Natl. Acad. Sci. U S A.

    (1986)
  • Cited by (0)

    Present address: College of Natural Sciences, the University of Texas at Austin, Austin, TX 78712, USA.

    View full text