The flow of genetic information from DNA to protein requires polymerase-II-transcribed RNA characterized by the presence of a 5′-cap. The cap-binding complex (CBC), consisting of the nuclear cap-binding protein (NCBP) 2 and its adaptor NCBP1, is believed to bind all capped RNA and to be necessary for its processing and intracellular localization. Here we show that NCBP1, but not NCBP2, is required for cell viability and poly(A) RNA export. We identify C17orf85 (here named NCBP3) as a cap-binding protein that together with NCBP1 forms an alternative CBC in higher eukaryotes. NCBP3 binds mRNA, associates with components of the mRNA processing machinery and contributes to poly(A) RNA export. Loss of NCBP3 can be compensated by NCBP2 under steady-state conditions. However, NCBP3 becomes pivotal under stress conditions, such as virus infection. We propose the existence of an alternative CBC involving NCBP1 and NCBP3 that plays a key role in mRNA biogenesis. The processing of RNAs transcribed by RNA polymerase II requires a cap-binding complex (CBC), consisting of NCBP1 and NCBP2. Here, the authors report an alternative CBC formed by NCBP1 and a previously uncharacterized protein, NCBP3 that is critical for RNA processing under cellular stress conditions. Polymerase II transcripts, such as messenger (mRNA), antisense (asRNA), long intergenic non-coding (lincRNA) and small nuclear RNA (snRNA) are marked by an N7-methylated guanine  as a novel genuine cap-binding protein that directly interacts with NCBP1 and binds cellular mRNA. Similar to NCBP2, NCBP3 is non-essential under steady-state conditions. However, simultaneous depletion of NCBP2 and -3 mimics the phenotype of NCBP1 knockdown. Notably, NCBP3 becomes pivotal under cellular stress conditions, such as virus infections. We propose the existence of a canonical and an alternative CBC that is fundamental for mRNA biogenesis of higher eukaryotes.in situ hybridization (RNA-FISH)  severely affected cell growth . SurprisNA-FISH) . In contNA-FISH) .Muscomorpha and Saccharomycetaceae, which include Drosophila melanogaster and Saccharomyces cerevisiae, respectively. Recent data on distribution of proteins in human tissues suggest that C17orf85 is expressed in all tissues23A protein with redundant function to NCBP2 should have the ability to associate with the RNA cap structure and to bind to NCBP1 to engage factors required for RNA biogenesis. To identify potential candidate proteins, we performed affinity purification followed by liquid chromatography tandem MS (AP–LC–MS/MS) using lysates from human and mouse cell lines and capped and non-capped RNA as baits . Capped 207GTP)-coupled beads , confirming specificity of this assay.To confirm cap-dependent binding of NCBP3, we used capped and non-capped RNA as bait for AP, followed by western blotting. NCBP3 precipitated with capped RNA in human THP-1 and murine RAW 264.7 cells comparably well as the cap-binding protein EIF4E . The caned beads . m7GTP aTo functionally assess the potential contribution of NCBP3 to compensate for loss of NCBP2, we employed siRNA-based knockdown and tested for cell growth in HeLa cells. Depletion of either NCBP2 or NCBP3 alone did not considerably affect cell growth  despite 7GpppG. The RRM of NCBP3 shares high sequence homology (identity of 21.4% for residues 121–191) and critical residues with the RRM of PARN  fold (β1–α1–β2–β3–α2–β4) of PARN 272829 asPARN 2728. To studt manner . The hal not GTP . A highl not GTP . Microsc 7GpppG28.7GpppG binding. N7-methylated GTP is bound to a surface pocket on the C-terminal RRM of PARN mainly through π-interaction with tryptophan W468 and polar interactions with two aspartic acids, D471 and D470 . In agreement with the ability of NCBP3 and NCBP2 to associate with the cap structure, both proteins enriched for capped RNAs as compared with control precipitates  RNA distribution . RemarkaPerturbation of the RNA export machinery components often shows loss-of-function phenotypes under challenging conditions39in vivo antibody-mediated inhibition of NCBP2 only affected nuclear export of U snRNAs but not general export of mRNAThe canonical CBC consisting of NCBP1 and -2 is considered to be one of the most fundamental parts of the biogenesis machinery of all capped RNAs. This notion is based on the involvement of the CBC in pre-mRNA splicing, 3′-end processing, intranuclear transport, nuclear–cytoplasmic transport of RNA and nonsense-mediated decay and translation10147GTP. Co-depletion of NCBP2 and -3 is synthetically lethal in human und murine fibroblasts, suggesting redundancy between both proteins. This redundancy can be explained by RIP-seq experiments, showing that NCBP2 and -3 share affinity for the majority of mRNA. In addition to mRNA binding, NCBP2 but not NCBP3 has the ability to bind snRNA. Consistent with the selective affinity for snRNA, only NCBP2 binds the snRNA transport protein PHAX. Our results consolidate reports that are incompatible with the current model of a single CBC for RNA export: while inhibition of NCBP2 selectively impairs U snRNA nuclear–cytoplasmic export, but not of mRNAStructure-guided binding studies using recombinant NCBP3 clearly established direct cap-binding through a canonical RRM bearing a WLDD motif at the centre of the RNA-binding grove. This motif is also conserved in the canonical cap-RNA-binding protein PARN and mediates affinity to cap-RNA in the low micromolar range. In addition, NCBP3 directly binds NCBP1 when co-expressed in bacteria and thus allows formation of a protein complex with affinity to capped RNA. A direct interaction between NCBP1 and -3 is further suggested from large-scale yeast two-hybrid interaction dataIt is commonly accepted that EJC components, the TREX complex, PHAX and the NELF complex are recruited to RNAs by virtue of the CBC. We here show that EJC and TREX precipitate superior with NCBP3 as compared with NCBP2, whereas PHAX and the NELF complex are exclusively enriched in NCBP2 precipitates. It is tempting to speculate that binding of either NCBP2 or -3 induces a conformational change of NCBP1 that in turn allows differential binding of adaptor proteins. Structural analysis of NCBP1 in the context of NCBP2 and -3 bound to capped RNA, as well as binding assays with purified proteins will be necessary to study this in detail.Saccharomyces cervisiae. Notably, in S. cervisiae only 3% of genes contain introns and only six genes contain two intronsSchizosaccharomyces pombe expresses an NCBP3 homologue and codes for introns in 43% of its genesS. cerevisiae may therefore be a result of evolutionary adaptation.Given the association with proteins of the EJC and TREX complex, NCBP3 may play a primary role in biogenesis of spliced mRNA. Such a role is supported by genetic correlation: although NCBP3 is remarkably conserved during evolution from fungi to humans, we could not find homologues for NCBP3 in The canonical CBC (consisting of NCBP1/-2) guides mRNA from the nucleus into the cytoplasm. Similarly, NCBP3 localizes to the nucleus and partially to the cytoplasm, suggesting that the alternative CBC drives mRNA export to the cytoplasm. RNA bound to the canonical CBC or EIF4E is sensitive to the nonsense-mediated mRNA decay (NMD) pathway4445Given that RNA metabolism is of central importance for all physiological and pathophysiological processes, we envisage a central role of the alternative CBC in various diseases, as known for many proteins involved in RNA splicing, 3′-end processing and degradation241−1 penicillin and 100 μg ml−1 streptomycin). Streptavidin-agarose beads were obtained from Novagen, GFP-Trap-coupled agarose beads were from Chromotek, and γ-Aminohexyl m7GTP- and GTP-agarose beads from Jena Bioscience and Biorbyt, respectively. Primary antibodies (1:1000 dilution for western blotting if not stated otherwise) used in this study were as follows: C17orf85 , NCBP1, SRRT, EIF4A3 and ALYREF , THOC5, PHAX and KPNA3 , GFP , PABP1 and β-tubulin (1:500 dilution for western blotting) , histone H3  and EIF4E . Antibodies against NCBP2 (1:500 dilution for western blotting) were raised by immunizing rabbits with recombinant full-length protein purified from E. coli. Antibodies against β-actin , His-tag  and secondary antibodies detecting mouse or rabbit IgG (Jackson ImmunoResearch) were horseradish peroxidase-coupled. 4′,6-diamidino-2-phenylindole (DAPI) and secondary antibodies used for immunofluorescence were purchased from Invitrogen. Vesicular stomatitis virus M2 (mutant VSV with the M51R substitution in the matrix protein), SFV and EMCV have been described previouslyHeLa S3 (CCL-2.2) and Vero E6 cells (CRL-1586) were purchased from ATCC. Human THP-1, NIH3T3 and murine RAW 264.7 macrophages have been described previously6 cells and repeated transfection 48 h later under the same conditions. For experiments where two genes were silenced simultaneously, we added scrambled-siRNA to single-gene knockdown controls. Cells were analysed at the indicated time points after the second transfection. Duplex siRNAs were either purchased from Qiagen or synthesized by the Core Facility at the MPI of Biochemistry. siRNA target sequences were as follows: human NCBP3 , mouse NCBP3 , human NCBP1 , mouse NCBP1 , human NCBP2 , mouse NCBP2 , huNXF1 , GFP (5′-AAGCAGCACGACUUCUUCAAG-3′) and scrambled (5′-AAGGTAATTGCGCGTGCAACT-3′).Duplex siRNAs were transfected using either siPrime transfection reagent  or the Neon Transfection System (Invitrogen) for target gene knockdowns. Transfection was performed according to the manufacturer's instructions for HeLa or NIH3T3 cells. Briefly, we transfected 200 pmol of siRNA per 1 × 104 cells were seeded in 24-well dishes and cell titres determined after the indicated time points using CellTiter-Glo (Promega) according to the manufacturer's instructions with the following modifications: cells were washed once with 1 × PBS and incubated with 100 μl CellTiter-Glo reagent (diluted 1:5 in 1 × PBS) for 10 min. Luminescence was measured using an Infinite 200 PRO series microplate reader (Tecan).For cell growth assays, 1 × 10For immunofluorescence, HeLa cells grown on coverslips were fixed with 4% (w/v) paraformaldehyde for 10 min, permeabilized with 0.1% (v/v) Triton X-100 for 10 min and washed three times with blocking buffer ). Cells were then incubated with primary antibody (1:500 dilution), followed by incubation with secondary antibodies (1:200 dilution) covalently linked to fluorophores and DAPI. Coverslips were mounted on microscope slides using ProLong Gold Antifade Reagent (Molecular Probes). Confocal imaging was performed using a LSM780 confocal laser scanning microscope  equipped with a Plan-APO × 63/numerical aperture 1.46 oil immersion objective (Zeiss).−1 5′-Cy5-labelled oligo (dT)45 (Sigma), 0.5 μg μl−1 transfer RNA (Ambion), 1% (v/v) bovine serum albumin, 10% (v/v) dextran sulphate, 20% (v/v) deionized formamide and 2 × sodium saline citrate (SSC)) for 3 h at 30 °C in a humid chamber. After washing once with 4 × SSC and twice with 2 × SSC, cells were incubated with DAPI diluted in 2 × SSC containing 0.1% (v/v) Triton X-100. Finally, cells were washed twice with 2 × SSC and coverslips placed on a microscope slide. Confocal analysis was performed as described above. Quantification of area and intensity of cytoplasm versus nuclei was performed with Volocity 6.3 analysis software . With the protocol four populations of objects were defined: (1) Nuclei were masked using a DAPI channel with a threshold range of 13–100% and restriction to a minimum object size of 50 μm2. ‘Fill holes in objects' was applied to remove holes in the mask; (2) cytoplasm including nuclei was masked using a Cy5 channel with an intensity-based threshold of 6–255 and restriction to a minimum object size of 100 μm2. (3) A mask for measurements in the cytoplasm was created using ‘Exclusively combine' of the first two masks. (4) Finally, a mask for measurements in the Cy5-stained nuclei was created by subtracting (3) from (2). Within the mask (3) and (4), area and summarized intensity were measured and intensities were normalized to area. The nuclear to cytoplasmic ratio was calculated using the intensities from a minimum of 125 cells.For RNA-FISH analysis, cells grown on coverslips were fixed with 4% (v/v) PFA for 10 min, followed by 100% (v/v) ethanol for 10 min and washed in 70% (v/v) ethanol. Subsequently, cells were incubated with 1 M Tris-HCl pH 8 for 10 min, followed by incubation with hybridization buffer (2 ng μlN-hydroxysuccinimide ester (Epicentre) or fluorophore using DyLight 488 NHS ester according to the manufacturer's instructions and purified by reverse-phase-HPLC. Biotinylated 7SK-as RNA bearing a 5′-CAP or –OH group, respectively, were synthesized by in vitro transcription and enzymatic modification of the 5′ terminiSynthetic oligoribonucleotides (Chemgenes Corporation) with a 3′-terminal amino linker harbouring either an N7-methylated cap structure (CAP) or a 5′-hydroxyl group (OH) were generated as described previously2 and protease inhibitor cocktail ) in the presence of 40 U RNase inhibitor (Fermentas) for 60 min at 4 °C on a rotary wheel and excess RNA removed by three washes with TAP buffer. Cell lysates were prepared by snap-freezing cells in liquid nitrogen, incubation in TAP buffer for 30 min on ice and clarification of the lysate by centrifugation at 16,000g. RNA-coated beads were incubated with 2 mg of clarified lysate for 60 min at 4 °C, washed three times with TAP buffer, boiled in Laemmli buffer for 10 min at 95 °C and subjected to SDS–polyacrylamide gel electrophoresis and western blot analysis. Uncropped western blots are provided in For APs with biotin-labelled RNA, streptavidin-affinity resin was first incubated with RNA in TAP buffer  glycerol, 0.2% (v/v) Nonidet-P40, 1.5 mM MgCl7GTP- and GTP-coupled beads, cell lysate was sonicated using a Branson Sonifier 250 with 15 pulses at a duty cycle of 50% and an output control of 1. The lysate was further incubated on ice for 15 min and clarified by centrifugation at 16,000g for 10 min at 4 °C. Similarly, we used GFP-Trap beads (Chromotek) for affinity purification of proteins binding to GFP-tagged baits. The sonicated lysate was further treated with 10 U of Benzonase  for 30 min on ice before clarification of the lysate.To detect proteins binding to mE. coli expression of recombinant proteins was induced overnight at 18 °C in E. coli strain BL21-AI using 1 mM isopropyl-β-D-thiogalactoside (Thermo) and 0.2% L-(+)-Arabinose (Santa Cruz). Cells were lysed in HIS buffer , including 50 U ml−1 of Benzonase  and protease inhibitor cocktail  using sonication and lysates clarified by centrifugation at 16,000g for 10 min at 4 °C. Lysates were used for AP using Ni-NTA spin columns according to the manufacturer's instructions (Qiagen). Ni-NTA-bound proteins were eluted using HIS buffer containing 500 mM imidazole. Eluates were boiled in Laemmli buffer for 10 min at 95 °C and subjected to SDS–polyacrylamide gel electrophoresis and western blot analysis.For co-precipitation of proteins expressed in t-tests with permutation-based false discovery rate (FDR) statistics, performing 250 permutations. FDR thresholds and S0 parameters were empirically set to separate background from specifically enriched proteins.To detect and quantify RNA-binding proteins and proteins bound to GFP-tagged baits by AP and MS, samples were prepared as described above. After the final three washes in TAP buffer, samples were, in addition, washed twice with TAP buffer lacking Nonidet-P40 to remove residual detergent. Three independent APs were performed for each bait. Sample preparations and LC–MS/MS analysis was performed as described previouslyFor total proteome analysis, HeLa cells were electroporated twice with siScrambled or siRNAs against NCBP1, NCBP2, NCBP3 or NCBP2 and NCBP3 and lysed 5 days after knockdown. Three knockdowns were performed in parallel. Cells were pulsed with SILAC Arg10- and Lys8-containing medium for the last 6 h ref. . Cells whttp://david.abcc.ncifcrf.gov). Proteomics data were analysed using Perseus, results were plotted using R (www.R-project.org) and GraphPad Prism version 5.02 and visually adapted using Adobe Illustrator.For functional annotation analysis, we used DAVIDg for 10 min at 4 °C and washed twice with ice-cold PBS. Cells were lysed in an equal volume of RIP lysis buffer  Nonidet-P40, 1 mM dithiothreitol, protease inhibitor cocktail ) for 30 min on ice in the presence of 100 U ml−1 RNase inhibitor (Fermentas) and lysates clarified by centrifugation at 9,000g and 4 °C for 10 min. Clarified lysates were incubated with GFP-Trap beads (Chromotek) in RIP wash buffer  Nonidet-P40) containing 25 mM EDTA, protease inhibitors and 100 U ml−1 RNase inhibitor for 4 h at 4 °C on a rotary wheel, followed by five washes with RIP wash buffer. Beads were then resuspended in Trizol (Invitrogen) and RNA isolated using the Direct-zol RNA MiniPrep kit (Zymo Research) according to the manufacturer's instructions. Three RNA immunoprecipitations per bait were carried out in parallel. RNA quality was assessed on a Genetic Analyzer (Agilent) and TruSeq RNA library construction and next-generation sequencing were performed by the Max Planck-Genome-Center Cologne, Germany (http://mpgc.mpipz.mpg.de/home/). All samples were sequenced on an Illumina HiSeq2500 platform at 15 million 100-bp single reads per sample. After quality control of the sequencing libraries, reads were trimmed and mapped against the Ensembl genome annotation and the human genome assembly (hg19/GRCh37) using Tophat2  and quantified by quantitative RT–PCR using the QuantiFast SYBR Green RT-PCR kit (Qiagen) and a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Each cycle consisted of 10 s at 95 °C and 30 s at 60 °C, followed by melting curve analysis. Primer sequences were as follows: huMYC (5′-CAGTGGGCTGTGAGGAGGTT-3′ and 5′-CAGGCTCCTGGCAAAAGGT-3′), huSLC43A3 (5′-CATGACATTCCCCACTGGCT-3′ and 5′-GATTCCCCCAATGGTGAGCA-3′), huRNU1-1 (5′-ACTTACCTGGCAGGGGAGATAC-3′ and 5′-ACATCCGGAGTGCAATGGATAA-3′), huRNU4-1 (5′-GCGCAGTGGCAGTATCGTAG-3′ and 5′-GGCGGGGTATTGGGAAA-3′), huNCBP1 (5′-GGCTGCAGCAGATCTTCCTA-3′ and 5′-TCTCCAGGGTCACCATGTACT-3′), huNCBP2 (5′-TTTCCAACATATAACGTACAGCTTTT-3′ and 5′-CTACGTGGAGCTGAGCCAGT-3′), huNCBP3 (5′-GCAGGAAGACAGTTCAGATG-3′ and 5′-ACTTCTTCTGGCTGCTCCAA-3′).http://chimborazo.biochem.mpg.de/morfeus). The multiple sequence alignment was generated using mafftOrthologous sequences were collected using the web-server morFeushttp://toolkit.tuebingen.mpg.de)) with the RRM of human poly(A) adenylate-binding protein 1 (PDB code 4f02) as a template model. The final best model was chosen according to DOPE (Descrete Optimized Protein Energy) scoresA structural model of the RRM domain of C17orf85/NCBP3 (UniProt entry Q53F19) was obtained using the HHpred server2, 10 mM EDTA and 0.5% (v/v) NP-40) containing protease inhibitor  and Benzonase Nuclease (Novagen). For western blot analysis, we loaded 4% of the total cell lysate and 20% of the cytoplasmic and nuclear fractions.Cells grown in a 10-cm dish were fractionated as described previouslyE. coli strain BL21-AI using 1 mM isopropyl-β-D-thiogalactoside (Thermo) and 0.2% L-(+)-Arabinose (Santa Cruz). Cells were lysed in BL buffer ) and the cleared lysate was used for further experiments or AP using a HisTrap HP column . Recombinant untagged wild-type and mutant (ALAA) NCBP3 1–282aa used for MST and CD measurements was purified by the Core Facility at the MPI of Biochemistry. Identity of recombinant NCBP3 was confirmed by MS.The long isoform of C17orf85/NCBP3 (GenBank NM_001114118.2) was cloned from human complementary DNA (cDNA) into vector pDONR221 (Invitrogen). Mutations and truncations in C17orf85/NCBP3 were introduced by site-directed mutagenesis and PCR, respectively. Expression constructs containing N-terminal His-tagged full-length C17orf85/NCBP3 (pETG10A-NCBP3) or its mutant versions (pETG10A-NCBP3-D134A and -ALAA) were generated using the Gateway system (Invitrogen) as described earlier2, 0.05% (v/v) Tween-20) to a final concentration of 50 nM. A twofold dilution series (16 samples) of NCBP3 1–282aa starting from 204 μM were performed in MST-2 buffer  glycerol). The two components were mixed in equal volumes and the mixture (∼8 μl) was then measured in the Monolith NT.015T at room temperature using a blue light-emitting diode (LED). The MST settings were: LED power (20%), MST laser power (80%), fluorescence before (5 s), MST on (30 s) and fluorescence after (5 s). The normalized fluorescence signal  was analysed and plotted by Monolith NT.015T analysis software. Three individual measurements were combined to calculate the Kd values with s.e.For MST affinity measurements, labelled RNA oligos (CAP0- and OH-RNA-DyLight) were diluted in MST-1 buffer , four scans were recorded and averaged. The averaged baseline spectrum was subtracted from the averaged sample spectra and the resulting spectra were smoothed using an FFT (Fast Fourier Transform) filter (as part of the software package). Measurements were only made down to wavelengths where the instrument dynode voltage indicated the detector was still in its linear range (190 nm). Spectra are shown as the mean residue elipticity. Secondary structure compositions were estimated using the CONTINLL program5 HeLa cells that had been transfected for 48 h with siRNA targeting NCBP3 or GFP as control were infected with VSV-M2, EMCV or SFV at a multiplicity of infection of 0.01. At 24 h post infection, virus titres in supernatants were quantified by 50% tissue culture-infective dose (TCID50) assays on Vero E6 cells.To determine the impact of siRNA-mediated knockdown of NCBP3 on virus growth, aliquots of 1 × 10Accession codes: RIP-seq data sets are available from Gene Expression Omnibus (GEO) at the National Center for Biotechnical Information under the accession code GSE71552.How to cite this article: Gebhardt, A. et al. mRNA export through an additional cap-binding complex consisting of NCBP1 and NCBP3. Nat. Commun. 6:8192 doi: 10.1038/ncomms9192 (2015).Supplementary Figures 1-8 and Supplementary ReferencesLC-MS/MS identified proteins binding to 5'modified RNAs.Abbreviations and accession numbers of NCBP3 orthologous proteins.Quantitative proteomics after NCBP knockdown.LC-MS/MS identified proteins in NCBP3 and NCBP2 precipitates.Data tables for the quantification of transcript binding to NCBP3 and NCBP2 measured by RIP-Seq.


Chi3l1 is induced by a variety of cancers where it portends a poor prognosis and plays a key role in the generation of metastasis. However, the mechanisms that Chi3l1 uses to mediate these responses and the pathways that control Chi3l1-induced tumor responses are poorly understood. We characterized the mechanisms that Chi3l1 uses to foster tumor progression and the ability of the RIG-like helicase (RLH) innate immune response to control Chi3l1 elaboration and pulmonary metastasis. Here we demonstrate that RLH activation inhibits tumor induction of Chi3l1 and the expression of receptor IL-13Rα2 and pulmonary metastasis while restoring NK cell accumulation and activation, augmenting the expression of IFN-α/β, chemerin and its receptor ChemR23, p-cofilin, LIMK2 and PTEN and inhibiting BRAF and NLRX1 in a MAVS-dependent manner. These studies demonstrate that Chi3l1 is a multifaceted immune stimulator of tumor progression and metastasis whose elaboration and tissue effects are abrogated by RLH innate immune responses. The prototypic chitinase-like protein (CLP), Chitinase 3-like-1 (Chi3l1)  is a member of the 18 glycosyl hydrolase (GH 18) gene family, which binds to but does not degrade chitin24567891071121314151617181920Metastatic spread is an ominous prognostic event in cancer biology. This can be readily appreciated in malignant melanoma where there is a good chance of recovery if the primary lesion is detected early and the 5 year survival is less than 10% in patients with distant melanoma metastases (stages III and IV)2217We hypothesized that interventions that alter the induction of Chi3l1/YKL-40/BRP-39 decrease the metastatic spread of malignant melanoma and other tumors. We also hypothesized that the RIG-like helicase (RLH) innate immune response is a powerful inhibitor of Chi3l1 and pulmonary metastasis. To test these hypotheses we characterized the ability of the known pulmonary RLH activator Poly(I:C)2526As shown in the previous studies from our laboratoryin vivo studies demonstrated that Chi3l1 expression and production are augmented after the administration of B16 melanoma cells and that Poly(I:C) is a potent inhibitor of these metastasis permitting responses  uses innate immunity to inhibit melanoma metastasis, we evaluated the roles of the Toll-like receptor 3 (TLR3) and RIG-helicase (RLH) innate immune pathways. In these experiments, we compared the effects of Poly(I:C) in wild type, TLR3 null and mitochondrial antiviral signaling molecule (MAVS) null mice. MAVS was chosen because it is the central integrator of the RLH pathway that links the viral nucleic acid sensing RNA helicases like retinoic acid inducible gene-I (RIG-I) and melanoma differentiation antigen 5 (Mda5) to anti-viral effector responses28The studies noted above demonstrate that RLH pathway activation via MAVS plays an important role in the control of tumor spread. Because virulent tumors have been repeatedly shown to coopt antitumor responses3132Because RLH pathway activation has been linked to Type I IFNs, we compared the production of IFN-α and IFN-β in the lungs from mice treated with vehicle or Poly(I:C) and the metastasis-inhibitory effects of RLH activation in WT and Type I IFN receptor (IFNAR1) null mice. Poly(I:C)-induced RLH activation was a potent stimulator of both Type I IFNs in these animals . These iPrevious studies from our laboratory demonstrated that melanoma cells stimulate Chi3l1 via a semaphorin 7a (Sema7a)-dependent mechanism with Sema7a stimulating Chi3l1 when it interacts with its β1 integrin receptor and inhibiting Chi3l1 when it interacts with its Plexin C1 receptor+ B cells with and without Poly(I:C) stimulation  cells have been shown to regulate melanoma growth and metastasis in a number of systemsmulation . This wamulation  and was mulation . B16 celmulation . In accomulation  and thesmulation . The NK mulation . These sTo understand the mechanisms that underlie the augmented NK cell accumulation described above, we evaluated the expression of the endogenous NK cell chemoattractant chemerin in our experimental system. These studies demonstrate that the levels of chemerin accumulation were decreased in bronchoalveolar lavage (BAL) from tumor challenged WT mice . They alPrevious studies demonstrated that melanoma metastasis and invasion are associated with decreased expression of LIM kinase (LIMK) and reduced levels of phosphorylated cofilin (p-cofilin)Chi3l1 is produced by and present in elevated quantities in the circulation of patients with a variety of tumors where it is a poor prognostic sign16373839414243The RLH pathway is believed to play a major role in antiviral responses based on its ability to detect double-stranded (ds) RNA (which is produced during the replication of many viruses) and 5′-triphosphorylated single-stranded RNA (which are produced by many single stranded RNA virus including influenza). The antiviral immune responses are triggered by cytoplasmic RNA sensors including RIG-I and Mda5 which are linked via MAVS to downstream signaling molecules and the induction of Type I IFNs28It has recently been appreciated that the RLH innate immune system is finely regulated to allow for appropriate antiviral activation without inappropriate activation and subsequent tissue injury31+ or CD4+ T cell or CD19+ B cells, accumulation and that both are mediated by a MAVS- and Chi3l1-dependent mechanism(s). These studies suggest that Chi3l1 inhibition of the chemerin system and NK cell function and activation are important events in the ability of Chi3l1 to foster metastasis and that RLH inhibits pulmonary metastasis, at least in part, by abrogating these NK cell effects.Chemerin is a chemoattractant protein for NK cells that has been implicated in melanoma homeostasis. It is down-regulated in melanoma cells. In addition, it can elicit antitumor responses via an NK cell dependent mechanism(s) and high levels of expression correlate with improved disease outcomesPrevious studies from our laboratory demonstrated that melanoma and other metastasis stimulate Chi3l1 via a novel Sema7a containing pathway in which Sema7a stimulates and inhibits Chi3l1 when it interacts with its β1 integrin and Plexin C1 receptors respectivelyPTEN is a powerful multifaceted tumor suppressor that is functionally involved in many different hallmarks of cancer36The B-Raf oncoprotein is a serine threonine kinase that regulates MAPK/ERK signaling and, in turn, cell growth, differentiation and apoptosis51In conclusion, these studies define the many ways that tumor-induced Chi3l1 contributes to the generation of a metastasis permissive microenvironment and highlight the ability of RLH innate immunity to abrogate these responses. Additional investigation of the effects of Chi3l1 in the tumor microenvironment and the ways that RLH innate immunity can be manipulated to control metastasis is warranted.−/−) and Chi3l1/YKL-40 transgenic (Tg) mice were generated and characterized in our laboratory as previously described7−/− mice were provided by Dr. A.L. Kolodkin . TLR3−/− mice were gifts from Richard A. Flavell −/− and IFNAR1−/− mice were provided from Dr. ZJ Chen Guide for the Care and Use of Laboratory Animals and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).Chi3l1/BRP-39 null mutant (Chi3l16 cells/ml and delivered to the mice by tail vein injection (2 × 105 cells/mouse in 200 μl of DMEM)4 cells/moues in 200 μl of DMEM) were delivered via tail vein injection to BALB/c WT mice, because these cells only survive in BALB/c animalsThe mouse melanoma cell line (B16-F10) established from C57BL6/J mouse melanoma was purchased from ATCC (Cat#: CRL-6475). After being cultured to confluence in Dulbecco’s Modified Eagles Medium (DMEM), the cells were collected, adjusted to a concentrations of 10Melanoma lung metastasis was quantified by counting the number of melanoma colonies (which appear as black dots) on the surface of the lung. For the evaluation of EMT6 metastasis, the lungs were harvested, treated with Bouin’s fixative overnight, then washed with 70% alcohol and the number of colonies on the surface of the lungs was counted under a surgical microscope.Poly(I:C)  was delivered to WT and genetically modified mice via intranasal (i.n.) administration (1 μg/kg/mouse) as describedThe levels of pulmonary Chi3l1/BRP-39, Sema7a, β1 integrin, Plexin C1, cytokines, RIG-I, Mda-5, LGP-2, MAVS, NLRX1, Type I IFN, granzyme B, perforin, chemerin, ChemR23, p-cofilin, LIMK2, PTEN and BRAF mRNAs and or proteins were assessed using real-time RT-PCR (RT-PCR), Western blotting, ELISA or slot blotting as previously described by our laboratory724−CD11bhiGr-1+ cells as previously reported+ CD4+ CD8−, CD3+ CD4−, CD8+ and CD3− CD19+ as previously describedFACS analysis of NK cells were evaluated as previously describedWhole-lung lysates were prepared and the total protein content of each was measured using the DC protein assay reagents . Equal amounts of sample proteins were fractionated on 4%–15% SDS-PAGE gels under reducing conditions. These were individual gels that were prepared and run at the same time. The sample proteins were transferred to polyvinylidene difluoride membranes and incubated in blocking buffer (5% w/v nonfat dry milk in TBS/0.05% Tween 20) for 1 hour at room temperature. They were then incubated with primary antibodies overnight at 4 °C, washed 3 times in TBS/0.05% Tween 20, and incubated for 2 hours at room temperature with appropriate secondary antibodies. Immunoreactive signal was detected using a chemiluminescent procedure  according to the manufacturer’s instructions.t test or with nonparametric Mann-Whitney U test. Values are expressed as mean ± SEM. Statistical significance was defined as a level of P < 0.05.Statistical evaluations were undertaken with SPSS software. As appropriate, groups were compared with 2-tailed Student’s How to cite this article: Ma, B. et al. RIG-like Helicase Regulation of Chitinase 3-like-1 Axis and Pulmonary Metastasis. Sci. Rep.6, 26299; doi: 10.1038/srep26299 (2016).


BRR2 and PRP16 suppresses or enhances physical interaction and growth defects in an allele-specific manner, signifying functional interactions. Notably, we show that Brr2p Sec63-2 domain can modulate the ATPase activity of Prp16p in vitro by interfering with its ability to bind RNA. We therefore propose that the carboxy-terminal helicase module of Brr2p acquired a regulatory function that allows Brr2p to modulate the ATPase activity of Prp16p in the spliceosome by controlling access to its RNA substrate/cofactor.RNA helicases are essential for virtually all cellular processes, however, their regulation is poorly understood. The activities of eight RNA helicases are required for pre-mRNA splicing. Amongst these, Brr2p is unusual in having two helicase modules, of which only the amino-terminal helicase domain appears to be catalytically active. Using genetic and biochemical approaches, we investigated interaction of the carboxy-terminal helicase module, in particular the carboxy-terminal Sec63-2 domain, with the splicing RNA helicase Prp16p. Combining mutations in  Nuclear pre-mRNA splicing is catalyzed by the spliceosome complex, composed of five small nuclear ribonucleoprotein particles  and a large number of non-snRNP proteins . DExD/H in vitro, inhibiting RNA binding there  domain and a Sec63 domain, separated by a winged helix (WH) domain Figure  17–19).–19.17–19In a yeast two-hybrid (Y2H) screen, the C-terminal part of budding yeast Brr2p was found to interact with the spliceosomal helicases Prp2p and Prp16p and with other splicing factors, possibly as a protein interaction platform ,22. It hin vivo and in vitro approaches to further characterize the interaction of Brr2p C-terminus with Prp16p. First, we identified several mutations in Brr2p Sec63-2p that alter the interaction with Prp2p and/or Prp16p. Notably, these cluster in a particular structural element in the Sec63-2 domain. Focusing on the relationship between Brr2p C-terminus and Prp16p, we find that certain combinations of prp16 and brr2 mutant alleles result in synthetic growth phenotypes, supporting the functional significance of the physical interactions. We show that Prp16p interacts directly with the Sec63-2 domain of Brr2p. This interaction interferes with RNA binding to Prp16p and the RNA stimulation of ATP hydrolysis by Prp16p. We therefore propose that Brr2p Sec63-2 domain could participate in regulating the activation of Prp16p at the catalytic center of the spliceosome by controlling access to its RNA substrate/cofactor.Here we present a combination of Plasmids and strains are listed in Supplementary Tables S1 and S2, respectively.PRP2 and PRP16 were generated by InFusion cloning (Takara). The Gal4AD-prey fusion for H2-Sec63-2p was constructed in pACTII-stop using XmaI/BamHI sites. Mutations in PRP16 were created by site-directed mutagenesis .Brr2p domains H2-Sec63-2 and Sec63-2 were polymerase chain reaction (PCR)-amplified and cloned into pBlueScript using SpeI/XhoI restriction sites. LexA-bait fusions of HIS3 reporter gene (growth on medium lacking leucine and tryptophan (-LW) to select for both plasmids and lacking histidine (-H) to select for HIS3 expression; -LWH medium)  and anti-LexA  ,24 and inti-LexA  antibodi2, then integrated into pACTII-stop plasmid by Megaprimer PCR. DpnI-treated PCR products were transformed to Escherichia coli XL10 Gold (Stratagene). Plasmids from 50 clones were sequenced to determine the mutation-frequency prior to pooling the transformants.Amino-acids 1762 to 2163 of Brr2p were randomly mutated by error-prone PCR using non-proofreading TaKaRa LA TaqTM polymerase with 0.75-mM MnClYeast L40ΔG cells were transformed with a LexA-bait fusion (Prp2p or Prp16p) and the library of mutated H2-Sec63-2 plasmids. A Singer ROTOR yeast handling robot spotted stationary phase cultures of single yeast clones in 384-sample format on agar plates. After 2 days at 30°C, clones were replica-plated onto selective medium (-LWH), supplemented with different concentrations of 3-AT, and incubated at 30 or 14°C before comparison of colony size with wild-type (WT). Clones with a ‘stronger’ Y2H interaction were able to grow on medium containing 3-AT concentrations  that were toxic with WT H2-Sec63. Clones with a ‘weaker’ interaction grew on selective medium (minus histidine) at 14°C but not at 30°C and/or showed reduced or no resistance to 3-AT. Sequencing H2-Sec63-2 prey constructs identified the mutations in selected mutants. To confirm the observed phenotypes, prey plasmids were recovered, re-transformed in the Y2H strains and the interaction with the original baits or other bait constructs assessed.brr2Δ strain was constructed by replacing the entire open reading frame (ORF) from one chromosomal copy of BRR2 with the kanMX6 cassette in W303 diploid cells (BRR2 (URA3), sporulation and isolation of brr2Δ haploids carrying pRS316-BRR2. Mutant derivatives of pRS315-BRR2 were generated by site-directed mutagenesis  and their phenotypes analyzed after plasmid shuffle on 5-FOA to select for clones that had lost pRS316-BRR2 , using plasmids described in Supplementary Table S1. Cultures were grown in LB with ampicillin (50 μg/ml) and chloramphenicol (30 μg/ml) at 30°C to OD600 0.6, then transferred to 18°C for 1 h before addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to 0.4 mM and incubation overnight at 18°C. Cell pellets were resuspended in 25-ml lysis buffer . After incubation with egg-white Lysozyme on ice for 30 min, lysates were sonicated , centrifuged for 30 min at 17 000 rpm (Beckman Avanti J-25) then filtered using a 0.22 μm syringe filter and loaded on a Hi-Trap IMAC HP  charged with NiCl. The column was washed with 20 volumes of lysis buffer, and the target proteins were eluted using a linear gradient of imidazole (10 to 500 mM) in lysis buffer. After desalting, buffer exchange  and concentration, glycerol concentration was adjusted to 50% and the purified protein snap frozen in liquid nitrogen. Prp16p was purified using NiNTA-agarose resin (Qiagen) following the manufacturer's recommendations. Ded1p and Prp22p were purified as previously described  NP-40) containing bovine serum albumin (BSA) (100 μg/ml), glycogen (50 μg/ml) and tRNA (100 μg/ml). For co-immunoprecipitations treated with RNase A (30 ng/μl), tRNA was omitted. After extensive washing with IP buffer, beads were loaded onto NuPAGE 4–12% gradient gels (Invitrogen), followed by western blotting using anti-His-HRP-conjugated antibodies .Prp16p (3 μg) and Sec63-2p (1.5 μg) were incubated overnight at 4°C with protein A dynabeads (Roche) and anti-Prp16p-029  at 1/100 dilution in IP buffer , purified and labeled as in  and analyzed using AIDA software (Raytest). To account for the background, RNA bound was calculated as  ×100.A 44-nt RNA (5′ GGG CGA AUU CAA AAC AAA ACA AAA CUA GCA CCG UAA AGC AAG CU 3′) was produced by ed as in . Electroed as in  using th4, 1-mM DTT, 0.1-μg/μl BSA, 1-mM ATP, 50-ng/μl total yeast RNA and enzyme at 10, 20 and 10 nM respectively. Ded1p assays contained 5-nM enzyme, 20-mM Tris–HCl pH 7.5, 50-mM KAc, 1-mM MgOAc, 0.1-μg/μl BSA, 2-mM DTT, 1-mM ATP and 50-ng/μl total yeast RNA. Incubations were conducted at 30°C for up to 45 min. Results were converted to ‘hydrolyzed Pi’ using a Pi standard curve specific for each experiment and plotted using GraphPad Prism 5.0.ATP hydrolysis was measured as in  with minbrr2 mutations that reduce or enhance interaction with either Prp16p or Prp2p in two independent Y2H screens . From ∼4000 clones that were tested, 272 were selected that showed altered growth with different concentrations of 3-aminotriazole  at 14, 30 or 37°C. These were sequenced and 18 brr2 H2-Sec63-2 domain mutants that have only a single amino acid substitution were selected for further characterization . These results indicate that the second Sec63 domain of Brr2p is important for Brr2p activity in vivo and that some structural elements, such as helix α5, are necessary for Brr2p stability and/or function.To address whether mutations in Sec63-2 affect the function of Brr2p ts Table . Five alts Table  and had brr2-L1930P or brr2-L1951P at 30°C identified several splicing factor mutations as genetic interactors. Amongst those, we identified isy1Δ as one of the strongest interactors, being synthetic lethal with both brr2 alleles when tested at 37°C . Significantly, ISY1 also has a close genetic interaction with PRP16. Deletion of ISY1 rescues the growth defect caused by the allele prp16-302 and alleviates the stalling of spliceosomes that contain Prp16-302p (A Genetic Interaction Mapping (GIM) screen  to identp16-302p .in vivo, we first tested the effects of previously characterized mutations in the active site of Prp16p  and prp16-302 , that strongly reduced interaction with H2-Sec63-2p. However, brr2 H2-Sec63-2 R1899G, originally isolated as a stronger interactor of Prp16p, rescued the defective interaction with the mutant Prp16-R686I protein and partially rescued the interaction with the Prp16-302 protein . In the BRR2 background, prp16-Q685H caused very poor growth at all temperatures tested, the best growth being at 35°C  but, strikingly, growth was improved in the presence of brr2-R1899G (which improved the Y2H interaction). The prp16-R686I allele . As the most striking effect on growth was seen on combining the brr2-R1889G and prp16-Q685H mutations, we analyzed splicing of several intron-containing transcripts by RT-qPCR in the single and double mutant strains as well as in the well characterized prp16-2 mutant. Figure ACT1 intron-containing gene in cultures grown at 25°C and 40 min after shifting to 35°C. The prp16-2 strain showed no pre-mRNA or lariat accumulation relative to WT at 25°C but accumulated high levels of lariat-exon2 after shifting to 35°C, with pre-mRNA also building up by 40 min . Similar analyses of the intron-containing DBP2 and RPL28 transcripts showed only a similarly mild or no splicing defect (data not shown).Typically, g defect . Howeverin vitro assays (data not shown). We therefore purified recombinant Brr2 Sec63-2p, Sec63-2 R1899Gp (other mutant versions of Sec63-2 protein were largely insoluble), Prp16p and several mutant versions of Prp16p. WT and two mutant Prp16 proteins (DE473-4AA and R686I) were immunoprecipitated with similar efficiencies with an anti-Prp16p antibody , and they efficiently co-immunoprecipitated Brr2 Sec63-2p irrespective of RNase A treatment , indicating that the interaction is direct and not mediated by contaminating RNA. The interaction is also largely unaffected by an excess of ATP . Selecting a concentration of Prp16p (300 nM) that bound between 30 and 40% of the RNA substrate, we added increasing amounts of WT Sec63-2p, which greatly reduced the association of Prp16p with the RNA  or Pnslation . As prevnslation , Prp16p nslation . In the nslation . TherefoTP Table . Sec63-2KMapp,RNA increased as the concentration of Sec63-2p in the reaction increased, while it remained stable in the presence of increasing amounts of Sec63-2 R1899Gp , confirming the inhibitory effect of Sec63-2p on ATP hydrolysis by Prp16p, and a lack of this effect with the Brr2-R1899G mutant protein.To characterize further the functional relationship between RNA and Sec63-2p, we measured the ATPase activity of Prp16p in the presence of increasing amounts of Sec63-2p and of RNA . Significantly, Activities of splicing RNA helicases must be finely controlled in order to ensure timely rearrangements of RNA–RNA and RNA–protein interactions during the splicing cycle ,3,40. FoPRP16 when present in full-length BRR2, validating this focused genetic approach. However, as discussed below, an increased or decreased Y2H interaction does not predict a corresponding effect on splicing activity. The various genetic and physical interactions observed between the brr2 and prp16 alleles are summarized in Table Using mutagenesis and a Y2H screen we uncovered determinants in the Brr2p C-terminus that are important for its interaction with the Prp16 helicase, and identified the first known mutants of the second helicase module of Brr2p that cause a splicing defect. Importantly, mutations that were identified based on Y2H interactions also affect the genetic interaction with in vitro. In addition, the observation that RNA inhibits interaction of the Prp16 R686I mutant protein with recombinant WT Sec63-2p argues for a competition between RNA and Sec63-2p for interaction with Prp16p (but not necessarily for the same binding site in Prp16p). The strong genetic interactions observed between brr2 alleles and prp16 motif VI alleles  suggest that modulation of Prp16p activity by Brr2p occurs at the level of RNA stimulation of the ATPase activity, a known function of motif VI in RNA helicases of the DExD/H-box families  on Prp2p activity prior to step 1. Somewhat surprisingly, the prp16 alleles, prp16-Q685H, prp16-R686I and prp16-R686Q, displayed only very mild splicing defects for ACT1, DBP2 and RPL28 transcripts that are insufficient to explain the severe growth defect. These prp16 alleles carry mutations in motif VI that has been proposed to affect RNA stimulation of the ATPase activity of this family of RNA helicases. Koodathingal et al.  might indirectly alter the position of the 5′HP and/or the OB-fold domain of Prp16p, allowing RNA to bind and stimulate ATP hydrolysis, albeit less efficiently than in the absence of Sec63-2p.How might Brr2 affect RNA binding to Prp16p? The crystal structure of the DEAH-box RNA helicase Prp43p reveals the presence of a 5′ hairpin (5′HP) and an OB-fold domain that are conserved among DEAH-box helicases ,42,43. Iin vivo. With the WT Brr2p, it is possible that modifying the position of these structures relative to each other, e.g. upon protein–protein interaction, post-translational modification or ATP binding to Brr2p H2 domain, impacts on the overall folding of the Brr2p C-terminus, thereby altering its specificity for interacting partners, such as Prp2p or Prp16p, in the spliceosome. This suggests a very complex interplay between RNA helicases in the splicesome's catalytic center, with Brr2p playing a pivotal role that is very difficult to dissect in vivo and extremely challenging to analyze in vitro.The inhibition by Sec63-2p of the ATPase activity of Prp16p but not of Prp22p, suggests that Brr2 Sec63-2p interaction with Prp16p is mediated by specific structures in the DEAH-box proteins. Although the C-termini of DEAH-box proteins share high similarity, they also present clear differences  that couSupplementary Data are available at NAR Online.


In eukaryotes, mRNA polyadenylation is a well-known modification that is essential for many aspects of the protein-coding RNAs life cycle. However, modification of the 3′ terminal nucleotide within various RNA molecules is a general and conserved process that broadly modulates RNA function in all kingdoms of life. Numerous types of modifications have been characterized, which are generally specific for a given type of RNA such as the CCA addition found in tRNAs. In recent years, the addition of nontemplated uridine nucleotides or uridylation has been shown to occur in various types of RNA molecules and in various cellular compartments with significantly different outcomes. Indeed, uridylation is able to alter RNA half-life both in positive and in negative ways, highlighting the importance of the enzymes in charge of performing this modification. The present review aims at summarizing the current knowledge on the various processes leading to RNA 3′-end uridylation and on their potential impacts in various diseases. RNA 3′-end processing or modification plays an important role in determining their biological fate –3. One mEukaryotic mRNAs start to be modified during their transcription, where capping and polyadenylation take place at their 5′- and 3′-ends, respectively, except for histone and some viral mRNAs . Pre-mRNRecently, another player in the mRNA decay pathways has come into focus: the cytoplasmic poly(U) polymerases. These enzymes add uridine residues to the 3′-end of either coding RNAs or ncRNAs. Even though this modification has been known since the late fifties, its significance had been underestimated –45. In tIn this review, we focus on the latest research about the terminal polyuridylation by a specific group of noncanonical ribonucleotidyl transferases, a long time underestimated 3′-end posttranscriptional modification found in various RNAs and influencing RNA half-life and functions. The review will be divided in the following sections including a brief overview of the nucleotidyl transferase family followed by a review of the functional consequences of RNA polyuridylation in the different cell compartments. Finally, we will touch upon the multiple implications of polyuridylation mechanisms in diseases.β- (Pol β-) like nucleotidyl transferase superfamily and more specifically to the group of template-independent polymerases that covalently add nucleotides to the 3′-end of RNA molecules. This protein family was precisely defined a few years ago . Th. Th47]. It is noteworthy that RNAs with poly(U) tails have also been observed in human mitochondria under certain conditions –89. In sCytoplasmic polyuridylation occurs on a variety of RNA molecules ranging from polyadenylated to nonpolyadenylated RNA molecules including mRNAs, small RNAs, miRNAs, or piRNAs  7, 17, , 73–75.  S. pombe by the poly(U) polymerase Cid1  polymerizing activities [Nonpolyadenylated mRNAs are also uridylated in the cytoplasm . This istivities . One coutivities . The Lsmtivities . Again, tivities . C. elegans and H. sapiens, polyuridylation of pre-let-7-miRNA has been reported and is performed by the proteins PUP-2 and ZCCHC11, respectively [A variety of ncRNAs from diverse organisms have recently been shown to carry mono- or multiple non-templated uridine residues at their 3′-end  19, 73,–98. The ectively , 104. Asectively , 106. Hoectively . Furtherectively . So, uriectively . Finallyectively . Furtherectively . Recentlectively . FurtherO-methylation in these organisms [ C. elegans, CSR-1 is an Ago protein necessary for proper chromosome segregation rather than regulation of mRNA levels [ C. elegans PUP, uridylates unmethylated siRNAs of the CSR-1 pathway [ Chlamydomonas reinhardtii, MUT68 was first known to adenylate 5′ cleavage fragments of mRNAs targeted by the RNA-induced silencing complexes (RISC), thereby promoting their decay [Other types of ncRNAs subject to 3′ uridylation are siRNA and piRNAs . In nemarganisms –19, 22: rganisms –19, 22. A levels , 112. CD pathway . Mutatio pathway . Uridylair decay . Furtherir decay . 3′ uridir decay . In zebrir decay . Taken tThe HESO-1 enzyme, like MUT68, is also shown to act on atypical substrates, that is, the product of the miRNA-directed mRNA cleavage . In this Arabidopsis thaliana, uridylation of oligoadenylated mRNAs has been suggested to prevent their 3′ trimming and rather establish a preferential 5′-to-3′ mRNA degradation manner [ in vivo and its absence contributed to the degradation of oligoadenylated mRNAs highlighting a new role of uridylation in mRNA stabilization. The ZCCHC11 enzyme, besides its role in histone mRNA and pre-miRNA decay, has also been implicated in indirect mRNA stabilization by uridylation of mature miRNAs (At last, polyuridylation has also been reported to stabilize RNAs, rather than destabilize them. Inn manner . Indeed,e miRNAs . ZCCHC11These data together support a crucial role of cytoplasmic polyuridylation in the regulation of gene expression and stability control of both coding and noncoding RNAs in diverse eukaryotic species.RNA uridylation in the cytoplasm has been shown to induce tumorogenesis in mammals. Uridylation at the 3′-end of the tumor suppressor pre-let-7 microRNA by cytoplasmic ZCCHC11 and ZCCHC6 enzymes blocks let-7 miRNA maturation, which in turn stimulates tumor growth . Lin28 iPolyuridylation was for a long time an underestimated 3′-end modification; most probably because sequencing techniques were focused on polyadenylated RNAs. With the development of new and adapted techniques to detect 3′ uridylation, this event is starting to gain strength with impacting roles in RNA degradation and stability , 119. RN


Choices are not only communicated via explicit actions but also passively through inaction. In this study we investigated how active or passive choice impacts upon the choice process itself as well as a preference change induced by choice. Subjects were tasked to select a preference for unfamiliar photographs by action or inaction, before and after they gave valuation ratings for all photographs. We replicate a finding that valuation increases for chosen items and decreases for unchosen items compared to a control condition in which the choice was made post re-evaluation. Whether choice was expressed actively or passively affected the dynamics of revaluation differently for positive and negatively valenced items. Additionally, the choice itself was biased towards action such that subjects tended to choose a photograph obtained by action more often than a photographed obtained through inaction. These results highlight intrinsic biases consistent with a tight coupling of action and reward and add to an emerging understanding of how the mode of action itself, and not just an associated outcome, modulates the decision making process. This asymmetry of regret for action and inaction has been linked to a number of possible explanations and mediating factors including sense of agency [Choosing according to our preferences is an everyday occurrence in our lives. Some choices are expressed explicitly through action while others are expressed passively by accepting a default or not altering an existing choice. Interestingly, an xkcd comic titled “Regrets”  of 30 subjects participated in the study. Participants were recruited through a UCL participant pool, were self-reported right-handers, had normal or corrected to normal vision and were paid for their participation. The study was approved by the UCL local research ethics committee (PWB/ED/11–10–12b and 3450/002) and subjects gave informed written consent. To exclude subjects with poor compliance with task instructions subjects with more than 10 missed responses were removed from the analysis leaving 24 in the first group  and 27 in the second group .The two experimental groups only differed in the stimuli with which they were presented and what the outcome would be incentivising accurate choice. In the positive valence group, the pictures comprised diverse colour photographs gathered from various sources on the internet including landscapes and juvenile animals , Condition (Session 1 Choice/Session 2 Choice) and Action (Go/No Go) and a between subject factor of group . Choice refers to the choice the subjects made for Session 1 Choice pairs before the second rating session and for Session 2 Choice items after the second rating session. The interaction of Choice and Condition revealed whether the rating change differed when the choice preceded the second rating (rate-choose-rate) or did not exceed rating variation due to noise (rate-rate-choose). In other words the interaction term (Choice by Condition) represents the choice-induced preference change. We then investigated whether this choice-induced preference change was modulated by Action (Choice x Condition x Action), Valence  or the interaction of the two . To slice the interaction, the interaction term representing choice-induced value change was analysed with paired t-tests between the Chosen by Go/Chosen by No Go conditions in both valence groups. We also investigated whether the raw choice spread (main effect of Choice) was modulated by Action or Valence . In order to estimate the size of the choice-induced value change without control condition, the rate-choose-rate condition was analysed in a 2x2 within (Action and Choice) x2 between (group) design. See Additionally, our paradigm allowed us to test whether there is an intrinsic choice preference associated with Go or No Go. Pictures are paired with matched ratings and a picture is presented to be chosen by Go or No Go by virtue of a random allocation. Therefore the frequency of subject’s choosing to perform a Go or No Go response should be equal. If the ratio of Go and No Go choices deviates from 1 then this reflects the fact that subjects have an intrinsic preference to express choice by either action or inaction, which may cause deviation from optimal choice. This was assessed with a one sample t-test against 1 for the ratio of Go and No Go responses calculated for each subject. A two sample t-test was used to test whether this bias differs across the two valence groups. To determine whether one group actually rated the pictures lower, a two sample t-test was used to test the mean of all ratings from one group against the other. Trials in which subjects failed to respond were excluded from the analysis. Statistical analysis was carried out with Matlab R2012b and SPSS 19. As the size of the effect of action and valence on choice-induced value change is not known the sample size was determined beforehand based on existing studies on choice-induced preference change and action ,16,17,21The mean rating of the images given by subjects differed across the two groups and were higher in the positive valence group  = 4.98, p<.001; 95% CI. 64–1.5). Note that this difference occurred even though subjects were instructed to scale their ratings relatively to the stimuli in the set and use the whole range of the scale.2 = .86). The strong effect of choice is not surprising as it captures both an enhancement by choice, as well as alignment of choice with the drift of the noise contained in the initial measurements [2 = .45). This is illustrated by values being larger than 0 in 2 = .866).As displayed in urements . The cru2 = .11), which went in the opposite direction  = 1.39, p = .18, 95% CI-.59-.12). As visible in As displayed in 2 = .211), yet not the genuine choice induced-preference change  = 1.84, p = .18, partial η2 = .036). As visible in Additionally, stimulus valence (group) affected the aforementioned choice spread  = 13.11, p = .001, partial ηAnalysing frequency of choice itself showed that subjects were biased towards choices obtained by Go  is significantly larger than zero; t(50) = 2.83, p = .007; 95% CI. 039-.23) and notably this Go bias did not differ across the valence groups (t(49) = .41, p = .69; 95% CI-.15-.23).The current study measured choice spread in value change in two conditions; before and after a choice was made between stimuli. We found that choosing between two images before revaluation resulted in a larger relative increase in valuation for the chosen option than making the choice after the revaluation. This replicated findings showing that a significant portion of choice-induced preference change is not a methodological artefact  was higher when stimuli were of positive valence. This choice consistency is not merely revaluation caused by the choice but also includes conforming the choice to fluctuations in valuation. The fact that this effect was stronger for positive stimuli shows a choice bias that is sensitive to stimulus valence. Additionally, the magnitude of the choice-induced preference change was not modulated by action or valence per se, but by their interaction. This effect was driven by changes across all conditions, including the control condition. Schonberg at al.  showed tThe results also demonstrate how choice can be biased towards action. The fact that choices associated with action are preferred over choices associated with inaction suggests a possible source for suboptimal decision making. Given that this bias is independent of valence it may reflect a general action bias observed in instrumental learning under uncertainty ,30–32. TIn conclusion, our findings indicate that actions are not only the expression of a decision process but also impact on both revaluation and choice.S1 File(XLSX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Self-rated health (SRH) has strong predictive value for mortality in different contexts and cultures, but there is inconsistent evidence on ethnoracial disparities in SRH in Latin America, possibly due to the complexity surrounding ethnoracial self-classification.We used 370,539 Single Nucleotide Polymorphisms (SNPs) to examine the association between individual genomic proportions of African, European and Native American ancestry, and ethnoracial self-classification, with baseline and 10-year SRH trajectories in 1,311 community dwelling older Brazilians. We also examined whether genomic ancestry and ethnoracial self-classification affect the predictive value of SRH for subsequent mortality.European ancestry predominated among participants, followed by African and Native American ; the prevalence of Non-White (Mixed and Black) was 39.8%. Persons at higher levels of African and Native American genomic ancestry, and those self-identified as Non-White, were more likely to report poor health than other groups, even after controlling for socioeconomic conditions and an array of self-reported and objective physical health measures. Increased risks for mortality associated with worse SRH trajectories were strong and remarkably similar (hazard ratio ~3) across all genomic ancestry and ethno-racial groups.Our results demonstrated for the first time that higher levels of African and Native American genomic ancestry—and the inverse for European ancestry—were strongly correlated with worse SRH in a Latin American admixed population. Both genomic ancestry and ethnoracial self-classification did not modify the strong association between baseline SRH or SRH trajectory, and subsequent mortality. Self-rated health (SRH) is one of the most widely used epidemiologic variables because of its simplicity, its validity in different contexts and cultures, and its strong predictive power for future mortality. –4 While Brazil, the world’s fifth most populous nation , offers To our knowledge, no previous study has examined the association between genome-wide ancestry and SRH in an admixed Western population. We used 370,539 SNPs to examine the association between individual genomic proportions of African, European and Native American ancestry, as well as that of ethnoracial self-classification, with SRH in a population-based cohort of older adults in Brazil. Additionally, we examined whether genomic ancestry and ethnoracial self-classification affect the predictive value of SRH for subsequent mortality.The Bambui cohort study of aging was fully approved by the Comissão de Ética em Pesquisa  at Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. Written informed consent was obtained from all participants at baseline and at all follow-up interviews. Genotyping was approved by Brazil’s national research ethics committee, as part of the Epigen-Brazil protocol .The Bambui cohort study of aging is ongoing in Bambuí, a city of approximately 15,000 inhabitants in the state of Minas Gerais in Southeast Brazil.  The popuInformation on SRH was obtained during the household interview at baseline and at each subsequent wave. SRH was measured by asking the question, ‘‘In general, would you say your health is …,” and five response categories ranged from ‘‘excellent,” to “very poor”. Language plays a role in an individual´s choice on the SRH scale , 16 and Participants were genotyped by the Illumina facility, using the Omni 2.5M array . We performed the unsupervised tri-hybrid (k = 3) admixture analyses based on 370,539 SNPs shared between samples from the Epigen-Brazil study population, the HapMap Project, and the Human Genome Diversity Project (HGDP). , 19 As eThe Brazilian census uses ethnoracial self-classification with five groups: White, Brown/Mixed , Black, “Yellow” (Asian) and Indigenous (Native American).  At baselSRH is affected by several main factors  and variBased on its distribution, we categorized schooling into incomplete primary school (<4 years) and complete primary and higher (4 years and more). We categorized monthly household income per capita into equal or superior to the median value (median = 1.5 Brazilian minimum wage or USD 180.00 at baseline) and lower. Current smokers were persons who had smoked at least 100 cigarettes during their lifetime and who were still smokers. Leisure-time physical activity was defined as activity of any intensity for 20–30 minutes at least 3 times a week during the previous 3 months. Assessment of common mental disorders was based on participants’ answers to the 12-item version of the General Health Questionnaire. A score of 4/5 was used to define exposure status, as recommended for the study population. Trypanosoma cruzi was assessed by means of 1 hemagglutination assay  and 2 enzyme-linked immunosorbent assays  performed concurrently. Blood samples were collected after a 12-hour fast. Further details are described elsewhere. [Objective measures of health were those previously found to be associated with increased risk of mortality among cohort participants. –25 Systosewhere. To estimate the genetic ancestry of the study population, we applied the methodology implemented in the software Admixture  and usedT. cruzi infection (yes or no). Scores may range from - ∞ to + ∞. Higher scores indicated worse health.Principal component analysis  was usedWe used negative binomial regression (for dichotomous variables) and ordinary least squares regression (for continuous variables) to examine the age and sex (plus family structure) adjusted association between genomic ancestry and ethnoracial self-classification with schooling, household income, health behaviors, physical functioning  and the physical health score (a continuous variable) defined above.We used mixed-effects logistic regression to estimate odds ratios and 95% confidence intervals to model baseline and 10-year SRH trajectory, using poor SRH as the reference category.  To take We estimated adjusted hazard ratios and 95% confidence intervals for the association between baseline SRH and time dependent SRH, and subsequent mortality, using Cox proportional hazards models  after coThe multivariable models (described above) were build separately for each tertile of genomic ancestry and for ethnoracial self-classification. Separate analyses were performed because African, Native American and European ancestry proportions are complementary measures, whose sum is equal to 1. Further, ethnoracial self-classification showed a strong correlation with each of the genomic ancestries in exploratory analysis. Thus, we opted for not controlling self-classification for ancestry and vice-versa.Statistical analyses were conducted using Stata 13.1 statistical software. All p-values were 2-tailed .Of the 1,606 baseline cohort participants, complete data on all study variables were available for 1,311 persons, who were included in the current analysis. As can be seen in Overall, 18.5% of participants rated their health as poor or very poor. As shown in As shown in Over the 10-year follow-up, 9,721 measures of SRH were made (median = 7.4 per participant). With regards to SRH trajectories, During a mean follow-up period of 8.9 years, 522 participants died and 84 were lost, leading to 11,725 person-years of observation. Overall, the mortality rate was 65.6 per 1,000 person-years among persons who reported poor SRH at baseline compared to 43.3 per 1,000 among those who reported better health. As shown in 2 = 0.41; 95% CI 0.35, 0.49) compared with a single measure of SRH at the baseline . The corresponding values ranged from 0.38  to 0.45  across genome-wide ancestry and ethno-racial self identified groups for SRH trajectory, and from 0.25  to 0.34  for a single baseline measure of SRH.Overall, the variation in mortality  explained by repeated measures of SRH was about 40% higher , the importation of slaves from Africa, and colonization by Europeans and their descendants.  Our resuPrevious research has shown that adult Brazilians who self-report as Black and/or Mixed are more likely to report worse health outcomes. , 10, 35 th century. [A major explanation for socioeconomic disparities associated with African ancestry in Brazil is the cumulative effect of the lack of social policies designed to support the newly freed slaves and their descendants since the abolition of slavery in 1888 up to the last decades of the 20century. ,36 As a century. . They alcentury. , 38 The century.  Our resupardo”in Portuguese or mestizo in Spanish), especially if they live in urban areas. [As a consequence of sustained marginalization, indigenous populations through Latin America tend to live in more isolated communities and experience higher rates of mortality and morbidity than the general population. , 38 Indin areas. , 38 Thern areas. ), as weln areas. , 38 Our Self-rated health appears to be a universal predictor of mortality. This association has been reported in very different populations and countries –3, incluThis study has strengths and limitations. Strengths of the study include the community-based sample, standardized and systematic measurement of parameters at baseline, annual measures of SRH, continuous surveillance of mortality according to standardized criteria, and minimal loss of participants to follow-up. Another major strength is the use of genome-wide measures of ancestry. Genomic ancestry does not change over time, while self-classification is prone to misclassification, particularly in admixed populations, and can even fluctuate over time, due to changing social norms.  PreviousIn conclusion, our analyses provide new insights based on solid data on the association between genomic ancestry/ethnoracial and SRH, as well as on its prognostic value for mortality. Our results demonstrate for the first time that higher levels of African and Native American genomic ancestry–and the inverse for European ancestry–were strongly correlated with worse SRH in a Latin American admixed population. Further, genomic ancestry/ethnoracial classification did not modify the strong predictive power of SRH for mortality. Further research is needed to identify the set of mechanisms by which ethnoracial ancestry and the social constructions of ethnoracial ethnic identities may interact to cause the observed disparities in SRH.


As they assemble, retroviruses encapsidate both their genomic RNAs and several types of host RNA. Whereas limited amounts of messenger RNA (mRNA) are detectable within virion populations, the predominant classes of encapsidated host RNAs do not encode proteins, but instead include endogenous retroelements and several classes of non-coding RNA (ncRNA), some of which are packaged in significant molar excess to the viral genome. Surprisingly, although the most abundant host RNAs in retroviruses are also abundant in cells, unusual forms of these RNAs are packaged preferentially, suggesting that these RNAs are recruited early in their biogenesis: before associating with their cognate protein partners, and/or from transient or rare RNA populations. These RNAs’ packaging determinants differ from the viral genome’s, and several of the abundantly packaged host ncRNAs serve cells as the scaffolds of ribonucleoprotein particles. Because virion assembly is equally efficient whether or not genomic RNA is available, yet RNA appears critical to the structural integrity of retroviral particles, it seems possible that the selectively encapsidated host ncRNAs might play roles in assembly. Indeed, some host ncRNAs appear to act during replication, as some transfer RNA (tRNA) species may contribute to nuclear import of human immunodeficiency virus 1 (HIV-1) reverse transcription complexes, and other tRNA interactions with the viral Gag protein aid correct trafficking to plasma membrane assembly sites. However, despite high conservation of packaging for certain host RNAs, replication roles for most of these selectively encapsidated RNAs—if any—have remained elusive. In the late 1960s, before RNA tumor viruses were known to reverse-transcribe and at a time when experiments characterizing RNA required radioactive cell labeling and cumbersome sedimentation gradients, it was already clear that RNA tumor viruses contained multiple RNA species: a high molecular weight class that was presumed to be the viral genome, and a heterogeneous pool of smaller RNAs with sedimentation coefficients from 4–10 S and unknown functions ,3. HowevBy mass, 30% or more of the RNA in retroviruses consists of host RNAs ,5. A smaIn contrast, specific classes of small host non-coding RNAs—most of which are transcription products of RNA polymerase III (Pol III)—are abundant in retroviral particles. For example, one study that quantified relative levels of certain RNA species in virions and cells found that the host non-coding 7SL RNA is 250-fold more highly enriched in virions than actin mRNA . Based oSubsets of endogenous retroelement RNAs expressed in virion producer cells also are abundantly packaged. These include intact endogenous retroviral transcripts like the murine VL30 elements that are packaged by MLV, and fragments of non-long terminal repeats (LTR) retroelements such as the transcripts of divergent and truncated Long INterspersed Elements (LINEs) packaged by human immunodeficiency virus 1 (HIV-1) ,21. RecoWhat about microRNAs (miRNAs)? Deep sequencing studies of the shorter RNAs in HIV-1 particles report that some miRNAs are selectively packaged . HoweverThe roles of specific encapsidated tRNAs in reverse transcription were first described in the 1970s ,13,14, aThe spectra of packaged tRNAs differ from those observed in cells, with both the tRNA used to prime reverse transcription and certain non-primer tRNAs enriched over other tRNAs about 10-fold relative to their intracellular levels for HIV-1, and differing levels of enrichment among retroviral genera ,34. For Early analysis of RSV suggested that about a quarter of its total host RNA content consisted of a single 7S RNA species in 5- to 10-fold molar excess to the 70S presumptive genome . The obs7SL is the RNA component of signal recognition particles, the ribonucleoprotein (RNP) that ferries nascent signal peptides to the endoplasmic reticulum and mediates co-translational export of secretory proteins . AlthougIntriguingly, the Alu family of repetitive sequence elements that pepper our genomes are comprised of portions of the 7SL gene . Alu eleBesides 7SL and tRNAs, packaged host RNAs that are present in at least one copy per virion for one retrovirus or another include the 60 kDa Ro autoantigen-associated RNAs Y1 and Y3, which are each present at about four copies per MLV virion , the splThe trafficking and molecular associations of replication intermediates that form between the time of provirus transcription and the appearance of assembly intermediates at the plasma membrane remain poorly understood and are areas of active investigation. Multimerization of a small number of Gag molecules on genomic RNA is reported to precede gRNA arrival at the plasma membrane ,55. HoweLys3 detectable in cells is aminoacylated, this host RNA—as found within HIV-1 virions—is not aminoacylated [In a simplified view of HIV-1 and murine and avian retroviruses’ assembly, virions form when coalesced Gag molecules on the plasma membrane pinch off to form membrane-bound particles. Describing retroviral assembly in this way suggests the possibility that packaged host RNAs might represent a random sampling of the cytoplasm. However, it is clear from a comparison of virion and cytoplasmic RNA content that retroviruses are not merely packets of cytoplasmic contents . For exaacylated .+/+ cells, even when Y RNA expression is not directly affected [+/+ cells, despite the virtual absence of Y RNA within knockout producer cells [Another striking example of the non-randomness of host RNA packaging comes from studies of host Y RNA packaging by MLV . Y RNAs,affected ,61. Whener cells . This suThe determinants of ncRNA packaging differ from those of gRNA. For example, MLV particles formed by viral NC protein mutants with alterations in residues responsible for specific recruitment of gRNA retain wild-type levels of 7SL ,62. AddiThe Y RNA findings from Ro knockout cells described above suggested that Ro was not necessary for Y RNA encapsidation, and indeed Ro protein is not detectable in MLV particles . The infStudies of 7SL packaging support a model in which 7SL interacts with multiple regions in Gag. Eukaryotic 7SL RNA is comprised of two folded domains called the Alu and S domains, separated by a linker region . Becausecis-acting determinants of 7SL packaging by HIV-1 have been addressed by examining the packaging of exogenously expressed 7SL RNA mutants [The  mutants . These a mutants .A possible significance of even the substoichiometric ncRNAs found in virions is that their recruitment may be mementos of the trafficking pathways taken by viral subassemblies, and that their study may illuminate early pathways in retroviral assembly. Observations that the spectra of packaged RNAs change under conditions wherein host ncRNA traffic or RNP assembly pathways are altered may indicate that the RNAs are recruited into assembling viral RNPs in competition with their normal biogenesis and decay pathways ,9. As fuIn at least some reports, the spectra of host ncRNAs identifiable in extracellular vesicles released by uninfected cells are similar to those reported for retroviruses , leavingAt least some host ncRNAs are demonstrably packaged as nascent RNAs ,9. A higHowever, despite molecular signatures suggesting they are nuclear products, at least some of these RNA processing intermediates appear to be recruited for packaging from the cytoplasm. Specifically, by blocking nuclear re-entry of snRNA U2 precursors, which undergo nuclear export and subsequent re-import as part of their normal biogenesis, packaging of these very rare ncRNA species increased. Similarly, depletion of receptors required for nuclear export of certain ncRNAs decreased encapsidation of the corresponding ncRNAs ,9. AdditAs described above, host tRNAs function in the initiation of reverse transcription by serving as primers. Although no other essential replication roles have been demonstrated for the encapsidated host RNAs, the following discusses occasional and postulated roles.A key step in retroviral replication is the generation of a DNA copy of encapsidated viral RNA. Ordinarily, viral gRNA serves as the reverse transcription template, but exceptions have been observed. For example, reverse transcription products of encapsidated VL30 retroelements are readily detectable among MLV endogenous reaction products . RNAs thReverse transcription products of 7SL have been observed in endogenous reactions and patched into an experimental recombinant ,81. AlthSometimes portions of encapsidated host RNAs contribute to reverse transcription products via recombination. Retroviral recombination is exceptionally frequent, and results from template switching between RNA templates during reverse transcription . A classThere are marked differences in the sensitivity to ribonuclease (RNase) of immature and mature virions, and among retroviruses. For example, uncoated immature MLV particles are sensitive to RNase treatment but those of HIV-1 are not ,91,92. NRetroviral particles are released from cells with indistinguishable kinetics whether or not gRNA is available, but retroviral assembly requires RNA ,97. ThusGag undergoes a complex series of RNA binding events during assembly ,93. WithDespite lack of evidence for any encapsidated host RNAs functions, recent findings demonstrate that specific host ncRNA do play important replication roles prior to HIV-1 virion release . CrossliThe finding that most intracellular interactions between Gag and RNA map to MA was surprising. For retroviruses like HIV-1 and simple murine and avian retroviruses, Gag selectively multimerizes at the plasma membrane and not on intracellular membranes such as those of endosomes. This targeting maps to the highly basic region of the matrix domain of Gag and requires interactions with specific membrane phospholipids ,103,104.The assembly steps that precede membrane binding are among the least-well understood stages of retroviral replication, and likely not only include the stages where host RNAs are recruited, but may also include steps that are affected by encapsidated host RNAs. With the absence of visible intracellular Gag foci, arguably most evidence suggests Gag multimerization occurs primarily at the plasma membrane , with gRSome host tRNA species have also been implicated in facilitating an early step in HIV-1 replication. Specifically, in one study that sought to identify host factors that promoted nuclear entry of HIV-1 preintegrative reverse transcription complexes, (possibly aberrant) tRNAs were shown to be the active component of a HeLa cytosolic extract fraction . InteresThe endogenous retroelement RNAs that retroviruses package are undoubtedly not necessary for virus replication, since they are encapsidated at less than one copy per virion when produced by cells that express them, and infectious virions can be produced by cells that do not express these RNAs. However, some evidence suggests retroelement RNA sequences may be able to substitute for required assembly functions under certain circumstances. Specifically, in one study aimed at identifying host factors required for retroviral assembly that involved selecting mutagenized cell pools for clones resistant to murine leukemia virus infection, subsequent selections identified suppressor genes capable of restoring virus susceptibility ,112. SurThe discovery that members of the retroviral restriction factor apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3) family of cytidine deaminases—most notably APOBEC3G (A3G)—are RNA binding proteins ignited interest in deciphering which kind of RNA these intrinsic antiviral factors use to gain access to the virion. There is general agreement that the incorporation of A3G requires RNA binding and that this RNA is ordinarily recruited via interaction with Gag’s NC domain ,113,114.Another speculative role of encapsidated host RNAs is in host sensing of retroviral infections. If so, it is possible that the conserved packaging of host Pol III products does not reflect a selective advantage to the virus but instead is an evolved defense by the host. The 5’ ends of Pol III transcripts are not 7-methylguanylate capped, but rather triphosphorylated, and thus should be recognized by cytosolic RNA sensors such as retinoic acid inducible gene 1 (RIG-I), which activate signaling cascades that lead to the expression of cytokines such as interferon (IFN). IFN-α can suppress HIV replication in patients, and IFN-stimulated genes are upregulated in infection, including several antiviral restriction factors that limit HIV replication .Recent screens for the mediators of HIV-1 molecular sensing have principally implicated sensors of reverse transcription products (DNA) ,118,119.cis-acting determinants of 7SL packaging, it was observed that overexpressed S domain RNAs were packaged in addition to, rather than in competition with, endogenous 7SL [With little evidence of relevance to retroviral biology, is there any significance to the packaging of host ncRNAs? In the course of defining nous 7SL . When vanous 7SL . This suWhen retroviruses assemble, the RNA they incorporate is 30%–50% host derived by mass, likely representing roughly 100 host RNA molecules per virion. Some of these are tRNAs recruited directly or as a consequence of the machinery necessary for primer tRNA packaging. Others are endogenous retroelement RNAs that likely share packaging mechanisms with the virus genome. The most abundantly packaged host ncRNAs are unusual forms of RNA Pol III transcripts: these lack their normal protein binding partners and often are processing intermediates or show other signatures of recruitment from early phases of their biosynthesis. Despite their abundance and the high conservation of packaging of some host ncRNAs in a uniform stoichiometry relative to Gag, the replication roles these RNAs remain elusive.