RESUMO
Reactive aldehydes are abundant endogenous metabolites that challenge homeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA damage requires repair to prevent cancer and premature aging, but it is unknown whether cells also possess mechanisms that resolve aldehyde-induced RNA lesions. Here, we establish photoactivatable ribonucleoside-enhanced crosslinking (PAR-CL) as a model system to study RNA crosslinking damage in the absence of confounding DNA damage in human cells. We find that such RNA damage causes translation stress by stalling elongating ribosomes, which leads to collisions with trailing ribosomes and activation of multiple stress response pathways. Moreover, we discovered a translation-coupled quality control mechanism that resolves covalent RNA-protein crosslinks. Collisions between translating ribosomes and crosslinked mRNA-binding proteins trigger their modification with atypical K6- and K48-linked ubiquitin chains. Ubiquitylation requires the E3 ligase RNF14 and leads to proteasomal degradation of the protein adduct. Our findings identify RNA lesion-induced translational stress as a central component of crosslinking damage.
Assuntos
RNA , Ubiquitina , Humanos , RNA/metabolismo , Ubiquitinação , Ubiquitina/metabolismo , Ribossomos/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Aldeídos , Biossíntese de ProteínasRESUMO
Translation of messenger RNAs (mRNAs) with premature termination codons produces truncated proteins with potentially deleterious effects. This is prevented by nonsense-mediated mRNA decay (NMD) of these mRNAs. NMD is triggered by ribosomes terminating upstream of a splice site marked by an exon-junction complex (EJC), but also acts on many mRNAs lacking a splice junction after their termination codon. We developed a genome-wide CRISPR flow cytometry screen to identify regulators of mRNAs with premature termination codons in K562 cells. This screen recovered essentially all core NMD factors and suggested a role for EJC factors in degradation of PTCs without downstream splicing. Among the strongest hits were the translational repressors GIGYF2 and EIF4E2. GIGYF2 and EIF4E2 mediate translational repression but not mRNA decay of a subset of NMD targets and interact with NMD factors genetically and physically. Our results suggest a model wherein recognition of a stop codon as premature can lead to its translational repression through GIGYF2 and EIF4E2.
Assuntos
Proteínas de Transporte/genética , Fator de Iniciação 4E em Eucariotos/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Regiões 3' não Traduzidas/genética , Linhagem Celular , Linhagem Celular Tumoral , Códon sem Sentido/genética , Códon de Terminação/genética , Éxons/genética , Células HEK293 , Humanos , Células K562 , Splicing de RNA/genéticaRESUMO
Ribosome-associated quality control (RQC) pathways protect cells from toxicity caused by incomplete protein products resulting from translation of damaged or problematic mRNAs. Extensive work in yeast has identified highly conserved mechanisms that lead to degradation of faulty mRNA and partially synthesized polypeptides. Here we used CRISPR-Cas9-based screening to search for additional RQC strategies in mammals. We found that failed translation leads to specific inhibition of translation initiation on that message. This negative feedback loop is mediated by two translation inhibitors, GIGYF2 and 4EHP. Model substrates and growth-based assays established that inhibition of additional rounds of translation acts in concert with known RQC pathways to prevent buildup of toxic proteins. Inability to block translation of faulty mRNAs and subsequent accumulation of partially synthesized polypeptides could explain the neurodevelopmental and neuropsychiatric disorders observed in mice and humans with compromised GIGYF2 function.
Assuntos
Proteínas de Transporte/genética , Fator de Iniciação 4E em Eucariotos/genética , Iniciação Traducional da Cadeia Peptídica , Ribossomos/genética , Animais , Sistemas CRISPR-Cas/genética , Humanos , Camundongos , Biossíntese de Proteínas/genética , Processamento de Proteína Pós-Traducional/genética , Controle de Qualidade , RNA Mensageiro/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Invertebrates rely on Dicer to cleave viral double-stranded RNA (dsRNA), and Drosophila Dicer-2 distinguishes dsRNA substrates by their termini. Blunt termini promote processive cleavage, while 3' overhanging termini are cleaved distributively. To understand this discrimination, we used cryo-electron microscopy to solve structures of Drosophila Dicer-2 alone and in complex with blunt dsRNA. Whereas the Platform-PAZ domains have been considered the only Dicer domains that bind dsRNA termini, unexpectedly, we found that the helicase domain is required for binding blunt, but not 3' overhanging, termini. We further showed that blunt dsRNA is locally unwound and threaded through the helicase domain in an adenosine triphosphate-dependent manner. Our studies reveal a previously unrecognized mechanism for optimizing antiviral defense and set the stage for the discovery of helicase-dependent functions in other Dicers.
Assuntos
Proteínas de Drosophila/química , RNA Helicases/química , RNA de Cadeia Dupla/química , Ribonuclease III/química , Trifosfato de Adenosina/química , Animais , Microscopia Crioeletrônica , Proteínas de Drosophila/ultraestrutura , Ligação Proteica , Estrutura Terciária de Proteína , Clivagem do RNA , RNA Helicases/ultraestrutura , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Ribonuclease III/ultraestrutura , Especificidade por SubstratoRESUMO
In previous studies we observed that the helicase domain of Drosophila Dicer-2 (dmDcr-2) governs substrate recognition and cleavage efficiency, and that dsRNA termini are key to this discrimination. We now provide a mechanistic basis for these observations. We show that discrimination of termini occurs during initial binding. Without ATP, dmDcr-2 binds 3' overhanging, but not blunt, termini. By contrast, with ATP, dmDcr-2 binds both types of termini, with highest-affinity binding observed with blunt dsRNA. In the presence of ATP, binding, cleavage, and ATP hydrolysis are optimal with BLT termini compared to 3'ovr termini. Limited proteolysis experiments suggest the optimal reactivity of BLT dsRNA is mediated by a conformational change that is dependent on ATP and the helicase domain. We find that dmDcr-2's partner protein, Loquacious-PD, alters termini dependence, enabling dmDcr-2 to cleave substrates normally refractory to cleavage, such as dsRNA with blocked, structured, or frayed ends.