Elongation Factor 1β' Gene from Spodoptera exigua: Characterization and Function Identification through RNA Interference

ABSTRACT Distinct mammalian RNA viruses trigger Dicer-mediated production of virus-derived small-interfering RNAs (vsiRNA) and encode unrelated proteins to suppress vsiRNA biogenesis. However, the mechanism and function of the mammalian RNA interference (RNAi) response are poorly understood. Here, we characterized antiviral RNAi in a mouse model of infection with Nodamura virus (NoV), a mosquito-transmissible positive-strand RNA virus encoding a known double-stranded RNA (dsRNA)-binding viral suppressor of RNAi (VSR), the B2 protein. We show that inhibition of NoV RNA replication by antiviral RNAi in mouse embryonic fibroblasts (MEFs) requires Dicer-dependent vsiRNA biogenesis and Argonaute-2 slicer activity. We found that VSR-B2 of NoV enhances viral RNA replication in wild-type but not RNAi-defective MEFs such as Argonaute-2 catalytic-dead MEFs and Dicer or Argonaute-2 knockout MEFs, indicating that VSR-B2 acts mainly by suppressing antiviral RNAi in the differentiated murine cells. Consistently, VSR-B2 expression in MEFs has no detectable effect on the induction of interferon-stimulated genes or the activation of global RNA cleavages by RNase L. Moreover, we demonstrate that NoV infection of adult mice induces production of abundant vsiRNA active to guide RNA slicing by Argonaute-2. Notably, VSR-B2 suppresses the biogenesis of both vsiRNA and the slicing-competent vsiRNA-Argonaute-2 complex without detectable inhibition of Argonaute-2 slicing guided by endogenous microRNA, which dramatically enhances viral load and promotes lethal NoV infection in adult mice either intact or defective in the signaling by type I, II, and III interferons. Together, our findings suggest that the mouse RNAi response confers essential protective antiviral immunity in both the presence and absence of the interferon response. IMPORTANCE Innate immune sensing of viral nucleic acids in mammals triggers potent antiviral responses regulated by interferons known to antagonize the induction of RNA interference (RNAi) by synthetic long double-stranded RNA (dsRNA). Here, we show that Nodamura virus (NoV) infection in adult mice activates processing of the viral dsRNA replicative intermediates into small interfering RNAs (siRNAs) active to guide RNA slicing by Argonaute-2. Genetic studies demonstrate that NoV RNA replication in mouse embryonic fibroblasts is inhibited by the RNAi pathway and enhanced by the B2 viral RNAi suppressor only in RNAi-competent cells. When B2 is rendered nonexpressing or nonfunctional, the resulting mutant viruses become nonpathogenic and are cleared in adult mice either intact or defective in the signaling by type I, II, and III interferons. Our findings suggest that mouse antiviral RNAi is active and necessary for the in vivo defense against viral infection in both the presence and absence of the interferon response.

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Energetic dependencies dictate folding mechanism in a complex protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914366116 ◽

2019 ◽

Vol 116 (51) ◽

pp. 25641-25648 ◽

Cited By ~ 7

Author(s):

Kaixian Liu ◽

Xiuqi Chen ◽

Christian M. Kaiser

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Large Scale ◽

Elongation Factor ◽

Multidomain Protein ◽

Domain Interactions ◽

The Stability ◽

Domain Iii ◽

And Function ◽

Terminal Domains

Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.

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DBC1, p300, HDAC3, and Siah1 coordinately regulate ELL stability and function for expression of its target genes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1912375117 ◽

2020 ◽

Vol 117 (12) ◽

pp. 6509-6520 ◽

Cited By ~ 1

Author(s):

Subham Basu ◽

Mahesh Barad ◽

Dipika Yadav ◽

Arijit Nandy ◽

Bidisha Mukherjee ◽

...

Keyword(s):

Type 2 Diabetes ◽

Rna Polymerase Ii ◽

Target Genes ◽

Mononuclear Cells ◽

Elongation Factor ◽

Diabetes Patient ◽

Elongation Complex ◽

Peripheral Blood Mononuclear ◽

And Function

Among all of the Super Elongation Complex (SEC) components, ELL1 (also known as ELL) is the only bona fide elongation factor that directly stimulates transcription elongation by RNA polymerase II. However, the mechanism(s) of functional regulation of ELL1 (referred to as ELL hereafter), through its stabilization, is completely unknown. Here, we report a function of human DBC1 in regulating ELL stability involving HDAC3, p300, and Siah1. Mechanistically, we show that p300-mediated site-specific acetylation increases, whereas HDAC3-mediated deacetylation decreases, ELL stability through polyubiquitylation by the E3 ubiquitin ligase Siah1. DBC1 competes with HDAC3 for the same binding sites on ELL and thus increases its acetylation and stability. Knockdown of DBC1 reduces ELL levels and expression of a significant number of genes, including those involved in glucose metabolism. Consistently, Type 2 diabetes patient-derived peripheral blood mononuclear cells show reduced expression of DBC1 and ELL and associated key target genes required for glucose homeostasis. Thus, we describe a pathway of regulating stability and functions of key elongation factor ELL for expression of diverse sets of genes, including ones that are linked to Type 2 diabetes pathogenesis.

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DICER1 and PRKRA in Colon Adenocarcinoma

Biomarker Insights ◽

10.4137/bmi.s600 ◽

2008 ◽

Vol 3 ◽

pp. BMI.S600 ◽

Cited By ~ 6

Author(s):

S. Chiosea ◽

M. Acquafondata ◽

J. Luo ◽

SF. Kuan ◽

RR. Seethala

Keyword(s):

Rna Interference ◽

Gene Array ◽

Colon Adenocarcinoma ◽

Clinicopathologic Features ◽

Array Analysis ◽

Gene Array Analysis ◽

Microrna Profile ◽

The Status ◽

And Function ◽

Dicer1 Expression

Differential microRNA expression in colon adenocarcinoma (CA) was previously reported. MicroRNA biogenesis and function requires a set of proteins designated as the microRNA machinery, which includes DICER1 and PRKRA. Loss of heterozygosity at 14q32.13 DICER1 locus was detected in up to 60% of CA cases. The in silico gene array analysis of CA showed down-regulation of DICER1 and an up-regulation of PRKRA. Immunohistochemically, DICER1 expression was abnormal in 65% of CA (95 of 147 cases). PRKRA was deregulated in 70% of CA (32 of 46 cases). Expression of DICER1 and PRKRA was correlated with clinicopathologic features of CA. DICER1 up-regulation was seen more commonly in women. Only 10 of 46 cases immunostained for both DICER1 and PRKRA showed normal levels of both DICER1 and PRKRA. Microsatellite status of 32 cases was determined. Microsatellite instable cases showed DICER1 up-regulation more commonly when compared to microsatellite stable cases; however, this trend was not statistically significant. Abnormal DICER1 and/or PRKRA expression might explain the observed changes in microRNA profile. The status of the endogenous DICER1 and PRKRA in CA may help to predict the response to future RNA interference-based therapy.

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