Jan Rehwinkel

Activation of MDA5 requires higher-order RNA structures generated during virus infection.

ABSTRACT Recognition of virus presence via RIG-I (retinoic acid inducible gene I) and/or MDA5 (melanoma differentiation-associated protein 5) initiates a signaling cascade that culminates in transcription of innate response genes such as those encoding the alpha/beta interferon (IFN-α/β) cytokines. It is generally assumed that MDA5 is activated by long molecules of double-stranded RNA (dsRNA) produced by annealing of complementary RNAs generated during viral infection. Here, we used an antibody to dsRNA to show that the presence of immunoreactivity in virus-infected cells does indeed correlate with the ability of RNA extracted from these cells to activate MDA5. Furthermore, RNA from cells infected with encephalomyocarditis virus or with vaccinia virus and precipitated with the anti-dsRNA antibody can bind to MDA5 and induce MDA5-dependent IFN-α/β production upon transfection into indicator cells. However, a prominent band of dsRNA apparent in cells infected with either virus does not stimulate IFN-α/β production. Instead, stimulatory activity resides in higher-order structured RNA that contains single-stranded RNA and dsRNA. These results suggest that MDA5 activation requires an RNA web rather than simply long molecules of dsRNA.

The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin alpha.

Addition of N-acetylglucosamine (GlcNAc) is a ubiquitous form of intracellular glycosylation catalyzed by the conserved O-linked GlcNAc transferase (OGT). OGT contains an N-terminal domain of tetratricopeptide (TPR) repeats that mediates the recognition of a broad range of target proteins. Components of the nuclear pore complex are major OGT targets, as OGT depletion by RNA interference (RNAi) results in the loss of GlcNAc modification at the nuclear envelope. To gain insight into the mechanism of target recognition, we solved the crystal structure of the homodimeric TPR domain of human OGT, which contains 11.5 TPR repeats. The repeats form an elongated superhelix. The concave surface of the superhelix is lined by absolutely conserved asparagines, in a manner reminiscent of the peptide-binding site of importin α. Based on this structural similarity, we propose that OGT uses an analogous molecular mechanism to recognize its targets.

Aicardi-Goutières syndrome and related phenotypes: linking nucleic acid metabolism with autoimmunity

Aicardi-Goutières syndrome (AGS) is a genetically determined encephalopathy demonstrating phenotypic overlap both with the sequelae of congenital infection and with systemic lupus erythematosus (SLE). Recent molecular advances have revealed that AGS can be caused by mutations in any one of five genes, most commonly on a recessive basis but occasionally as a dominant trait. Like AGS, SLE is associated with a perturbation of type I interferon metabolism. Interestingly then, heterozygous mutations in the AGS1 gene TREX1 underlie a cutaneous subtype of SLE-called familial chilblain lupus, and mutations in TREX1 represent the single most common cause of monogenic SLE identified to date. Evidence is emerging to show that the nucleases defective in AGS are involved in removing endogenously produced nucleic acid (NA) species, and that a failure of this removal results in activation of the immune system. This hypothesis explains the phenotypic overlap of AGS with congenital infection and some aspects of SLE, where an equivalent type I interferon-mediated innate immune response is triggered by viral and self NAs, respectively. The combined efforts of clinicians, geneticists, immunologists and cell biologists are producing rapid progress in the understanding of AGS and overlapping autoimmune disorders. These studies provide important insights into the pathogenesis of SLE and beg urgent questions about the development and use of immunosuppressive therapies in AGS and related phenotypes.

Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5'-diphosphates.

Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and β; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5′-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5′-diphosphates (5′pp). Genomes from mammalian reoviruses with 5′pp termini, 5′pp-RNA isolated from yeast L-A virus, and base-paired 5′pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5′pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5′pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5′pp-RNA, like that of 5′ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.

Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster

In yeast cells, the THO complex has been implicated in mitotic recombination, transcription elongation and mRNA nuclear export. The stable core of THO consists of Tho2p, Hpr1p, Mft1p and Thp2p. Whether a complex with similar functions assembles in metazoa has not yet been established. Here we report that Drosophila melanogaster THO consists of THO2, HPR1 and three proteins, THOC5–THOC7, which have no orthologs in budding yeast. Gene expression profiling in cells depleted of THO components revealed that <20% of the transcriptome was regulated by THO. Nonetheless, export of heat-shock mRNAs under heat stress was strictly dependent on THO function. Notably, 8% of upregulated genes encode proteins involved in DNA repair. Thus, although THO function seems to be conserved, the vast majority of mRNAs are transcribed and exported independently of THO in D. melanogaster.

mRNA quality control: an ancient machinery recognizes and degrades mRNAs with nonsense codons.

Nonsense‐mediated mRNA decay (NMD) is an mRNA surveillance pathway which ensures the rapid degradation of mRNAs containing premature translation termination codons (PTCs or nonsense codons), thereby preventing the accumulation of truncated and potentially harmful proteins. In this way, the NMD pathway contributes to suppressing or exacerbating the clinical manifestations of specific human genetic disorders. Studies in model organisms have led to the identification of the effectors of the NMD pathway, and illuminated the mechanisms by which premature stops are discriminated from natural stops, so that only the former trigger rapid mRNA degradation. These studies are providing important insights that will aid the development of new treatments for at least some human genetic diseases.

A conserved role for cytoplasmic poly(A)-binding protein 1 (PABPC1) in nonsense-mediated mRNA decay

The nonsense‐mediated mRNA decay (NMD) pathway degrades mRNAs with premature translation termination codons (PTCs). The mechanisms by which PTCs and natural stop codons are discriminated remain unclear. We show that the position of stops relative to the poly(A) tail (and thus of PABPC1) is a critical determinant for PTC definition in Drosophila melanogaster. Indeed, tethering of PABPC1 downstream of a PTC abolishes NMD. Conversely, natural stops trigger NMD when the length of the 3′ UTR is increased. However, many endogenous transcripts with exceptionally long 3′ UTRs escape NMD, suggesting that the increase in 3′ UTR length has co‐evolved with the acquisition of features that suppress NMD. We provide evidence for the existence of 3′ UTRs conferring immunity to NMD. We also show that PABPC1 binding is sufficient for PTC recognition, regardless of cleavage or polyadenylation. The role of PABPC1 in NMD must go beyond that of providing positional information for PTC definition, because its depletion suppresses NMD under conditions in which translation efficiency is not affected. These findings reveal a conserved role for PABPC1 in mRNA surveillance.

Genome-wide analysis of mRNAs regulated by drosha and argonaute proteins in Drosophila melanogaster

ABSTRACT RNA silencing pathways are conserved gene regulation mechanisms that elicit decay and/or translational repression of mRNAs complementary to short interfering RNAs and microRNAs (miRNAs). The fraction of the transcriptome regulated by these pathways is not known, but it is thought that each miRNA may have hundreds of targets. To identify transcripts regulated by silencing pathways at the genomic level, we examined mRNA expression profiles in Drosophila melanogaster cells depleted of four Argonaute paralogs (i.e., AGO1, AGO2, PIWI, or Aubergine) that play essential roles in RNA silencing. We also profiled cells depleted of the miRNA-processing enzyme Drosha. The results reveal that transcripts differentially expressed in Drosha-depleted cells have highly correlated expression in the AGO1 knockdown and are significantly enriched in predicted and validated miRNA targets. The levels of a subset of miRNA targets are also regulated by AGO2. Moreover, AGO1 and AGO2 silence the expression of a common set of mobile genetic elements. Together, these results indicate that the functional overlap between AGO1 and AGO2 in Drosophila is more important than previously thought.

RIGorous Detection: Exposing Virus Through RNA Sensing

Virus infection in mammals elicits a variety of defense responses that are initiated by signals from virus-sensing receptors expressed by the host. These receptors include the ubiquitously expressed RIG-I–like receptor (RLR) family of RNA helicases. RLRs are cytoplasmic proteins that act in cell-intrinsic antiviral defense by recognizing RNAs indicative of virus presence. Here, we highlight recent progress in understanding how RLRs discriminate between the RNA content of healthy versus virus-infected cells, functioning as accurate sensors of virus invasion.

SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and is involved in response to DNA damage

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase and a nuclease that restricts HIV-1 in noncycling cells. Germ-line mutations in SAMHD1 have been described in patients with Aicardi-Goutières syndrome (AGS), a congenital autoimmune disease. In a previous longitudinal whole genome sequencing study of chronic lymphocytic leukemia (CLL), we revealed a SAMHD1 mutation as a potential founding event. Here, we describe an AGS patient carrying a pathogenic germ-line SAMHD1 mutation who developed CLL at 24 years of age. Using clinical trial samples, we show that acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed/refractory CLL patients. We provide evidence that SAMHD1 regulates cell proliferation and survival and engages in specific protein interactions in response to DNA damage. We propose that SAMHD1 may have a function in DNA repair and that the presence of SAMHD1 mutations in CLL promotes leukemia development.