A global view of the nonprotein-coding transcriptome in Plasmodium falciparum

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense–antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors

Experimental identification and characterization of 97 novel npcRNA candidates in Salmonella enterica serovar Typhi

We experimentally identified and characterized 97 novel, non-protein-coding RNA candidates (npcRNAs) from the human pathogen Salmonella enterica serovar Typhi (hereafter referred to as S. typhi). Three were specific to S. typhi, 22 were restricted to Salmonella species and 33 were differentially expressed during S. typhi growth. We also identified Salmonella Pathogenicity Island-derived npcRNAs that might be involved in regulatory mechanisms of virulence, antibiotic resistance and pathogenic specificity of S. typhi. An in-depth characterization of S. typhi StyR-3 npcRNA showed that it specifically interacts with RamR, the transcriptional repressor of the ramA gene, which is involved in the multidrug resistance (MDR) of Salmonella. StyR-3 interfered with RamR–DNA binding activity and thus potentially plays a role in regulating ramA gene expression, resulting in the MDR phenotype. Our study also revealed a large number of cis-encoded antisense npcRNA candidates, supporting previous observations of global sense–antisense regulatory networks in bacteria. Finally, at least six of the npcRNA candidates interacted with the S. typhi Hfq protein, supporting an important role of Hfq in npcRNA networks. This study points to novel functional npcRNA candidates potentially involved in various regulatory roles including the pathogenicity of S. typhi

Evidence for a Novel Mechanism of Influenza Virus-Induced Type I Interferon Expression by a Defective RNA-Encoded Protein

Influenza A virus (IAV) defective RNAs are generated as byproducts of error-prone viral RNA replication. They are commonly derived from the larger segments of the viral genome and harbor deletions of various sizes resulting in the generation of replication incompatible viral particles. Furthermore, small subgenomic RNAs are known to be strong inducers of pattern recognition receptor RIG-I-dependent type I interferon (IFN) responses. The present study identifies a novel IAV-induced defective RNA derived from the PB2 segment of A/Thailand/1(KAN-1)/2004 (H5N1). It encodes a 10 kDa protein (PB2∆) sharing the N-terminal amino acid sequence of the parental PB2 protein followed by frame shift after internal deletion. PB2∆ induces the expression of IFNβ and IFN-stimulated genes by direct interaction with the cellular adapter protein MAVS, thereby reducing viral replication of IFN-sensitive viruses such as IAV or vesicular stomatitis virus. This induction of IFN is completely independent of the defective RNA itself that usually serves as pathogen-associated pattern and thus does not require the cytoplasmic sensor RIG-I. These data suggest that not only defective RNAs, but also some defective RNA-encoded proteins can act immunostimulatory. In this particular case, the KAN-1-induced defective RNA-encoded protein PB2∆ enhances the overwhelming immune response characteristic for highly pathogenic H5N1 viruses, leading to a more severe phenotype in vivo

The rocks and shallows of deep RNA sequencing: Examples in the Vibrio cholerae RNome

New deep RNA sequencing methodologies in transcriptome analyses identified a wealth of novel nonprotein-coding RNAs (npcRNAs). Recently, deep sequencing was used to delineate the small npcRNA transcriptome of the human pathogen Vibrio cholerae and 627 novel npcRNA candidates were identified. Here, we report the detection of 223 npcRNA candidates in V. cholerae by different cDNA library construction and conventional sequencing methods. Remarkably, only 39 of the candidates were common to both surveys. We therefore examined possible biasing influences in the transcriptome analyses. Key steps, including tailing and adapter ligations for generating cDNA, contribute qualitatively and quantitatively to the discrepancies between data sets. In addition, the state of 5′-end phosphorylation influences the efficiency of adapter ligation and C-tailing at the 3′-end of the RNA. Finally, our data indicate that the inclusion of sample-specific molecular identifier sequences during ligation steps also leads to biases in cDNA representation. In summary, even deep sequencing is unlikely to identify all RNA species, and caution should be used for meta-analyses among alternatively generated data sets

PB2_∆ interacts with MAVS at mitochondria thereby inducing IFNβ expression.

<p><b>A)</b> Hek293 cells were transfected with HA-MAVS or empty vector in combination with Myc-PB2_<b>∆</b>, Myc-PB2 or empty vector. 24 h p.t. co-immunoprecipitation was performed by using HA-specific antibodies. Analysis of the co-immunoprecipitation of PB2 and PB2_<b>∆</b> was performed by detection of the Myc-tag. Blots are representative of three independent experiments. <b>B)</b> A549 cells were transfected with MAVS-specific or scrambled siRNA and subsequently infected with 5 MOI KAN-1. At the indicated time points, mitochondria were isolated and the presence of viral proteins PB2_<b>Δ</b> and PB2 was analyzed by Western blot. Knockdown efficiency was verified by using MAVS-specific antibodies. Tubulin expression served as loading control. <b>C)</b> Hek293 cells were transfected with the IFNβ promoter in combination with HA-MAVS or empty vector as well as with PB2_<b>∆</b>, PB2, PB2_<b>∆</b>/PB2 or empty vector. 24 h p.t. promoter activity was measured by luciferase assay. Depicted are mean percentages (±SD) of four independent experiments. *p≤0.05, **p≤0.01; two-way ANOVA, Tukey’s multiple comparisons test. <b>D)</b> A549 cells were transfected with MAVS-specific or scrambled siRNA and subsequently infected with 0.5 MOI rKAN-1 WT or PB2_<b>Δ</b> for 24 h. Expression levels of cytokines and ISGs were detected by qRT-PCR and are depicted as mean <i>n</i>-fold (±SD) of one representative out of three independent experiments normalized to non-infected control cells. *p≤0.05, two-way ANOVA, Sidak’s multiple comparisons test. Knockdown efficiency was verified by Western blot analysis using MAVS-specific antibodies. Presence of PB2_<b>∆</b> was analyzed by PB2-specific antibodies and Tubulin expression served as loading control. Blots are representative of two independent experiments.</p

The PB2_∆ mRNA encodes a 10 kDa protein that impairs viral gene expression.

<p><b>A)</b> Sequence alignment of the PB2_<b>∆</b> protein in comparison to PB2. <b>B)</b> Western blot analysis of total lysates of A549 infected with 5 MOI of different influenza viruses of the subtypes H5N1 (KAN-1, Mallard, Vietnam; <i>right</i>), H7N7 (FPV) and H1N1 (PR8) (<i>left</i>). PB2_<b>∆</b> and PB2 were detected 3, 5, 8 and 12 h p.i.. Equal loading was verified by the detection of total ERK2. <b>C, D)</b> A549 cells were transfected with PB2_<b>∆</b>-specific or scrambled siRNA and subsequently infected with KAN-1 (<b>C</b>: MOI 5, <b>D</b>: MOI 2) for the indicated time points. <b>C)</b> Expression of viral proteins PB1, PB2, NP, M1 and NS1 upon PB2_<b>∆</b> knockdown was analyzed by Western blot. Knockdown efficiency was verified by using PB2-specific antibodies. ERK2 expression served as loading control. <b>D)</b> Changes in infectivity titers upon PB2_<b>∆</b> knockdown were determined by standard plaque assay and are depicted as mean (±SD) of six independent experiments (<i>above</i>). **p≤0.01, unpaired two-tailed Student’s t-test. Efficient knockdown of PB2_<b>∆</b> was verified by Western blot analysis (<i>below)</i>. Efficient infection was confirmed by immunodetection of viral PB2 and equal loading was verified by analysis of total ERK2 expression. <b>E, F)</b> A549 cells were transfected with PB2_<b>∆</b> or empty vector and subsequently infected with 5 MOI KAN-1 for the indicated time points. <b>E)</b> Expression of viral proteins PB1, PB2, NP, M1 and NS1 was analyzed by Western blot. Overexpression of PB2_<b>∆</b> was confirmed by using Myc-specific antibodies. Equal loading was verified by detection of total ERK2. <b>F)</b> Expression levels of viral m/cRNAs were detected by qRT-PCR and are depicted as mean <i>n</i>-fold (±SD) of one representative out of two independent experiments normalized to 15 min infection of empty vector-transfected cells. *p≤0.05, two-way ANOVA, Sidak’s multiple comparisons test. <b>G)</b> A549 cells were infected with 5 MOI KAN-1 for 8 h. Subsequently, co-immunoprecipitation of the viral polymerase complex was performed by using NP-specific antibodies. Efficient immunoprecipitation was confirmed by detection of viral proteins NP, PB1 and PB2. Presence of PB2_<b>∆</b> was analyzed by PB2-specific antibodies. <b>B, C, E, G)</b> Blots are representative of three independent experiments.</p

Integrative genomic analysis of pediatric T-cell lymphoblastic lymphoma reveals candidates of clinical significance

T-cell lymphoblastic lymphoma (T-LBL) is a heterogeneous malignancy of lymphoblasts committed to T-cell lineage. The dismal outcomes (15%-30%) after T-LBL relapse warrant establishing risk-based treatment. To our knowledge, this study presents the first comprehensive, systematic, integrated, genome-wide analysis including relapsed cases that identifies molecular markers of prognostic relevance for T-LBL. NOTCH1 was identified as the putative driver for T-LBL. An activated NOTCH/PI3K-AKT signaling axis and alterations in cell cycle regulators constitute the core oncogenic program for T-LBL. Mutated KMT2D was identified as a prognostic marker. The cumulative incidence of relapse was 47% +/- 17% in patients with KMT2D mutations, compared with 14% +/- 3% in wild-type KMT2D. Structural analysis of the mutated domains of KMT2D revealed a plausible impact on structure and functional consequences. These findings provide new insights into the pathogenesis of T-LBL, including high translational potential. The ongoing LBL 2018 trial (www.clinicaltrials.gov #NCT04043494) allows for prospective validation and subsequent fine tuning of the stratification criteria for T-LBL risk groups to improve survival of pediatric patients