Topically delivered 22 nt siRNAs enhance RNAi silencing of endogenous genes in two species.

MAIN CONCLUSION: 22 nt siRNAs applied to leaves induce production of transitive sRNAs for targeted genes and can enhance local silencing. Systemic silencing was only observed for a GFP transgene. RNA interference (RNAi) is a gene silencing mechanism important in regulating gene expression during plant development, response to the environment and defense. Better understanding of the molecular mechanisms of this pathway may lead to future strategies to improve crop traits of value. An abrasion method to deliver siRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves of N. benthamiana, but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for three endogenous genes: magnesium cheletase subunit I (CHL-I), magnesium cheletase subunit H (CHL-H), and GENOMES UNCOUPLED4 (GUN4). However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also produced transitive siRNAs in response to 22 nt siRNAs. 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for three of the genes. These special properties of 22 nt siRNAs were also observed for the CHL-H gene in A. cruentus. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.

Metastable differentially methylated regions within Arabidopsis inbred populations are associated with modified expression of non-coding transcripts.

Individual plants within a population may vary at both genetic and epigenetic levels. The rate of genetic divergence and its underlying mechanisms is well understood. Less is known about the factors contributing to epigenetic divergence among isogenic populations except that, despite the presence of mechanisms that faithfully maintain epigenetic marks, epigenetic differences are more frequent than genetic variation. Epigenetically divergent stretches of isogenic DNA sequence are called epialleles. Currently, it is not clear why certain regions exhibit variable epigenetic status. We identified and characterised two long RNA transcripts with altered expression and DNA methylation in an ago5 mutant. However, further investigation revealed that these changes were not dependent upon AGO5. Rather, the variable transcription of these loci in Arabidopsis mutant and wild-type populations corresponds to spontaneous differential methylated regions (DMRs) or epialleles. These two DMRs are delineated by RNAs which are highly expressed when the DMR is hypomethylated. Furthermore, they control the expression of 5' transcriptional start site mRNA variants of nearby protein coding genes. Our data support the recent observations that meiotically stable DMRs exist within inbred populations. We further demonstrate that DMR boundaries can be defined by putative non-coding promoter-associated transcripts.

AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana.

RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO) proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points.

Detection of small RNAs and microRNAs using deep sequencing technology.

Small RNAs (sRNAs) have made a large impact on many recent scientific discoveries. MicroRNAs (miRNAs) are a type of sRNA molecule and, although usually just 20-22 nucleotides in length, they are potent regulators of gene expression. Therefore, identification of miRNAs and profiling their abundance are fundamental to understanding an organism's or tissue's gene regulatory network. Next-generation sequencing methods have allowed researchers to quickly sequence and profile sRNA populations. This chapter describes a cloning procedure to identify the sRNAs or miRNAs present in an RNA sample.

Evidence for large complex networks of plant short silencing RNAs.

BACKGROUND: In plants and animals there are many classes of short RNAs that carry out a wide range of functions within the cell; short silencing RNAs (ssRNAs) of 21-25 nucleotides in length are produced from double-stranded RNA precursors by the protein Dicer and guide nucleases and other proteins to their RNA targets through base pairing interactions. The consequence of this process is degradation of the targeted RNA, suppression of its translation or initiation of secondary ssRNA production. The secondary ssRNAs in turn could then initiate further layers of ssRNA production to form extensive cascades and networks of interacting RNA [1]. Previous empirical analysis in plants established the existence of small secondary ssRNA cascade [2], in which a single instance of this event occurred but it was not known whether there are other more extensive networks of secondary sRNA production. METHODOLOGY/PRINCIPAL FINDINGS: We generated a network by predicting targets of ssRNA populations obtained from high-throughput sequencing experiments. The topology of the network shows it to have power law connectivity distribution, to be dissortative, highly clustered and composed of multiple components. We also identify protein families, PPR and ULP1, that act as hubs within the network. Comparison of the repetition of genomic sub-sequences of ssRNA length between Arabidopsis and E.coli suggest that the network structure is made possible by the underlying repetitiveness in the genome sequence. CONCLUSIONS/SIGNIFICANCE: Together our results provide good evidence for the existence of a large, robust ssRNA interaction network with distinct regulatory function. Such a network could have a massive effect on the regulation of gene expression via mediation of transcript levels.

The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci.

Argonaute (AGO) effectors of RNA silencing bind small RNA (sRNA) molecules and mediate mRNA cleavage, translational repression, or epigenetic DNA modification. In many organisms, these targeting mechanisms are devolved to different products of AGO multigene families. To investigate the basis of AGO functional diversification, we characterized three closely related Arabidopsis thaliana AGOs (AGO4, AGO6, and AGO9) implicated in RNA-directed DNA methylation. All three AGOs bound 5' adenosine 24-nucleotide sRNAs, but each exhibited different preferences for sRNAs from different heterochromatin-associated loci. This difference was reduced when AGO6 and AGO9 were expressed from the AGO4 promoter, indicating that the functional diversification was partially due to differential expression of the corresponding genes. However, the AGO4-directed pattern of sRNA accumulation and DNA methylation was not fully recapitulated with AGO6 or AGO9 expressed from the AGO4 promoter. Here, we show that sRNA length and 5' nucleotide do not account for the observed functional diversification of these AGOs. Instead, the selectivity of sRNA binding is determined by the coincident expression of the AGO and sRNA-generating loci, and epigenetic modification is influenced by interactions between the AGO protein and the different target loci. These findings highlight the importance of tissue specificity and AGO-associated proteins in influencing epigenetic modifications.

The Sireviruses, a plant-specific lineage of the Ty1/copia retrotransposons, interact with a family of proteins related to dynein light chain 8.

Plant genomes are rich in long terminal repeat retrotransposons, and here we describe a plant-specific lineage of Ty1/copia elements called the Sireviruses. The Sireviruses vary greatly in their genomic organization, and many have acquired additional coding information in the form of an envelope-like open reading frame and an extended gag gene. Two-hybrid screens were conducted with the novel domain of Gag (the Gag extension) encoded by a representative Sirevirus from maize (Zea mays) called Hopie. The Hopie Gag extension interacts with a protein related to dynein light chain 8 (LC8). LC8 also interacts with the Gag extension from a Hopie homolog from rice (Oryza sativa). Amino acid motifs were identified in both Hopie Gag and LC8 that are responsible for the interaction. Two amino acids critical for Gag recognition map within the predicted LC8-binding cleft. Two-hybrid screens were also conducted with the Gag extension encoded by the soybean (Glycine max) SIRE1 element, and an interaction was found with light chain 6 (LC6), a member of the LC8 protein family. LC8 and LC6 proteins are components of the dynein microtubule motor, with LC8 being a versatile adapter that can bind many unrelated cellular proteins and viruses. Plant LC8 and LC6 genes are abundant and divergent, yet flowering plants do not encode other components of the dynein motor. Although, to our knowledge, no cellular roles for plant LC8 family members have been proposed, we hypothesize that binding of LC8 proteins to Gag aids in the movement of retrotransposon virus-like particles within the plant cell or possibly induces important conformational changes in the Gag protein.

The diversity of LTR retrotransposons.

Eukaryotic genomes are full of long terminal repeat (LTR) retrotransposons. Although most LTR retrotransposons have common structural features and encode similar genes, there is nonetheless considerable diversity in their genomic organization, reflecting the different strategies they use to proliferate within the genomes of their hosts.

Translational recoding signals between gag and pol in diverse LTR retrotransposons.

Because of their compact genomes, retroelements (including retrotransposons and retroviruses) employ a variety of translational recoding mechanisms to express Gag and Pol. To assess the diversity of recoding strategies, we surveyed gag/pol gene organization among retroelements from diverse host species, including elements exhaustively recovered from the genome sequences of Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Candida albicans, and Arabidopsis thaliana. In contrast to the retroviruses, which typically encode pol in the -1 frame relative to gag, nearly half of the retroelements surveyed encode a single gag-pol open reading frame. This was particularly true for the Ty1/copia group retroelements. Most animal Ty3/gypsy retroelements, on the other hand, encode gag and pol in separate reading frames, and likely express Pol through +1 or -1 frameshifting. Conserved sequences conforming to slippery sites that specify viral ribosomal frameshifting were identified among retroelements with pol in the -1 frame. None of the plant retroelements encoded pol in the -1 frame relative to gag; however, two closely related plant Ty3/gypsy elements encode pol in the +1 frame. Interestingly, a group of plant Ty1/copia retroelements encode pol either in a +1 frame relative to gag or in two nonoverlapping reading frames. These retroelements have a conserved stem-loop at the end of gag, and likely express pol either by a novel means of internal ribosomal entry or by a bypass mechanism.

SIRE1, an endogenous retrovirus family from Glycine max, is highly homogeneous and evolutionarily young.

SIRE1 is unusual among Ty1-copia retrotransposons in that it has an additional open reading frame with structural features similar to retroviral envelope proteins between pol and the 3' LTR. Here we report the characterization and comparison of eight different SIRE1 elements derived from a soybean genomic library, as well as SIRE1 reverse transcriptases from Glycine soja. The DNA sequences of the eight SIRE1 elements are highly homogeneous and share greater than 95% nucleotide identity. Partial sequences obtained from BAC ends are similarly conserved. Phylogenetic analyses resolve two closely related SIRE1 lineages, and nucleotide changes within and between SIRE1 lineages have occurred to preserve function. Both the gag and the env-like genes are evolving under similar levels of functional constraint. Considerable sequence heterogeneity in the form of short duplications was found within the LTRs and in the region between the envelope-like ORF and the 3' LTR. These duplications are suggestive of slippage by reverse transcriptase during replication. Sequence identity between LTRs of individual insertions suggests that they transposed within the past 70,000 years. Two of 10 SIRE1 insertions examined abut Ty3-gypsy retroelements. Since the soybean genome harbors more than 1,000 SIRE1 insertions, the collective data suggest that SIRE1 has undergone a very recent and robust amplification in soybean.

The soybean retroelement SIRE1 uses stop codon suppression to express its envelope-like protein.

The soybean SIRE1 family of Ty1/copia retrotransposons encodes an envelope-like gene (env-like). We analysed the DNA sequences of nine SIRE1 insertions and observed that the gag/pol and env-like genes are in the same reading frame and separated by a single UAG stop codon. The six nucleotides immediately downstream of the stop codon conform to a degenerate nucleotide motif, CARYYA, which is sufficient to facilitate stop codon suppression in tobacco mosaic virus. In vivo stop codon suppression assays indicate that SIRE1 sequences confer leakiness to the UAG stop codon at an efficiency of 5%. These data suggest that SIRE1 retro-elements use translational suppression to express their envelope-like protein; this is in contrast with all characterized retroviruses, which express the envelope protein from a spliced genomic messenger RNA.