PAPγ associates with PAXT nuclear exosome to control the abundance of PROMPT ncRNAs.

Pervasive transcription of the human genome generates an abundance of RNAs that must be processed and degraded. The nuclear RNA exosome is the main RNA degradation machinery in the nucleus. However, nuclear exosome must be recruited to its substrates by targeting complexes, such as NEXT or PAXT. By proteomic analysis, we identify additional subunits of PAXT, including many orthologs of MTREC found in S. pombe. In particular, we show that polyA polymerase gamma (PAPγ) associates with PAXT. Genome-wide mapping of the binding sites of ZFC3H1, RBM27 and PAPγ shows that PAXT is recruited to the TSS of hundreds of genes. Loss of ZFC3H1 abolishes recruitment of PAXT subunits including PAPγ to TSSs and concomitantly increases the abundance of PROMPTs at the same sites. Moreover, PAPγ, as well as MTR4 and ZFC3H1, is implicated in the polyadenylation of PROMPTs. Our results thus provide key insights into the direct targeting of PROMPT ncRNAs by PAXT at their genomic sites.

Murine leukemia virus (MLV) P50 protein induces cell transformation via transcriptional regulatory function.

BACKGROUND: The murine leukemia virus (MLV) has been a powerful model of pathogenesis for the discovery of genes involved in cancer. Its splice donor (SD')-associated retroelement (SDARE) is important for infectivity and tumorigenesis, but the mechanism remains poorly characterized. Here, we show for the first time that P50 protein, which is produced from SDARE, acts as an accessory protein that transregulates transcription and induces cell transformation. RESULTS: By infecting cells with MLV particles containing SDARE transcript alone (lacking genomic RNA), we show that SDARE can spread to neighbouring cells as shown by the presence of P50 in infected cells. Furthermore, a role for P50 in cell transformation was demonstrated by CCK8, TUNEL and anchorage-independent growth assays. We identified the integrase domain of P50 as being responsible for transregulation of the MLV promoter using luciferase assay and RTqPCR with P50 deleted mutants. Transcriptomic analysis furthermore revealed that the expression of hundreds of cellular RNAs involved in cancerogenesis were deregulated in the presence of P50, suggesting that P50 induces carcinogenic processes via its transcriptional regulatory function. CONCLUSION: We propose a novel SDARE-mediated mode of propagation of the P50 accessory protein in surrounding cells. Moreover, due to its transforming properties, P50 expression could lead to a cellular and tissue microenvironment that is conducive to cancer development.

NF90 interacts with components of RISC and modulates association of Ago2 with mRNA.

BACKGROUND: Nuclear factor 90 (NF90) is a double-stranded RNA-binding protein involved in a multitude of different cellular mechanisms such as transcription, translation, viral infection, and mRNA stability. Recent data suggest that NF90 might influence the abundance of target mRNAs in the cytoplasm through miRNA- and Argonaute 2 (Ago2)-dependent activity. RESULTS: Here, we identified the interactome of NF90 in the cytoplasm, which revealed several components of the RNA-induced silencing complex (RISC) and associated factors. Co-immunoprecipitation analysis confirmed the interaction of NF90 with the RISC-associated RNA helicase, Moloney leukemia virus 10 (MOV10), and other proteins involved in RISC-mediated silencing, including Ago2. Furthermore, NF90 association with MOV10 and Ago2 was found to be RNA-dependent. Glycerol gradient sedimentation of NF90 immune complexes indicates that these proteins occur in the same protein complex. At target RNAs predicted to bind both NF90 and MOV10 in their 3' UTRs, NF90 association was increased upon loss of MOV10 and vice versa. Interestingly, loss of NF90 led to an increase in association of Ago2 as well as a decrease in the abundance of the target mRNA. Similarly, during hypoxia, the binding of Ago2 to vascular endothelial growth factor (VEGF) mRNA increased after loss of NF90, while the level of VEGF mRNA decreased. CONCLUSIONS: These findings reveal that, in the cytoplasm, NF90 can associate with components of RISC such as Ago2 and MOV10. In addition, the data indicate that NF90 and MOV10 may compete for the binding of common target mRNAs, suggesting a role for NF90 in the regulation of RISC-mediated silencing by stabilizing target mRNAs, such as VEGF, during cancer-induced hypoxia.

Murine Leukemia Virus P50 Protein Counteracts APOBEC3 by Blocking Its Packaging.

Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to retrovirus infection. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) encode the viral infectivity factor (Vif) protein to counteract APOBEC3 proteins. Vif induces degradation of APOBEC3G and other APOBEC3 proteins and thereby prevents their packaging into virions. It is not known if murine leukemia virus (MLV) encodes a Vif-like protein. Here, we show that the MLV P50 protein, produced from an alternatively spliced gag RNA, interacts with the C terminus of mouse APOBEC3 and prevents its packaging without causing its degradation. By infecting APOBEC3 knockout (KO) and wild-type (WT) mice with Friend or Moloney MLV P50-deficient viruses, we found that APOBEC3 restricts the mutant viruses more than WT viruses in vivo Replication of P50-mutant viruses in an APOBEC3-expressing stable cell line was also much slower than that of WT viruses, and overexpressing P50 in this cell line enhanced mutant virus replication. Thus, MLV encodes a protein, P50, that overcomes APOBEC3 restriction by preventing its packaging into virions.IMPORTANCE MLV has existed in mice for at least a million years, in spite of the existence of host restriction factors that block infection. Although MLV is considered a simple retrovirus compared to lentiviruses, it does encode proteins generated from alternatively spliced RNAs. Here, we show that P50, generated from an alternatively spliced RNA encoded in gag, counteracts APOBEC3 by blocking its packaging. MLV also encodes a protein, glycoGag, that increases capsid stability and limits APOBEC3 access to the reverse transcription complex (RTC). Thus, MLV has evolved multiple means of preventing APOBEC3 from blocking infection, explaining its survival as an infectious pathogen in mice.

Unusually High Prevalence of Cosecretion of Ambler Class A and B Carbapenemases and Nonenzymatic Mechanisms in Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa in Lebanon.

The opportunistic pathogen, Pseudomonas aeruginosa, is a main cause of nosocomial infections in Lebanese hospitals. This pathogen is highly threatening due to its ability to develop multiresistance toward a large variety of antibiotics, including the carbapenem subgroup of ß-lactams. In this study, we surveyed the enzymatic and nonenzymatic mechanisms of carbapenem resistance in several multidrug-resistant (MDR) strains of P. aeruginosa isolated from patients suffering from nosocomial urinary tract infections in a Lebanese hospital. The occurrence of ß-lactamase-encoding genes notably GES, KPC, IMP, VIM, NDM, and OXA, which are characterized by a carbapenemase activity was checked by genomic analyses. Our results provide a first evidence of the occurrence of GES in clinical P. aeruginosa isolates resistant to carbapenems in Lebanon. More interestingly, we showed that almost 40% of the analyzed strains have acquired a dual-carbapenemase secretion of GES-6 and VIM-2 or IMP-15, this being a rare phenomenon among this type of multidrug resistance. Moreover, LC-MS/MS analyses revealed a high prevalence of another enzymatic mechanism of resistance; this is the coexistence of AmpC and Pdc-13 as well as a number of virulence proteins, for instance pilin, lytic transglycosylase, ecotin, chitin-binding protein (Cbp), and TolB-dependent receptor. It is to be noted that a mutation of the oprD2 gene encoding a porin selective for carbapenems has been detected in almost 66% of our strains. All in all, our study reveals by the use of different methods, unusual simultaneous enzymatic (GES, IMP, VIM, pdc13, and AmpC) and nonenzymatic mechanisms of resistance (reduction of OprD2 expression) for MDR Pseudomonas aeruginosa.

Role of outer membrane permeability, efflux mechanism, and carbapenemases in carbapenem-nonsusceptible Pseudomonas aeruginosa from Dubai hospitals: Results of the first cross-sectional survey.

OBJECTIVES: Carbapenem resistance in Pseudomonas aeruginosa is growing and results from variable mechanisms. The objectives of the current study were to investigate mechanisms of carbapenem resistance and genetic relatedness of P. aeruginosa isolates recovered in Dubai hospitals. METHODS: From June 2015 through June 2016, carbapenem-nonsusceptible P. aeruginosa were collected from 4 hospitals in Dubai, and subjected to antimicrobial susceptibility testing, molecular investigation of carbapenemases by PCR-sequencing, analysis of outer membrane porin OprD2 and multidrug efflux channel MexAB-OprM levels by qPCR, and fingerprinting by ERIC-PCR. RESULTS: Out of 1969 P. aeruginosa isolated during the study period, 471 (23.9%) showed reduced carbapenem susceptibility. Of these, 37 were analyzed and 32% of them produced VIM-type metallo-ß-lactamases, including VIM-2, VIM-30, VIM-31, and VIM-42, while GES-5 and GES-9 co-existed with VIM in 5.4% of isolates. Outer membrane impermeability was observed in 73% of isolates and 75.6% displayed overproduced MexAB-OprM. ERIC-PCR revealed one large clone including most carbapenemase-producing isolates indicating clonal dissemination. CONCLUSION: This is the first study on carbapenem-nonsusceptible P. aeruginosa from Dubai, incriminating VIM production as well as outer membrane permeability and efflux systems as resistance mechanisms. Further studies on carbapenem-nonsusceptible P. aeruginosa in Dubai are warranted for containment of such health hazard.