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1.
J Cell Biol ; 221(7)2022 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-35522180

RESUMO

The double-stranded RNA sensor kinase PKR is one of four integrated stress response (ISR) sensor kinases that phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF2α) in response to stress. The current model of PKR activation considers the formation of back-to-back PKR dimers as a prerequisite for signal propagation. Here we show that PKR signaling involves the assembly of dynamic PKR clusters. PKR clustering is driven by ligand binding to PKR's sensor domain and by front-to-front interfaces between PKR's kinase domains. PKR clusters are discrete, heterogeneous, autonomous coalescences that share some protein components with processing bodies. Strikingly, eIF2α is not recruited to PKR clusters, and PKR cluster disruption enhances eIF2α phosphorylation. Together, these results support a model in which PKR clustering may limit encounters between PKR and eIF2α to buffer downstream signaling and prevent the ISR from misfiring.


Assuntos
Fator de Iniciação 2 em Eucariotos , eIF-2 Quinase , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fosforilação , RNA de Cadeia Dupla , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
2.
J Clin Microbiol ; 59(4)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33478979

RESUMO

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly trained personnel, and large upfront investment. Here, we showcase an orthogonal pipeline we call CREST (Cas13-based, rugged, equitable, scalable testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows a binary interpretation of results. CREST may provide a point-of-care solution to increase the distribution of COVID-19 surveillance.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Pandemias , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase
3.
J Clin Microbiol ; 59(4)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33293367

RESUMO

Management of the coronavirus disease 2019 (COVID-19) pandemic requires widespread testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among them RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here, we propose an alternative method we call PEARL (precipitation-enhanced analyte retrieval) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers performance comparable to that of commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.


Assuntos
COVID-19 , SARS-CoV-2 , Teste para COVID-19 , Técnicas de Laboratório Clínico , DNA , Humanos , RNA Viral/genética
4.
Proc Natl Acad Sci U S A ; 116(43): 21769-21779, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31591196

RESUMO

Translational frameshifting involves the repositioning of ribosomes on their messages into decoding frames that differ from those dictated during initiation. Some messenger RNAs (mRNAs) contain motifs that promote deliberate frameshifting to regulate production of the encoded proteins. The mechanisms of frameshifting have been investigated in many systems, and the resulting models generally involve single ribosomes responding to stimulator sequences in their engaged mRNAs. We discovered that the abundance of ribosomes on messages containing the IS3, dnaX, and prfB frameshift motifs significantly influences the levels of frameshifting. We show that this phenomenon results from ribosome collisions that occur during translational stalling, which can alter frameshifting in both the stalled and trailing ribosomes. Bacteria missing ribosomal protein bL9 are known to exhibit a reduction in reading frame maintenance and to have a strong dependence on elongation factor P (EFP). We discovered that ribosomes lacking bL9 become compacted closer together during collisions and that the E-sites of the stalled ribosomes appear to become blocked, which suggests subsequent transpeptidation in transiently stalled ribosomes may become compromised in the absence of bL9. In addition, we determined that bL9 can suppress frameshifting of its host ribosome, likely by regulating E-site dynamics. These findings provide mechanistic insight into the behavior of colliding ribosomes during translation and suggest naturally occurring frameshift elements may be regulated by the abundance of ribosomes relative to an mRNA pool.


Assuntos
Escherichia coli/genética , Mudança da Fase de Leitura do Gene Ribossômico/genética , RNA Mensageiro/genética , Fases de Leitura/genética , Ribossomos/metabolismo , Escherichia coli/metabolismo , Mutação da Fase de Leitura/genética , Conformação de Ácido Nucleico , Fatores de Alongamento de Peptídeos/metabolismo , Biossíntese de Proteínas/genética , Proteínas Ribossômicas/metabolismo
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