The tRNA methyltransferase 1 (TRMT1) enzyme catalyzes the N2,N2-dimethylguanosine (m2,2G) modification in tRNAs. Intriguingly, vertebrates encode an additional tRNA methyltransferase 1-like (TRMT1L) paralog. Here, we use a comprehensive tRNA sequencing approach to decipher targets of human TRMT1 and TRMT1L. We find that TRMT1 methylates all known tRNAs containing guanosine at position 26, while TRMT1L represents the elusive enzyme catalyzing m2,2G at position 27 in tyrosine tRNAs. Surprisingly, TRMT1L is also necessary for maintaining 3-(3-amino-3-carboxypropyl)uridine (acp3U) modifications in a subset of tRNAs through a process that can be uncoupled from methyltransferase activity. We also demonstrate that tyrosine and serine tRNAs are dependent upon m2,2G modifications for their stability and function in translation. Notably, human patient cells with disease-associated TRMT1 variants exhibit reduced levels of tyrosine and serine tRNAs. These findings uncover unexpected roles for TRMT1 paralogs, decipher functions for m2,2G modifications, and pinpoint tRNAs dysregulated in human disorders caused by tRNA modification deficiency.

Inflammasome activation leads release of IL-1b, a proinflammatory cytokine that drives antimicrobial immune responses. Toxoplasma gondii has been shown to activate the NLRP3 inflammasome but the trigger has not yet been identified. Here we provide evidence that vacuolar disruption is a prerequisite for NLRP3 activation. T. gondii type I ROP5 and ROP18 protect the parasitophorous vacuolar membrane (PVM) and thereby inhibit inflammasome activation and IL-1b release. Besides protection of the PVM, we demonstrate an additional function of ROP5 and ROP18 for NLRP3 inhibition. We demonstrate the molecular mechanism of this inhibition includes direct interaction with GBP5. In conclusion, T. gondii ROP5 and ROP18 inhibit IL-1β production by protection of the intracellular replicative niche of the parasite and by actively subverting NLRP3 activation. Our findings provide further insight into the intricate mechanisms governing inflammasome activation and inhibition, enhancing our understanding of the complex dynamics during T. gondii infection.

Toxoplasma gondii ( T . gondii ) is a zoonotic apicomplexan parasite that is an important cause of clinical disability in humans. On a global scale, one third of the human population is infected with T . gondii . Mice and other small rodents are believed to be responsible for transmission of T . gondii to the domestic cat, its definitive host. Interferon-inducible Immunity-Related GTPases (IRG proteins) are important for control of murine T . gondii infections. Virulence differences between T . gondii strains are linked to polymorphic rhoptry proteins (ROPs) that cooperate to inactivate individual IRG family members. In particular, the pseudokinase ROP5 isoform B is critically important in laboratory strains of mice. We identified T . gondii ROP39 in complex with ROP5B and demonstrate its contribution to acute T . gondii virulence. ROP39 directly targets Irgb10 and inhibits homodimer formation of the GTPase leading to an overall reduction of IRG protein loading onto the parasitophorous vacuolar membrane (PVM). Maintenance of PVM integrity rescues the parasite from IRG protein-mediated clearance in vitro and in vivo . This study identifies a novel T . gondii effector that is important for specific inactivation of the IRG resistance system. Our data reveal that yet unknown T . gondii effectors can emerge from identification of direct interaction partners of ROP5B.

Recent high-throughput sequencing protocols have facilitated increased accuracy in measurements of transfer tRNAs (tRNAs) and tRNA-derived small RNAs (tDRs) from biological samples. However, commonly used RNA-seq analysis pipelines overlook special considerations given the unique features of tRNA metabolism. We present tRAX (tRNA Analysis of eXpression), a user-friendly analytic package for streamlined processing and graphic presentation of small-RNA sequencing data. Here, we apply it to both tRNAs and tDRs from mouse tissues to illustrate the extensive analysis and visualization features. Biologically compelling results demonstrate tRAX as an effective and accessible tool for in-depth characterization of tRNA and tDR transcriptomes.

The cellular response to stress is an important determinant of disease pathogenesis. Uncovering the molecular fingerprints of distinct stress responses may identify novel biomarkers and key signaling pathways for different diseases. Emerging evidence shows that transfer RNA-derived small RNAs (tDRs) play pivotal roles in stress responses. However, RNA modifications present on tDRs are barriers to accurately quantifying tDRs using traditional small RNA sequencing. Here, AlkB-facilitated methylation sequencing is used to generate a comprehensive landscape of cellular and extracellular tDR abundances in various cell types during different stress responses. Extracellular tDRs are found to have distinct fragmentation signatures from intracellular tDRs and these tDR signatures are better indicators of different stress responses than miRNAs. These distinct extracellular tDR fragmentation patterns and signatures are also observed in plasma from patients on cardiopulmonary bypass. It is additionally demonstrated that angiogenin and RNASE1 are themselves regulated by stressors and contribute to the stress-modulated abundance of sub-populations of cellular and extracellular tDRs. Finally, a sub-population of extracellular tDRs is identified for which AGO2 appears to be required for their expression. Together, these findings provide a detailed profile of stress-responsive tDRs and provide insight about tDR biogenesis and stability in response to cellular stressors.

Critical steps in resistance of mice against Toxoplasma gondii occur in the first 2 or 3 h after the pathogen has entered a cell that has been exposed to interferon gamma (IFN gamma). The newly formed parasitophorous vacuole is attacked by the IFN gamma-inducible IRG proteins and disrupted, resulting in death of the parasite and necrotic death of the cell. Here we describe some techniques that we have used to describe and quantify these events in different combinations of the host and the parasite.

Inflammatory caspase‐11 (rodent) and caspases‐4/5 (humans) detect the Gram‐negative bacterial component LPS within the host cell cytosol, promoting activation of the non‐canonical inflammasome. Although non‐canonical inflammasome‐induced pyroptosis and IL‐1‐related cytokine release are crucial to mount an efficient immune response against various bacteria, their unrestrained activation drives sepsis. This suggests that cellular components tightly control the threshold level of the non‐canonical inflammasome in order to ensure efficient but non‐deleterious inflammatory responses. Here, we show that the IFN‐inducible protein Irgm2 and the ATG8 family member Gate‐16 cooperatively counteract Gram‐negative bacteria‐induced non‐canonical inflammasome activation, both in cultured macrophages and in vivo. Specifically, the Irgm2/Gate‐16 axis dampens caspase‐11 targeting to intracellular bacteria, which lowers caspase‐11‐mediated pyroptosis and cytokine release. Deficiency in Irgm2 or Gate16 induces both guanylate binding protein (GBP)‐dependent and GBP‐independent routes for caspase‐11 targeting to intracellular bacteria. Our findings identify molecular effectors that fine‐tune bacteria‐activated non‐canonical inflammasome responses and shed light on the understanding of the immune pathways they control. Caspase‐11 targets cytosolic Gram‐negative bacteria, inducing pyroptosis and IL‐1 maturation. IFN‐inducible GTPases promote caspase‐11 targeting to bacterial membranes, whereas Irgm2 and the non‐canonical autophagy protein Gate‐16 restrain unnecessary caspase‐11 targeting. Caspase‐11 targets cytosolic Gram‐negative bacteria, inducing pyroptosis and IL‐1 maturation. IFN‐inducible GTPases promote caspase‐11 targeting to bacterial membranes, whereas Irgm2 and the non‐canonical autophagy protein Gate‐16 restrain unnecessary caspase‐11 targeting.

Inflammatory caspase-11 (rodent) and caspases-4 and -5 (human) detect gram-negative bacterial component LPS in the host cell cytosol, which promotes activation of the non-canonical inflammasome. Although non-canonical inflammasome-induced pyroptosis and IL-1 related cytokine release is of importance to mount an efficient immune response against various bacteria, its unrestrained activation drives sepsis. This suggests that cellular components might tightly control the threshold level of the non-canonical inflammasome in order to ensure efficient but not deleterious inflammatory response. Here we show that the IFN-inducible protein Irgm2 and the ATG8 family member Gate-16 cooperatively slow down non-canonical inflammasome activation both in macrophages and in vivo . Specifically, the Irgm2/Gate-16 axis dampens caspase-11 targeting to intracellular bacteria, which lower caspase-11-mediated pyroptosis and cytokine release. Specifically, deficiency in Irgm2 or Gate16 opens an alternative road for caspase-11 targeting to intracellular bacteria, independently of the classical pathway driven by the Guanylate Binding Proteins (GBPs). Thus, our findings provide new molecular effectors involved at fine-tuning the optimal non-canonical inflammasome response and add novel insights in the understanding of the immune pathways they control.

The original version of this Article contained an error in the Acknowledgements, which incorrectly omitted the following: ‘C.C., C.A., and J.C.H. were supported by the Fundação Calouste Gulbenkian through a grant from the Instituto Gulbenkian de Ciência and by the research infrastructure Congento, project LISBOA-01-0145-FEDER-022170, co-financed by Lisboa Regional Operational Programme (Lisboa 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and Foundation for Science and Technology (Portugal).’ This has been corrected in both the PDF and HTML versions of the Article.

Transfer RNA (tRNA) genes are among the most highly transcribed genes in the genome owing to their central role in protein synthesis. However, there is evidence for a broad range of gene expression across tRNA loci. This complexity, combined with difficulty in measuring transcript abundance and high sequence identity across transcripts, has severely limited our collective understanding of tRNA gene expression regulation and evolution. We establish sequence-based correlates to tRNA gene expression and develop a tRNA gene classification method that does not require, but benefits from, comparative genomic information and achieves accuracy comparable to molecular assays. We observe that guanine + cytosine (G + C) content and CpG density surrounding tRNA loci is exceptionally well correlated with tRNA gene activity, supporting a prominent regulatory role of the local genomic context in combination with internal sequence features. We use our tRNA gene activity predictions in conjunction with a comprehensive tRNA gene ortholog set spanning 29 placental mammals to estimate the evolutionary rate of functional changes among orthologs. Our method adds a new dimension to large-scale tRNA functional prediction and will help prioritize characterization of functional tRNA variants. Its simplicity and robustness should enable development of similar approaches for other clades, as well as exploration of functional diversification of members of large gene families.

Alternative pre-mRNA splicing plays a major role in expanding the transcript output of human genes. This process is regulated, in part, by the interplay of trans-acting RNA binding proteins (RBPs) with myriad cis-regulatory elements scattered throughout pre-mRNAs. These molecular recognition events are critical for defining the protein-coding sequences (exons) within pre-mRNAs and directing spliceosome assembly on noncoding regions (introns). One of the earliest events in this process is recognition of the 3' splice site (3'ss) by U2 small nuclear RNA auxiliary factor 2 (U2AF2). Splicing regulators, such as the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), influence spliceosome assembly both in vitro and in vivo, but their mechanisms of action remain poorly described on a global scale. HNRNPA1 also promotes proofreading of 3' ss sequences though a direct interaction with the U2AF heterodimer. To determine how HNRNPA1 regulates U2AF-RNA interactions in vivo, we analyzed U2AF2 RNA binding specificity using individual-nucleotide resolution cross-linking immunoprecipitation (iCLIP) in control and HNRNPA1 overexpression cells. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3'ss of alternative cassette exons but not constitutive exons upon HNRNPA1 overexpression. A subset of these events shows a concomitant increase of U2AF2 crosslinking at distal intronic regions, suggesting a shift of U2AF2 to "decoy" binding sites. Of the many noncanonical U2AF2 binding sites, Alu-derived RNA sequences represented one of the most abundant classes of HNRNPA1-dependent decoys. We propose that one way HNRNPA1 regulates exon definition is to modulate the interaction of U2AF2 with decoy or bona fide 3'ss.

BACKGROUND:Optical coherence tomography (OCT) and magnetic resonance imaging (MRI) can provide complementary information on visual system damage in multiple sclerosis (MS).OBJECTIVES:The objective of this paper is to determine whether a composite OCT/MRI score, reflecting cumulative damage along the entire visual pathway, can predict visual deficits in primary progressive multiple sclerosis (PPMS).METHODS:Twenty-five PPMS patients and 20 age-matched controls underwent neuro-ophthalmologic evaluation, spectral-domain OCT, and 3T brain MRI. Differences between groups were assessed by univariate general linear model and principal component analysis (PCA) grouped instrumental variables into main components. Linear regression analysis was used to assess the relationship between low-contrast visual acuity (LCVA), OCT/MRI-derived metrics and PCA-derived composite scores.RESULTS:PCA identified four main components explaining 80.69% of data variance. Considering each variable independently, LCVA 1.25% was significantly predicted by ganglion cell-inner plexiform layer (GCIPL) thickness, thalamic volume and optic radiation (OR) lesion volume (adjusted R2 0.328, p = 0.00004; adjusted R2 0.187, p = 0.002 and adjusted R2 0.180, p = 0.002). The PCA composite score of global visual pathway damage independently predicted both LCVA 1.25% (adjusted R2 value 0.361, p = 0.00001) and LCVA 2.50% (adjusted R2 value 0.323, p = 0.00003).CONCLUSION:A multiparametric score represents a more comprehensive and effective tool to explain visual disability than a single instrumental metric in PPMS.

The American alligator, Alligator mississippiensis, like all crocodilians, has temperature-dependent sex determination, in which the sex of an embryo is determined by the incubation temperature of the egg during a critical period of development. The lack of genetic differences between male and female alligators leaves open the question of how the genes responsible for sex determination and differentiation are regulated. Insight into this question comes from the fact that exposing an embryo incubated at male-producing temperature to estrogen causes it to develop ovaries. Because estrogen response elements are known to regulate genes over long distances, a contiguous genome assembly is crucial for predicting and understanding their impact. We present an improved assembly of the American alligator genome, scaffolded with in vitro proximity ligation (Chicago) data. We use this assembly to scaffold two other crocodilian genomes based on synteny. We perform RNA sequencing of tissues from American alligator embryos to find genes that are differentially expressed between embryos incubated at male- versus female-producing temperature. Finally, we use the improved contiguity of our assembly along with the current model of CTCF-mediated chromatin looping to predict regions of the genome likely to contain estrogen-responsive genes. We find that these regions are significantly enriched for genes with female-biased expression in developing gonads after the critical period during which sex is determined by incubation temperature. We thus conclude that estrogen signaling is a major driver of female-biased gene expression in the post-temperature sensitive period gonads.

April 28, 2017April 18, 2017Free AccessA composite MRI and OCT based measure to explore the visual pathways in primary-progressive Multiple Sclerosis (P6.405)Valentina Poretto, Maria Petracca, Catarina Saiote, Enricomaria Mormina, Jonathan Howard, Paolo Gallo, Aaron Miller, Fred Lublin, and Matilde IngleseAuthors Info & AffiliationsApril 18, 2017 issue88 (16_supplement)https://doi.org/10.1212/WNL.88.16_supplement.P6.405 Letters to the Editor

Irga6 co-localizes with LAMP1 in GMS transfected cells. Gene Switch 3T3 cells stably transfected with inducible Irga6 were stimulated with mifepristone and simultaneously transiently transfected with combinations of pGW1H-Irgm1, pGW1H-Irgm2, and pGW1H-Irgm3. Upon 24 hours of induction, samples were fixed and stained as in Fig. 2a. Representative microscopic images of Irga6 and lysosome co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: LAMP1, lower left: overlay, lower right: phase contrast. (TIF 10553 kb)

The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse.

Posttranscriptional control of gene expression is important for defining both normal and pathological cellular phenotypes. In vitro, RNA-binding proteins (RBPs) have recently been shown to play important roles in posttranscriptional regulation; however, the contribution of RBPs to cell specification is not well understood. Here, we determined that the RBP insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is specifically overexpressed in mixed lineage leukemia-rearranged (MLL-rearranged) B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and high risk of relapse. IGF2BP3 was required for the survival of B-ALL cell lines, as knockdown led to decreased proliferation and increased apoptosis. Enforced expression of IGF2BP3 provided murine BM cells with a strong survival advantage, led to proliferation of hematopoietic stem and progenitor cells, and skewed hematopoietic development to the B cell/myeloid lineage. Cross-link immunoprecipitation and high throughput sequencing uncovered the IGF2BP3-regulated transcriptome, which includes oncogenes MYC and CDK6 as direct targets. IGF2BP3 regulated transcripts via targeting elements within 3' untranslated regions (3'UTR), and enforced IGF2BP3 expression in mice resulted in enhanced expression of Myc and Cdk6 in BM. Together, our data suggest that IGF2BP3-mediated targeting of oncogenic transcripts may represent a critical pathogenetic mechanism in MLL-rearranged B-ALL and support IGF2BP3 and its cognate RNA-binding partners as potential therapeutic targets in this disease.

Nobody doubts that infections have imposed specialisations on the mammalian genome. However sufficient information is usually missing to attribute a specific genomic modification to pressure from a specific pathogen. Recent studies on mechanisms of mammalian resistance against the ubiquitous protozoan parasite, Toxoplasma gondii, have shown that the small rodents presumed to be largely responsible for transmission of the parasite to its definitive host, the domestic cat, possess distinctive recognition proteins, and interferon inducible effector proteins (IRG proteins) that limit the potential virulence of the parasite. The phylogenetic association of the recognition proteins, TLR11 and TLR12, with T. gondii resistance is weak, but there is evidence for reciprocal polymorphism between parasite virulence proteins and host IRG proteins that strongly suggests current or recent coevolution.