Induction, suppression and requirement of RNA silencing pathways in virulent Agrobacterium tumefaciens infections

Regulation of gene expression through microRNAs (miRNAs) and antiviral defense through small interfering RNAs (siRNAs) are aspects of RNA silencing, a process originally discovered as an unintended consequence of plant transformation by disarmed Agrobacterium tumefaciens strains. Although RNA silencing protects cells against foreign genetic elements, its defensive role against virulent, tumor-inducing bacteria has remained unexplored. Here, we show that siRNAs corresponding to transferred-DNA oncogenes initially accumulate in virulent A. tumefaciens-infected tissues and that RNA interference-deficient plants are hypersusceptible to the pathogen. Successful infection relies on a potent antisilencing state established in tumors whereby siRNA synthesis is specifically inhibited. This inhibition has only modest side effects on the miRNA pathway, shown here to be essential for disease development. The similarities and specificities of the A. tumefaciens RNA silencing interaction are discussed and contrasted with the situation encountered with plant viruses.     

Induction of an interferon response by RNAi vectors in mammalian cells

DNA vectors that express short hairpin RNAs (shRNAs) from RNA polymerase III (Pol III) promoters are a promising new tool to reduce gene expression in mammalian cells. shRNAs are processed to small interfering RNAs (siRNAs) of 21 nucleotides (nt) that guide the cleavage of the cognate mRNA by the RNA-induced silencing complex. Although siRNAs are thought to be too short to induce interferon expression, we report here that a substantial number of shRNA vectors can trigger an interferon response.     

Efficient delivery of siRNA for inhibition of gene expression in postnatal mice

It has recently been shown that RNA interference can be induced in cultured mammalian cells by delivery of short interfering RNAs (siRNAs). Here we describe a method for efficient in vivo delivery of siRNAs to organs of postnatal mice and demonstrate effective and specific inhibition of transgene expression in a variety of organs.     

Intra- and intercellular RNA interference in Arabidopsis thaliana requires components of the microRNA and heterochromatic silencing pathways

In RNA interference (RNAi), double-stranded RNA (dsRNA) is processed into short interfering RNA (siRNA) to mediate sequence-specific gene knockdown. The genetics of plant RNAi is not understood, nor are the bases for its spreading between cells. Here, we unravel the requirements for biogenesis and action of siRNAs directing RNAi in Arabidopsis thaliana and show how alternative routes redundantly mediate this process under extreme dsRNA dosages. We found that SMD1 and SMD2, required for intercellular but not intracellular RNAi, are allelic to RDR2 and NRPD1a, respectively, previously implicated in siRNA-directed heterochromatin formation through the action of DCL3 and AGO4. However, neither DCL3 nor AGO4 is required for non-cell autonomous RNAi, uncovering a new pathway for RNAi spreading or detection in recipient cells. Finally, we show that the genetics of RNAi is distinct from that of antiviral silencing and propose that this experimental silencing pathway has a direct endogenous plant counterpart.     

Homozygous L-SIGN (CLEC4M) plays a protective role in SARS coronavirus infection

Severe acute respiratory syndrome (SARS) is caused by infection of a previously undescribed coronavirus (CoV). L-SIGN, encoded by CLEC4M (also known as CD209L), is a SARS-CoV binding receptor that has polymorphism in its extracellular neck region encoded by the tandem repeat domain in exon 4. Our genetic risk association study shows that individuals homozygous for CLEC4M tandem repeats are less susceptible to SARS infection. L-SIGN is expressed in both non-SARS and SARS-CoV-infected lung. Compared with cells heterozygous for L-SIGN, cells homozygous for L-SIGN show higher binding capacity for SARS-CoV, higher proteasome-dependent viral degradation and a lower capacity for trans infection. Thus, homozygosity for L-SIGN plays a protective role during SARS infection.     

CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus

An expansion of a CTG repeat at the DM1 locus causes myotonic dystrophy (DM) by altering the expression of the two adjacent genes, DMPK and SIX5, and through a toxic effect of the repeat-containing RNA. Here we identify two CTCF-binding sites that flank the CTG repeat and form an insulator element between DMPK and SIX5. Methylation of these sites prevents binding of CTCF, indicating that the DM1 locus methylation in congenital DM would disrupt insulator function. Furthermore, CTCF-binding sites are associated with CTG/CAG repeats at several other loci. We suggest a general role for CTG/CAG repeats as components of insulator elements at multiple sites in the human genome.     

Restriction enzyme-generated siRNA (REGS) vectors and libraries

Small interfering RNA (siRNA) technology facilitates the study of loss of gene function in mammalian cells and animal models, but generating multiple siRNA vectors using oligonucleotides is slow, inefficient and costly. Here we describe a new, enzyme-mediated method for generating numerous functional siRNA constructs from any gene of interest or pool of genes. To test our restriction enzyme-generated siRNA (REGS) system, we silenced a transgene and two endogenous genes and obtained the predicted phenotypes. REGS generated on average 34 unique siRNAs per kilobase of sequence. REGS enabled us to create enzymatically a complex siRNA library (>4 x 10(5) clones) from double-stranded cDNA encompassing known and unknown genes with 96% of the clones containing inserts of the appropriate size.     

Charcot-Marie-Tooth type 1A duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomer unit.

We have constructed a 3.1 megabase (Mb) physical map of chromosome 17p11.2-p12, which contains a submicroscopic duplication in patients with Charcot-Marie-Tooth disease type 1A (CMT1A). We find that the CMT1A duplication is a tandem repeat of 1.5 Mb of DNA. A YAC contig encompassing the CMT1A duplication and spanning the endpoints was also developed. Several low copy repeats in 17p11.2-p12 were identified including the large (> 17 kb) CMT1A-REP unit which may be part of a mosaic repeat. CMT1A-REP flanks the 1.5 Mb CMT1A monomer unit on normal chromosome 17 and is present in an additional copy on the CMT1A duplicated chromosome. We propose that the de novo CMT1A duplication arises from unequal crossing over due to misalignment at these CMT1A-REP repeat sequences during meiosis.     

Heterogeneous mutation processes in human microsatellite DNA sequences

Although microsatellite polymorphisms are one of the most commonly used tools in genetic analyses, it remains to be understood how microsatellite DNA has evolved as a ubiquitous and highly abundant class of repetitive sequences in eukaryotic genomes. On the basis of analyses of spontaneous human microsatellite mutations of germline origin, I show here that different mutation biases underlie the evolution of microsatellite repeats. The within-locus mutation rate increases with allele length, but is not affected by the size difference between an individual's two alleles (allele span). Within loci, long alleles tend to mutate to shorter lengths, thereby acting to prevent infinite growth. Expansions are more common than contractions among dinucleotide repeats, whereas no such trend is evident among tetranucleotide repeats. This observation is consistent with the longer repeat lengths and higher frequency of di- compared with tetranucleotide repeats. An excess of paternally transmitted mutations (male-to-female ratio of 4.9) supports a male-biased mutation rate in the human genome.     

Intragenic tandem repeats generate functional variability

Tandemly repeated DNA sequences are highly dynamic components of genomes. Most repeats are in intergenic regions, but some are in coding sequences or pseudogenes. In humans, expansion of intragenic triplet repeats is associated with various diseases, including Huntington chorea and fragile X syndrome. The persistence of intragenic repeats in genomes suggests that there is a compensating benefit. Here we show that in the genome of Saccharomyces cerevisiae, most genes containing intragenic repeats encode cell-wall proteins. The repeats trigger frequent recombination events in the gene or between the gene and a pseudogene, causing expansion and contraction in the gene size. This size variation creates quantitative alterations in phenotypes (e.g., adhesion, flocculation or biofilm formation). We propose that variation in intragenic repeat number provides the functional diversity of cell surface antigens that, in fungi and other pathogens, allows rapid adaptation to the environment and elusion of the host immune system.