RNA editing in flowering plant mitochondria alters 400 to 500 nucleotides from C to U, changing the information content of most mRNAs and some tRNAs. So far, none of the specific or general factors responsible for RNA editing in plant mitochondria have been identified. Here, we characterize a nuclear-encoded gene that is involved in RNA editing of three specific sites in different mitochondrial mRNAs in Arabidopsis thaliana, editing sites rps4-956, nad7-963, and nad2-1160. The encoded protein MITOCHONDRIAL RNA EDITING FACTOR1 (MEF1) belongs to the DYW subfamily of pentatricopeptide repeat proteins. Amino acid identities altered in MEF1 from ecotype C24, in comparison to Columbia, lower the activity at these editing sites; single amino acid changes in mutant plants inactivate RNA editing. These variations most likely modify the affinity of the editing factor to the affected editing sites in C24 and in the mutant plants. Since lowered and even absent RNA editing is tolerated at these sites, the amino acid changes may be silent for the respective protein functions. Possibly more than these three identified editing sites are addressed by this first factor identified for RNA editing in plant mitochondria.

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SUMMARYPost-transcriptional RNA editing in flowering plant mitochondria alters several hundred nucleotides from cytidine to uridine, mostly in mRNAs. To characterize the factors involved in RNA editing in plant mitochondria, we initiated a screen for nuclear mutants defective in RNA editing at specific sites. Here we identify the nuclear-encoded gene MEF11, which is involved in RNA editing of the three sites cox3-422, nad4-124 and ccb203-344 in Arabidopsis thaliana. A T-DNA insertion line of this gene was previously characterized as showing enhanced tolerance to the compound lovastatin, an inhibitor of the mevalonate pathway of isoprenoid biosynthesis. The mef11-1 mutant described here shows similar tolerance to lovastatin. Identification of the function of the MEF11 protein in site-specific mitochondrial RNA editing suggests indirect effects of retrograde signalling from mitochondria to the cytoplasm to evoke alteration of the mevalonate pathway. The editing sites cox3-422 and ccb203-344 each alter amino acids that are conserved in the respective proteins, while the nad4-124 site is silent. The single amino acid change in the mef11-1 mutant occurs in the second pentatricopeptide repeat, suggesting that this motif is required for site-specific RNA editing.

Most of the 400 RNA editing sites in flowering plant mitochondria are found in mRNAs. Consequently, the sequence vicinities of homologous sites are highly conserved between different species and are presumably recognized by likewise conserved transfactors. To investigate the evolutionary adaptation to sequence variation, we have now analyzed the recognition elements of an editing site with divergent upstream sequences in the two species pea and cauliflower. This variation is tolerated at the site selected, because the upstream cis-elements reach into the 5 0 -UTR of the mRNA. To compare cis-recognition features in pea and cauliflower mitochondria, we developed a new in vitro RNA editing system for cauliflower. In vitro editing assays with deleted and mutated template RNAs show that the major recognition elements for both species are located within the conserved sequence. In cauliflower, however, the essential upstream nucleotides extend further upstream than they do in pea. In-depth analysis of single-nucleotide mutations reveals critical spacing of the editing site and the specific recognition elements, and shows that the +1 nucleotide identity is important in cauliflower, but not in pea.

IL-21 can induce both plasma cells and regulatory B cells. In this article, we demonstrate that untreated HIV patients display CD4+ T cells with enhanced IL-21 expression and high in vivo frequencies of regulatory B cells overexpressing the serine protease granzyme B. Granzyme B–expressing regulatory B cells (GraB cells) cells from HIV patients exhibit increased expression of CD5, CD43, CD86, and CD147 but do not produce IL-10. The main functional characteristic of their regulatory activity is direct granzyme B–dependent degradation of the TCR-ζ–chain, resulting in significantly decreased proliferative T cell responses. Although Th cells from HIV patients secrete IL-21 in a Nef-dependent manner, they barely express CD40L. When culturing such IL-21+CD40L− Th cells with B cells, the former directly induce B cell differentiation into GraB cells. In contrast, the addition of soluble CD40L multimers to T cell/B cell cultures redirects B cell differentiation toward plasma cells, indicating that CD40L determines the direction of IL-21–dependent B cell differentiation. As proof of principle, we confirmed this mechanism in a patient lacking intact CD40 signaling due to a NEMO mutation. The majority of peripheral B cells from this patient were GraB cells and strongly suppressed T cell proliferation. In conclusion, GraB cells represent potent regulatory B cells in humans that are phenotypically and functionally distinct from B10 cells and occur in early HIV infection. GraB cells may contribute significantly to immune dysfunction in HIV patients, and may also explain ineffective Ab responses after vaccination. The use of soluble CD40L multimers may help to improve vaccination responses in HIV patients.

Many viral pathogens target innate sensing cascades and/or cellular transcription factors to suppress antiviral immune responses. Here, we show that the accessory viral protein U (Vpu) of HIV-1 exerts broad immunosuppressive effects by inhibiting activation of the transcription factor NF-κB. Global transcriptional profiling of infected CD4 +T cells revealed that vpu-deficient HIV-1 strains induce substantially stronger immune responses than the respective wild type viruses. Gene set enrichment analyses and cytokine arrays showed that Vpu suppresses the expression of NF-κB targets including interferons and restriction factors. Mutational analyses demonstrated that this immunosuppressive activity of Vpu is independent of its ability to counteract the restriction factor and innate sensor tetherin. However, Vpu-mediated inhibition of immune activation required an arginine residue in the cytoplasmic domain that is critical for blocking NF-κB signaling downstream of tetherin. In summary, our findings demonstrate that HIV-1 Vpu potently suppresses NF-κB-elicited antiviral immune responses at the transcriptional level.

Autophagy is a cellular homeostatic pathway with functions ranging from cytoplasmic protein turnover to immune defense. Therapeutic modulation of autophagy has been demonstrated to positively impact the outcome of autophagy-dysregulated diseases such as cancer or microbial infections. However, currently available agents lack specificity, and new candidates for drug development or potential cellular targets need to be identified. Here, we present an improved method to robustly detect changes in autophagy in a high-throughput manner on a single cell level, allowing effective screening. This method quantifies eGFP-LC3B positive vesicles to accurately monitor autophagy. We have significantly streamlined the protocol and optimized it for rapid quantification of large numbers of cells in little time, while retaining accuracy and sensitivity. Z scores up to 0.91 without a loss of sensitivity demonstrate the robustness and aptness of this approach. Three exemplary applications outline the value of our protocols and cell lines: (I) Examining autophagy modulating compounds on four different cell types. (II) Monitoring of autophagy upon infection with e.g. measles or influenza A virus. (III) CRISPR/Cas9 screening for autophagy modulating factors in T cells. In summary, we offer ready-to-use protocols to generate sensitive autophagy reporter cells and quantify autophagy in high-throughput assays. Macroautophagy (hereafter called autophagy) is a catabolic cellular process 1-3. Basal levels of autophagy are required to maintain homeostasis of eukaryotic cells, mediating continuous turnover of organelles and proteins. Autophagy is active in almost all cells of the body. Upon induction of autophagy, cytoplasmic cargo is engulfed by double-layered membranes, which upon elongation and closure form so-called autophagosomes, eventually fusing with lysosomes. Their contents along with the inner membrane are degraded at a low pH by lysosomal peptidases and hydrolases. This process is either specifically (selective autophagy) targeting compounds recognized by so-called autophagy receptors like SQSTM1 (p62) or leads to bulk degradation of parts of the cytoplasm 4. Specific autophagy targets are usually earmarked for destruction by ubiquitin chains. Overall, autophagy primarily has a pro-survival, cytoprotective role 5. Diverse cellular signaling pathways are regulated by autophagy, including the turnover of key signaling components 6. Furthermore, cellular proteins processed by autophagy may lead e.g. to the release of anti-microbial peptides 7. In addition, autophagy targets foreign compounds such as viral proteins in the cytoplasm (Xenophagy). Thus, it acts as an innate immune defense pathway 8-10 and eventually provides peptides for display on major histocompatibility complex (MHC) molecules on antigen-presenting cells 11. Recent studies revealed that aberrant autophagy impacts several disorders 12,13. Diseases like cancer and neuropathies are characterized by dysregulation of autophagy 14,15. Hereditary diseases like Crohn's disease...

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