A viral suppressor of gene silencing in plants

Gene silencing is an important but little understood regulatory mechanism in plants. Here we report that a viral sequence, initially identified as a mediator of synergistic viral disease, acts to suppress the establishment of both transgene-induced and virus-induced posttranscriptional gene silencing. The viral suppressor of silencing comprises the 5′-proximal region of the tobacco etch potyviral genomic RNA encoding P1, helper component-proteinase (HC-Pro) and a small part of P3, and is termed the P1/HC-Pro sequence. A reversal of silencing assay was used to assess the effect of the P1/HC-Pro sequence on transgenic tobacco plants (line T4) that are posttranscriptionally silenced for the uidA reporter gene. Silencing was lifted in offspring of T4 crosses with four independent transgenic lines expressing P1/HC-Pro, but not in offspring of control crosses. Viral vectors were used to assess the effect of P1/HC-Pro expression on virus-induced gene silencing (VIGS). The ability of a potato virus X vector expressing green fluorescent protein to induce silencing of a green fluorescent protein transgene was eliminated or greatly reduced when P1/HC-Pro was expressed from the same vector or from coinfecting potato virus X vectors. Expression of the HC-Pro coding sequence alone was sufficient to suppress virus-induced gene silencing, and the HC-Pro protein product was required for the suppression. This discovery points to the role of gene silencing as a natural antiviral defense system in plants and offers different approaches to elucidate the molecular basis of gene silencing.

Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway

Certain plant viruses encode suppressors of posttranscriptional gene silencing (PTGS), an adaptive antiviral defense response that limits virus replication and spread. The tobacco etch potyvirus protein, helper component-proteinase (HC-Pro), suppresses PTGS of silenced transgenes. The effect of HC-Pro on different steps of the silencing pathway was analyzed by using both transient Agrobacterium tumefaciens-based delivery and transgenic systems. HC-Pro inactivated PTGS in plants containing a preexisting silenced β-glucuronidase (GUS) transgene. PTGS in this system was associated with both small RNA molecules (21–26 nt) corresponding to the 3′ proximal region of the transcribed GUS sequence and cytosine methylation of specific sites near the 3′ end of the GUS transgene. Introduction of HC-Pro into these plants resulted in loss of PTGS, loss of small RNAs, and partial loss of methylation. These results suggest that HC-Pro targets a PTGS maintenance (as opposed to an initiation or signaling) component at a point that affects accumulation of small RNAs and methylation of genomic DNA.

Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants

In transgenic and nontransgenic plants, viruses are both initiators and targets of a defense mechanism that is similar to posttranscriptional gene silencing (PTGS). Recently, it was found that potyviruses and cucumoviruses encode pathogenicity determinants that suppress this defense mechanism. Here, we test diverse virus types for the ability to suppress PTGS. Nicotiana benthamiana exhibiting PTGS of a green fluorescent protein transgene were infected with a range of unrelated viruses and various potato virus X vectors producing viral pathogenicity factors. Upon infection, suppression of PTGS was assessed in planta through reactivation of green fluorescence and confirmed by molecular analysis. These experiments led to the identification of three suppressors of PTGS and showed that suppression of PTGS is widely used as a counter-defense strategy by DNA and RNA viruses. However, the spatial pattern and degree of suppression varied extensively between viruses. At one extreme, there are viruses that suppress in all tissues of all infected leaves, whereas others are able to suppress only in the veins of new emerging leaves. This variation existed even between closely related members of the potexvirus group. Collectively, these results suggest that virus-encoded suppressors of gene silencing have distinct modes of action, are targeted against distinct components of the host gene-silencing machinery, and that there is dynamic evolution of the host and viral components associated with the gene-silencing mechanism.

A pyrimidine-rich exonic splicing suppressor binds multiple RNA splicing factors and inhibits spliceosome assembly

The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately downstream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3′ splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3′ splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind to the ESS showed that the U-rich 5′ region binds U2AF65 and polypyrimidine tract binding protein, the C-rich central part binds 35- and 54–55-kDa serine/arginine-rich (SR) proteins, and the AG-rich 3′ end binds alternative splicing factor/splicing factor 2. Mutational and functional studies indicated that the most critical region of the ESS maps to the central C-rich core (GGCUCCCCC). This core sequence, along with additional nonspecific downstream nucleotides, is sufficient for partial suppression of spliceosome assembly and splicing of BPV-1 pre-mRNAs. The inhibition of splicing by the ESS can be partially relieved by excess purified HeLa SR proteins, suggesting that the ESS suppresses pre-mRNA splicing by interfering with normal bridging and recruitment activities of SR proteins.

Gene dosage and stochastic effects determine the severity and direction of uniparental ribosomal RNA gene silencing (nucleolar dominance) in Arabidopsis allopolyploids

Nucleolar dominance is an epigenetic phenomenon in which one parental set of ribosomal RNA (rRNA) genes is silenced in an interspecific hybrid. In natural Arabidopsis suecica, an allotetraploid (amphidiploid) hybrid of Arabidopsis thaliana and Cardaminopsis arenosa, the A. thaliana rRNA genes are repressed. Interestingly, A. thaliana rRNA gene silencing is variable in synthetic Arabidopsis suecica F1 hybrids. Two generations are needed for A. thaliana rRNA genes to be silenced in all lines, revealing a species-biased direction but stochastic onset to nucleolar dominance. Backcrossing synthetic A. suecica to tetraploid A. thaliana yielded progeny with active A. thaliana rRNA genes and, in some cases, silenced C. arenosa rRNA genes, showing that the direction of dominance can be switched. The hypothesis that naturally dominant rRNA genes have a superior binding affinity for a limiting transcription factor is inconsistent with dominance switching. Inactivation of a species-specific transcription factor is argued against by showing that A. thaliana and C. arenosa rRNA genes can be expressed transiently in the other species. Transfected A. thaliana genes are also active in A. suecica protoplasts in which chromosomal A. thaliana genes are repressed. Collectively, these data suggest that nucleolar dominance is a chromosomal phenomenon that results in coordinate or cooperative silencing of rRNA genes.

Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA

Many examples of extreme virus resistance and posttranscriptional gene silencing of endogenous or reporter genes have been described in transgenic plants containing sense or antisense transgenes. In these cases of either cosuppression or antisense suppression, there appears to be induction of a surveillance system within the plant that specifically degrades both the transgene and target RNAs. We show that transforming plants with virus or reporter gene constructs that produce RNAs capable of duplex formation confer virus immunity or gene silencing on the plants. This was accomplished by using transcripts from one sense gene and one antisense gene colocated in the plant genome, a single transcript that has self-complementarity, or sense and antisense transcripts from genes brought together by crossing. A model is presented that is consistent with our data and those of other workers, describing the processes of induction and execution of posttranscriptional gene silencing.

RNA binding by the Wilms tumor suppressor zinc finger proteins.

The Wilms tumor suppressor gene WT1 is implicated in the ontogeny of genito-urinary abnormalities, including Denys-Drash syndrome and Wilms tumor of the kidney. WT1 encodes Kruppel-type zinc finger proteins that can regulate the expression of several growth-related genes, apparently by binding to specific DNA sites located within 5' untranslated leader regions as well as 5' promoter sequences. Both WT1 and a closely related early growth response factor, EGR1, can bind the same DNA sequences from the mouse gene encoding insulin-like growth factor 2 (Igf-2). We report that WT1, but not EGR1, can bind specific Igf-2 exonic RNA sequences, and that the zinc fingers are required for this interaction. WT1 zinc finger 1, which is not represented in EGR1, plays a more significant role in RNA binding than zinc finger 4, which does have a counterpart in EGR1. Furthermore, the normal subnuclear localization of WT1 proteins is shown to be RNase, but not DNase, sensitive. Therefore, WT1 might, like the Kruppel-type zinc finger protein TFIIIA, regulate gene expression by both transcriptional and posttranscriptional mechanisms.

Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas.

E-Cadherin, a cell adhesion molecule, which plays a key role in maintaining the epithelial phenotype, is regarded as an invasion-suppressor gene in light of accumulating evidence from in vitro experiments and clinical observations. In an attempt to clarify the mechanism responsible for inactivation of this gene in carcinomas, we investigated the methylation state around the promoter region by digestion of DNA with the methylation-sensitive restriction enzyme Hpa II, as CpG methylation of the promoter has been postulated to be a mechanism of transcriptional inactivation of some genes. We found that E-cadherin expression-negative carcinoma cell lines were accompanied by the hypermethylation state, whereas E-cadherin-positive cell lines were not. Furthermore, treatment of E-cadherin-negative carcinoma cells with the demethylating agent 5-azacytidine resulted in reexpression of the gene and reversion of scattered spindle-shaped cells to cells with epithelial morphology. These results suggest that hypermethylation around the promoter may be a mechanism of E-cadherin inactivation in human carcinomas and that treatment of E-cadherin-inactivated cells with a demethylating agent may cause gene expression reversion leading to epithelial morphogenesis with acquisition of the homophilic cell-cell adhesive property.

DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription

DsrA RNA regulates both transcription, by overcoming transcriptional silencing by the nucleoid-associated H-NS protein, and translation, by promoting efficient translation of the stress σ factor, RpoS. These two activities of DsrA can be separated by mutation: the first of three stem-loops of the 85 nucleotide RNA is necessary for RpoS translation but not for anti-H-NS action, while the second stem-loop is essential for antisilencing and less critical for RpoS translation. The third stem-loop, which behaves as a transcription terminator, can be substituted by the trp transcription terminator without loss of either DsrA function. The sequence of the first stem-loop of DsrA is complementary with the upstream leader portion of rpoS messenger RNA, suggesting that pairing of DsrA with the rpoS message might be important for translational regulation. Mutations in the Rpos leader and compensating mutations in DsrA confirm that this predicted pairing is necessary for DsrA stimulation of RpoS translation. We propose that DsrA pairing stimulates RpoS translation by acting as an anti-antisense RNA, freeing the translation initiation region from the cis-acting antisense RNA and allowing increased translation.

Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa

We report the isolation of 15 Neurospora crassa mutants defective in “quelling” or transgene-induced gene silencing. These quelling-defective mutants (qde) belonging to three complementation groups have provided insights into the mechanism of posttranscriptional gene silencing in N. crassa. The recessive nature of the qde mutations indicates that the encoded gene products act in trans. We show that when qde genes are mutated in a transgenic-induced silenced strain containing many copies of the transgene, the expression of the endogenous gene is maintained despite the presence of transgene sense RNA, the molecule proposed to trigger quelling. Moreover, the qde mutants failed to show quelling when tested with another gene, suggesting that they may be universally defective in transgene-induced gene silencing. As such, qde genes may be involved in sensing aberrant sense RNA and/or targeting/degrading the native mRNA. The qde mutations may be used to isolate the genes encoding the first components of the quelling mechanism. Moreover, these quelling mutants may be important in applied and basic research for the creation of strains able to overexpress a transgene.

Autoregulation at the level of mRNA 3′ end formation of the suppressor of forked gene of Drosophila melanogaster is conserved in Drosophila virilis

The Drosophila melanogaster Suppressor of forked [Su(f)] protein shares homology with the yeast RNA14 protein and the 77-kDa subunit of human cleavage stimulation factor, which are proteins involved in mRNA 3′ end formation. This suggests a role for Su(f) in mRNA 3′ end formation in Drosophila. The su(f) gene produces three transcripts; two of them are polyadenylated at the end of the transcription unit, and one is a truncated transcript, polyadenylated in intron 4. Using temperature-sensitive su(f) mutants, we show that accumulation of the truncated transcript requires wild-type Su(f) protein. This suggests that the Su(f) protein autoregulates negatively its accumulation by stimulating 3′ end formation of the truncated su(f) RNA. Cloning of su(f) from Drosophila virilis and analysis of its RNA profile suggest that su(f) autoregulation is conserved in this species. Sequence comparison between su(f) from both species allows us to point out three conserved regions in intron 4 downstream of the truncated RNA poly(A) site. These conserved regions include the GU-rich downstream sequence involved in poly(A) site definition. Using transgenes truncated within intron 4, we show that sequence up to the conserved GU-rich domain is sufficient for production of the truncated RNA and for regulation of this production by su(f). Our results indicate a role of su(f) in the regulation of poly(A) site utilization and an important role of the GU-rich sequence for this regulation to occur.

A CBF5 mutation that disrupts nucleolar localization of early tRNA biosynthesis in yeast also suppresses tRNA gene-mediated transcriptional silencing

In the budding yeast, Saccharomyces cerevisiae, actively transcribed tRNA genes can negatively regulate adjacent RNA polymerase II (pol II)-transcribed promoters. This tRNA gene-mediated silencing is independent of the orientation of the tRNA gene and does not require direct, steric interference with the binding of either upstream pol II factors or the pol II holoenzyme. A mutant was isolated in which this form of silencing is suppressed. The responsible point mutation affects expression of the Cbf5 protein, a small nucleolar ribonucleoprotein protein required for correct processing of rRNA. Because some early steps in the S. cerevisiae pre-tRNA biosynthetic pathway are nucleolar, we examined whether the CBF5 mutation might affect this localization. Nucleoli were slightly fragmented, and the pre-tRNAs went from their normal, mostly nucleolar location to being dispersed in the nucleoplasm. A possible mechanism for tRNA gene-mediated silencing is suggested in which subnuclear localization of tRNA genes antagonizes transcription of nearby genes by pol II.

Posttranscriptional Gene Silencing in Transgenic Sugarcane. Dissection of Homology-Dependent Virus Resistance in a Monocot That Has a Complex Polyploid Genome1

RNA-mediated, posttranscriptional gene silencing has been determined as the molecular mechanism underlying transgenic virus resistance in many plant virus-dicot host plant systems. In this paper we show that transgenic virus resistance in sugarcane (Saccharum spp. hybrid) is based on posttranscriptional gene silencing. The resistance is derived from an untranslatable form of the sorghum mosaic potyvirus strain SCH coat protein (CP) gene. Transgenic sugarcane plants challenged with sorghum mosaic potyvirus strain SCH had phenotypes that ranged from fully susceptible to completely resistant, and a recovery phenotype was also observed. Clones derived from the same transformation event or obtained after vegetative propagation could display different levels of virus resistance, suggesting the involvement of a quantitative component in the resistance response. Most resistant plants displayed low or undetectable steady-state CP transgene mRNA levels, although nuclear transcription rates were high. Increased DNA methylation was observed in the transcribed region of the CP transgenes in most of these plants. Collectively, these characteristics indicate that an RNA-mediated, homology-dependent mechanism is at the base of the virus resistance. This work extends posttranscriptional gene silencing and homology-dependent virus resistance, so far observed only in dicots, to an agronomically important, polyploid monocot.

Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs

Antisense-mediated gene silencing (ASGS) and posttranscriptional gene silencing (PTGS) with sense transgenes markedly reduce the steady-state mRNA levels of endogenous genes similar in transcribed sequence. RNase protection assays established that silencing in tobacco plants transformed with plant-defense-related class I sense and antisense chitinase (CHN) transgenes is at the posttranscriptional level. Infection of tobacco plants with cucumber mosaic virus strain FN and a necrotizing strain of potato virus Y, but not with potato virus X, effectively suppressed PTGS and ASGS of both the transgenes and homologous endogenes. This suggests that ASGS and PTGS share components associated with initiation and maintenance of the silent state. Small, ca. 25-nt RNAs (smRNA) of both polarities were associated with PTGS and ASGS in CHN transformants as reported for PTGS in other transgenic plants and for RNA interference in Drosophila. Similar results were obtained with an antisense class I β-1,3-glucanase transformant showing that viral suppression and smRNAs are a more general feature of ASGS. Several current models hold that diverse signals lead to production of double-stranded RNAs, which are processed to smRNAs that then trigger PTGS. Our results provide direct evidence for mechanistic links between ASGS and PTGS and suggest that ASGS could join a common PTGS pathway at the double-stranded RNA step.

The candidate tumor suppressor gene, RASSF1A, from human chromosome 3p21.3 is involved in kidney tumorigenesis

Clear cell-type renal cell carcinomas (clear RCC) are characterized almost universally by loss of heterozygosity on chromosome 3p, which usually involves any combination of three regions: 3p25-p26 (harboring the VHL gene), 3p12-p14.2 (containing the FHIT gene), and 3p21-p22, implying inactivation of the resident tumor-suppressor genes (TSGs). For the 3p21-p22 region, the affected TSGs remain, at present, unknown. Recently, the RAS association family 1 gene (isoform RASSF1A), located at 3p21.3, has been identified as a candidate lung and breast TSG. In this report, we demonstrate aberrant silencing by hypermethylation of RASSF1A in both VHL-caused clear RCC tumors and clear RCC without VHL inactivation. We found hypermethylation of RASSF1A's GC-rich putative promoter region in most of analyzed samples, including 39 of 43 primary tumors (91%). The promoter was methylated partially or completely in all 18 RCC cell lines analyzed. Methylation of the GC-rich putative RASSF1A promoter region and loss of transcription of the corresponding mRNA were related causally. RASSF1A expression was reactivated after treatment with 5-aza-2′-deoxycytidine. Forced expression of RASSF1A transcripts in KRC/Y, a renal carcinoma cell line containing a normal and expressed VHL gene, suppressed growth on plastic dishes and anchorage-independent colony formation in soft agar. Mutant RASSF1A had reduced growth suppression activity significantly. These data suggest that RASSF1A is the candidate renal TSG gene for the 3p21.3 region.