The Mimivirus L375 Nudix enzyme hydrolyzes the 5' mRNA cap.

The giant Mimivirus is a member of the nucleocytoplasmic large DNA viruses (NCLDV), a group of diverse viruses that contain double-stranded DNA (dsDNA) genomes that replicate primarily in eukaryotic hosts. Two members of the NCLDV, Vaccinia Virus (VACV) and African Swine Fever Virus (ASFV), both synthesize Nudix enzymes that have been shown to decap mRNA, a process thought to accelerate viral and host mRNA turnover and promote the shutoff of host protein synthesis. Mimivirus encodes two Nudix enzymes in its genome, denoted as L375 and L534. Importantly, L375 exhibits sequence similarity to ASFV-DP and eukaryotic Dcp2, two Nudix enzymes shown to possess mRNA decapping activity. In this work, we demonstrate that recombinant Mimivirus L375 cleaves the 5' m7GpppN mRNA cap, releasing m7GDP as a product. L375 did not significantly cleave mRNAs containing an unmethylated 5'GpppN cap, indicating that this enzyme specifically hydrolyzes methylated-capped transcripts. A point mutation in the L375 Nudix motif completely eliminated cap hydrolysis, showing that decapping activity is dependent on this motif. Addition of uncapped RNA significantly reduced L375 decapping activity, suggesting that L375 may recognize its substrate through interaction with the RNA body.

A central support system can facilitate implementation and sustainability of a Classroom-based Undergraduate Research Experience (CURE) in Genomics.

In their 2012 report, the President's Council of Advisors on Science and Technology advocated "replacing standard science laboratory courses with discovery-based research courses"-a challenging proposition that presents practical and pedagogical difficulties. In this paper, we describe our collective experiences working with the Genomics Education Partnership, a nationwide faculty consortium that aims to provide undergraduates with a research experience in genomics through a scheduled course (a classroom-based undergraduate research experience, or CURE). We examine the common barriers encountered in implementing a CURE, program elements of most value to faculty, ways in which a shared core support system can help, and the incentives for and rewards of establishing a CURE on our diverse campuses. While some of the barriers and rewards are specific to a research project utilizing a genomics approach, other lessons learned should be broadly applicable. We find that a central system that supports a shared investigation can mitigate some shortfalls in campus infrastructure (such as time for new curriculum development, availability of IT services) and provides collegial support for change. Our findings should be useful for designing similar supportive programs to facilitate change in the way we teach science for undergraduates.

The genomics education partnership: successful integration of research into laboratory classes at a diverse group of undergraduate institutions.

Genomics is not only essential for students to understand biology but also provides unprecedented opportunities for undergraduate research. The goal of the Genomics Education Partnership (GEP), a collaboration between a growing number of colleges and universities around the country and the Department of Biology and Genome Center of Washington University in St. Louis, is to provide such research opportunities. Using a versatile curriculum that has been adapted to many different class settings, GEP undergraduates undertake projects to bring draft-quality genomic sequence up to high quality and/or participate in the annotation of these sequences. GEP undergraduates have improved more than 2 million bases of draft genomic sequence from several species of Drosophila and have produced hundreds of gene models using evidence-based manual annotation. Students appreciate their ability to make a contribution to ongoing research, and report increased independence and a more active learning approach after participation in GEP projects. They show knowledge gains on pre- and postcourse quizzes about genes and genomes and in bioinformatic analysis. Participating faculty also report professional gains, increased access to genomics-related technology, and an overall positive experience. We have found that using a genomics research project as the core of a laboratory course is rewarding for both faculty and students.

The African swine fever virus g5R protein possesses mRNA decapping activity.

The African Swine Fever Virus (ASFV) encodes a single Nudix enzyme in its genome, termed the g5R protein (g5Rp). Nudix phosphohydrolases cleave a variety of substrates, such as nucleotides and diphosphoinositol polyphosphates. Previously, ASFV g5Rp was shown to hydrolyze diphosphoinositol polyphosphates and GTP, but was unable to cleave methylated mRNA cap analogues. In vaccinia virus (VACV), a distant relative of ASFV, the D9 and D10 Nudix enzymes were shown to cleave the mRNA cap, but only when the cap was attached to an RNA body. Here, we show that recombinant ASFV g5Rp hydrolyzes the mRNA cap when tethered to an RNA moiety, liberating m(7)GDP as a product. Mutations in the Nudix motif abolished mRNA decapping activity, confirming that g5Rp was responsible for cap cleavage. The decapping activity of g5Rp was potently inhibited by excess uncapped RNA but not by methylated cap analogues, suggesting that substrate recognition occurs by RNA binding.

Characterization of a second vaccinia virus mRNA-decapping enzyme conserved in poxviruses.

Vaccinia virus (VACV) encodes enzymes that cap the 5' end of viral mRNAs, which enhances their stability and translation. Nevertheless, recent studies demonstrated that the VACV D10 protein (VACV-WR_115) decaps mRNA, an enzymatic activity not previously shown to be encoded by a virus. The decapping activity of D10 is dependent on a Nudix hydrolase motif that is also present in the VACV D9 protein (VACV-WR_114), which shares 25% sequence identity with D10. Here, we showed that a purified recombinant VACV D9 fusion protein also decaps mRNA and that this activity was abolished by point mutations in the Nudix hydrolase motif. Decapping was specific for a methylated cap attached to RNA and resulted in the liberation of m7GDP. D9 differed from D10 in requiring a longer capped RNA substrate for optimal activity, having greater sensitivity to inhibition by uncapped RNA, and having lower sensitivity to inhibition by nucleotide cap analogs unattached to RNA. Since D9 is expressed early in infection and D10 late, we suggest that the two proteins enhance mRNA turnover and manipulate gene expression in a complementary and overlapping manner.

Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression.

Previous studies indicated that the vaccinia virus D10 protein, which is conserved in all sequenced poxviruses, participates in the rapid turnover of host and viral mRNAs. D10 contains a motif present in the family of Nudix/MutT enzymes, a subset of which has been shown to enhance mRNA turnover in eukaryotic cells through cleavage of the 5' cap (m7GpppNm-). Here, we demonstrate that a purified recombinant D10 fusion protein possesses an intrinsic activity that liberates m7GDP from capped RNA substrates. Furthermore, point mutations in the Nudix/MutT motif abolished decapping activity. D10 has a strong affinity for capped RNA substrates (Km approximately 3 nm). RNAs of 24-309 nt were decapped to comparable extents, whereas the cap of a 12-nt RNA was uncleaved. At large molar ratios relative to capped RNA substrate, competitor m7GpppG, m7GTP, or m7GDP inhibited decapping, whereas even higher concentrations of unmethylated analogs did not. High concentrations of uncapped RNA were also inhibitory, suggesting that D10 recognizes its substrate through interaction with both cap and RNA moieties. Thus far, poxviruses represent the only virus family shown to encode a Nudix hydrolase-decapping enzyme. Although it may seem self-destructive for a virus to encode a decapping and a capping enzyme, accelerated mRNA turnover helps eliminate competing host mRNAs and allows stage-specific synthesis of viral proteins.

Characterization of a vaccinia virus mutant with a deletion of the D10R gene encoding a putative negative regulator of gene expression.

The D9 and D10 proteins of vaccinia virus are 25% identical to each other, contain a mutT motif characteristic of nudix hydrolases, and are conserved in all sequenced poxviruses. Previous studies indicated that overexpression of D10 and, to a lesser extent, D9 decreased the levels of capped mRNAs and their translation products. Here, we further characterized the D10 protein and showed that only trace amounts are associated with purified virions and that it is expressed exclusively at late times after vaccinia virus infection. A viable deletion mutant (vdeltaD10) produced smaller plaques and lower virus yields than either wild-type virus or a D9R deletion mutant (vdeltaD9). Purified vdeltaD10 virions appeared normal by microscopic examination and biochemical analysis but produced 6- to 10-fold-fewer plaques at the same concentration as wild-type or vdeltaD9 virions. When 4 PFU per cell of wild-type or vdeltaD9 virions or equal numbers of vdeltaD10 virions were used for inoculation, nearly all cells were infected in each case, but viral early and late transcription was initiated more slowly in vdeltaD10-infected cells than in the others. However, viral early transcripts accumulated to higher levels in vdeltaD10-infected cells than in cells infected with the wild type or vdeltaD9. In addition, viral early and late mRNAs and cellular actin mRNA persisted longer in vdeltaD10-infected cells than in others. Furthermore, analysis of pulse-labeled proteins indicated prolonged synthesis of cellular and viral early proteins. These results are consistent with a role for D10 in regulating RNA levels in poxvirus-infected cells.

Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi.

RNA interference (RNAi) is a broadly used reverse genetics method in C. elegans. Unfortunately, RNAi does not inhibit all genes. We show that loss of function of a putative RNA-directed RNA polymerase (RdRP) of C. elegans, RRF-3, results in a substantial enhancement of sensitivity to RNAi in diverse tissues. This is particularly striking in the nervous system; neurons that are generally refractory to RNAi in a wild-type genetic background can respond effectively to interference in an rrf-3 mutant background. These data provide the first indication of physiological negative modulation of the RNAi response and implicate an RdRP-related factor in this effect. The rrf-3 strain can be useful to study genes that, in wild-type, do not show a phenotype after RNAi, and it is probably the strain of choice for genome-wide RNAi screens.