Bacterial Growth Inhibition Screen (BGIS): harnessing recombinant protein toxicity for rapid and unbiased interrogation of protein function.

In two proof-of-concept studies, we established and validated the Bacterial Growth Inhibition Screen (BGIS), which explores recombinant protein toxicity in Escherichia coli as a largely overlooked and alternative means for basic characterization of functional eukaryotic protein domains. By applying BGIS, we identified an unrecognized RNA-interacting domain in the DEK oncoprotein (this study) and successfully combined BGIS with random mutagenesis as a screening tool for loss-of-function mutants of the DNA modulating domain of DEK [1]. Collectively, our findings shed new light on the phenomenon of recombinant protein toxicity in E. coli. Given the easy and rapid implementation and wide applicability, BGIS will extend the repertoire of basic methods for the identification, analysis and unbiased manipulation of proteins.

Maximizing the ovarian reserve in mice by evading LINE-1 genotoxicity.

Female reproductive success critically depends on the size and quality of a finite ovarian reserve. Paradoxically, mammals eliminate up to 80% of the initial oocyte pool through the enigmatic process of fetal oocyte attrition (FOA). Here, we interrogate the striking correlation of FOA with retrotransposon LINE-1 (L1) expression in mice to understand how L1 activity influences FOA and its biological relevance. We report that L1 activity triggers FOA through DNA damage-driven apoptosis and the complement system of immunity. We demonstrate this by combined inhibition of L1 reverse transcriptase activity and the Chk2-dependent DNA damage checkpoint to prevent FOA. Remarkably, reverse transcriptase inhibitor AZT-treated Chk2 mutant oocytes that evade FOA initially accumulate, but subsequently resolve, L1-instigated genotoxic threats independent of piRNAs and differentiate, resulting in an increased functional ovarian reserve. We conclude that FOA serves as quality control for oocyte genome integrity, and is not obligatory for oogenesis nor fertility.

Kruppel-like factor 4-dependent Staufen1-mediated mRNA decay regulates cortical neurogenesis.

Kruppel-like factor 4 (Klf4) is a zinc-finger-containing protein that plays a critical role in diverse cellular physiology. While most of these functions attribute to its role as a transcription factor, it is postulated that Klf4 may play a role other than transcriptional regulation. Here we demonstrate that Klf4 loss in neural progenitor cells (NPCs) leads to increased neurogenesis and reduced self-renewal in mice. In addition, Klf4 interacts with RNA-binding protein Staufen1 (Stau1) and RNA helicase Ddx5/17. They function together as a complex to maintain NPC self-renewal. We report that Klf4 promotes Stau1 recruitment to the 3'-untranslated region of neurogenesis-associated mRNAs, increasing Stau1-mediated mRNA decay (SMD) of these transcripts. Stau1 depletion abrogated SMD of target mRNAs and rescued neurogenesis defects in Klf4-overexpressing NPCs. Furthermore, Ddx5/17 knockdown significantly blocked Klf4-mediated mRNA degradation. Our results highlight a novel molecular mechanism underlying stability of neurogenesis-associated mRNAs controlled by the Klf4/Ddx5/17/Stau1 axis during mammalian corticogenesis.

Identification of genetic modifiers of TDP-43 neurotoxicity in Drosophila.

Cytosolic aggregation of the nuclear RNA-binding protein TDP-43 is a histopathologic signature of degenerating neurons in amyotrophic lateral sclerosis (ALS), and mutations in the TARDBP gene encoding TDP-43 cause dominantly inherited forms of this condition. To understand the relationship between TDP-43 misregulation and neurotoxicity, we and others have used Drosophila as a model system, in which overexpression of either wild-type TDP-43 or its ALS-associated mutants in neurons is sufficient to induce neurotoxicity, paralysis, and early death. Using microarrays, we have examined gene expression patterns that accompany TDP-43-induced neurotoxicity in the fly system. Constitutive expression of TDP-43 in the Drosophila compound eye elicited widespread gene expression changes, with strong upregulation of cell cycle regulatory genes and genes functioning in the Notch intercellular communication pathway. Inducible expression of TDP-43 specifically in neurons elicited significant expression differences in a more restricted set of genes. Genes that were upregulated in both paradigms included SpindleB and the Notch target Hey, which appeared to be a direct TDP-43 target. Mutations that diminished activity of Notch or disrupted the function of downstream Notch target genes extended the lifespan of TDP-43 transgenic flies, suggesting that Notch activation was deleterious in this model. Finally, we showed that mutation of the nucleoporin Nup50 increased the lifespan of TDP-43 transgenic flies, suggesting that nuclear events contribute to TDP-43-dependent neurotoxicity. The combined findings identified pathways whose deregulation might contribute to TDP-43-induced neurotoxicity in Drosophila.

TDP-43 toxicity and the usefulness of junk.

A new study shows that loss of the lariat debranching enzyme Dbr1 suppresses TDP-43 toxicity. The accumulated intronic lariat RNAs, which are normally degraded after splicing, likely act as decoys to sequester TDP-43 away from binding to and disrupting functions of other RNAs.

Antagonizing activity of vaccinia virus E3L against human interferons in Huh7 cells.

The E3L protein of vaccinia virus (VV) is well known for its capacity to evade cellular innate antiviral immunity related to interferon (IFN), for example PKR and RNaseL mediated antiviral activities. However, due to the limited range of cells that support VV E3L deletion mutant replication, the full capacity of E3L inhibiting the innate immune response induced by IFNs remains to be examined. In this report, the inhibition activity of VV E3L against a wide spectrum of human IFNs, including type I IFNs (12 IFN-alpha subtypes, IFN-beta, and IFN-omega), and type II IFN (gamma), was comparatively examined using the Copenhagen strain E3L deletion mutant and its revertant control virus in a human hepatoma cell line, Huh7. Deletion of the E3L open reading frame rendered the mutant VV sensitive to all types of IFNs, while the revertant VV was strongly resistant to these treatments. Furthermore, we show that the inhibition of VV E3L deletion mutant by IFN occurs at the stage of intermediate gene translation, while the expression of early genes and transcription of intermediate genes are largely unaffected. Using specific siRNAs to suppress the classical IFN-induced antiviral pathways, we found that PKR is the key factor modulated by E3L, while the RNaseL and MxA pathways play limited roles in this Huh7 cell system. Thus, our data demonstrates that VV E3L can mediate strong inhibition activity against all human type I and type II IFNs, mainly through modulation of the PKR pathway in Huh7 cells.