Transcription termination by RNA polymerase II is coupled to transcript 3′ end formation. A large cleavage and polyadenylation complex containing the major poly(A) polymerase Pap1 produces mRNA 3′ ends, whereas those of nonpolyadenylated snoRNAs in yeast are formed either by endonucleolytic cleavage or by termination, followed by trimming by the nuclear exosome. We show that synthesis of independently transcribed snoRNAs involves default polyadenylation of two classes of precursors derived from termination at a main Nrd1/Nab3-dependent site or a “fail-safe” mRNA-like signal. Poly(A) tails are added by Pap1 to both forms, whereas the alternative poly(A) polymerase Tfr4 adenylates major precursors and processing intermediates to facilitate further polyadenylation by Pap1 and maturation by the exosome/Rrp6. A more important role of Trf4/TRAMP, however, is to enhance Nrd1 association with snoRNA genes. We propose a model in which polyadenylation of pre-snoRNAs is a key event linking their transcription termination, 3′ end processing, and degradation. Saccharomyces cerevisiae, two classes of snoRNAs, box C/D and box H/ACA, act in preribosomal RNA processing and modification. They have distinct secondary structures, associate with different sets of proteins, and guide unrelated modification reactions (2′-O-ribose methylation versus pseudouridylation)  require correct transcription termination, 3′ and 5′ processing, and assembly into ribonucleoprotein particles (RNPs). In ylation) . They arylation) . Transcrylation) . Some coylation) . This prylation) , but theylation) .trans-acting factors, lead to transcriptional readthrough of the terminators. Efficient termination requires cotranscriptional recruitment of Nrd1/Nab3 and cleavage and polyadenylation components to snoRNA genes. In addition, the Nrd1/Nab3-dependent pathway involves interaction between Nrd1 and the phosphorylated Ser-5 of CTD and direct binding of several Nrd1 and Nab3 molecules to multiple sites in the RNA  and a sequence resembling the mRNA cleavage/polyadenylation signal (site II) . Mutatio the RNA .Nuclear exosome, and above all its nuclear component Rrp6 and cofactor Rrp47, has long been known to participate in the formation of snoRNA 3′ ends. The absence of exosomal subunits leads to accumulation of 3′ unprocessed forms, carrying either a few additional residues or longer polyadenylated extensions . AlthougAlthough polyadenylation of intrinsically nonpolyadenylated sn/snoRNAs was attributed to the RNA quality control, polyadenylation-driven processing of precursors cleaved by Rnt1 has also been suggested . We haverrp6Δ strain. Circular RT-PCR (CR-RT-PCR) followed by sequencing shows that they carry one to six additional residues, of which only the first few overlap with those in the precursor, the remaining ones being one to four adenosines not present in the sequence + fraction selected on an oligo(dT) column. Two poly(A)+ populations were detected for box C/D snR65, snR13, snR64, snR68, and U14 as well as box H/ACA snR46 and snR33  sites and the number of added adenines in terminator II of pre-snR65 were determined by CR-RT-PCR. Of 13 sequenced clones, all contain from 3 to 107 As at different positions within this region (Longer polyadenylated snoRNA forms that accumulate in the nd snR33 D. Both pnd snR33 E. In thend snR33 D. In wilnd snR33 . An addis region F. For teThese data show that snoRNA species are not polyadenylated at random sites but at two discrete regions corresponding to terminators I and II. We predict that they do not represent stabilized exosome degradation intermediates but snoRNA precursors that are generated by polyadenylation linked to transcription.GAL1 promoter (GAL1::SNR65)  are visible after 30 min following the pulse. The heterogeneous II-pA is polyadenylated, as attested by deadenylation with RNase H and oligo(dT) (data not shown), and matches precursors resulting from termination in region II. The shorter RNA, which resembles species in rrp6Δ cells shown with arrows in ∗, also accumulates, only a few nucleotides longer than the mature snR65. It represents semimature snoRNA that is normally trimmed by Rrp6 and is predominant in its absence  and II-pA RNAs in the rrp6Δ are shortened and disappear with slightly different kinetics. This is accompanied by the buildup of the mature or semimature (M∗) snoRNAs in wild-type or rrp6Δ cells, respectively  A. Owing ence see A. AccumuΔ mutant B. I-pA iΔ mutant , showingectively , suggest∗ intermediates, which in turn are trimmed to mature species, also by Rrp6. The turnover of II-pA RNAs is much slower and is not carried out by Rrp6 alone, except for the final digestion. In the rrp6Δ mutant, semimature snoRNAs are produced from II-pA precursors independently of Rrp6, possibly by the core nuclear exosome (see below). I∗ and I-pA RNAs are synthesized but remain dead-end products that are not chased into mature snoRNAs. Digestion of adenines is not carried out by the CCR4-NOT and Pan2-Pan3 deadenylase complexes, as caf1Δ/ccr4Δ and ccr4Δ/pan2Δ mutants do not show any accumulation of poly(A)+ snoRNAs  tails at both terminators. In wild-type cells, snoRNAs are generated from either precursor but preferably from polyadenylated site I-associated transcripts processed by Rrp6 to M snoRNAs , designarrp6Δ strains lacking Trf4 or Trf5 + forms of pre-U18, generated by lariat debranching or Rnt1 cleavage, are not equally affected by deletion of TRF4, confirming that polyadenylation of other precursors is related to their termination. When deletion of RRP6 is combined with a trf4-236 catalytic site mutant with the abolished poly(A) polymerase activity (trf4Δ-like ladder of poly(A)− intermediates and major site I precursors accumulate  tails. These results are consistent with participation of Trf4, possibly by oligoadenylation, in the poly(A) tail synthesis of site I transcripts and, when Trf4 is missing, by shifting termination to site II that is polyadenylated by another polymerase. Weaker effects in cells lacking Trf5 point to a minor role of this protein.Trf4/5 polymerases polyadenylate Rrp6-dependent RNA targets, including snoRNA Rnt1 degradation and processing intermediates . Polyade or Trf5 A. High-mactivity , both pocumulate B. These + pre-snoRNAs was observed in trf4Δ cells  tail. Some accumulation of site I poly(A)+ species is visible for snR13 in trf4Δ/GAL1::TRF5 grown on galactose, where overexpression of Trf5 partially rescues trf4Δ phenotypes (trf4Δ cells poly(A) tail of the average length of 70 nt starts approximately 200 nt from the mature 3′ end (rrp6Δ (see Similar accumulation of longer poly(A)4Δ cells C. Since enotypes . RNaseH e 3′ end D. The lorp6Δ see E, and itrp6Δ see F + precursors + snR65 and snR13 precursors from site II, but not from site I, is elevated already before depletion of Dis3 in Tet::DIS3 cells and strongly increased after depletion in both strains  but are hardly detectable in the rrp6Δ/pap1-2 mutant and significantly decreased and shortened in rrp6Δ/pap1-5 cells after transfer to 37°C. Note that pap1-5 cells retain partial polyadenylation activity in nonpermissive conditions Δ strain . To veri strains A. As obs strains , some funditions . In contnditions . Phenotymutation B.trf4Δ/pap1-2  tails are significantly reduced after 30 min and virtually gone by 60 min following the shift to 37°C. These data provide further evidence that Pap1 participates in polyadenylation of pre-snoRNAs, particularly following termination at site II. Oligo(A) tails at 3′ ends of semimature snoRNAs in rrp6Δ cells are also not dependent on Trf4 or Trf5, as adenosines persist in rrp6Δ/trf4Δ or rrp6Δ/trf5Δ strains  or depleted (GAL1::MTR4), readthrough of SNR13 is detected by primer extension from the downstream TRS31 gene, though it is weaker than for the nrd1-102 mutant  was carried out for TAP-tagged Nrd1 in wild-type, trf4Δ, trf4-236, and GAL1::MTR4 strains  tails and sn/snoRNAs devoid of such a striking feature, are produced by the same Pol II in yeast. Our work corroborates that snoRNA termination is carried out by two complexes and occurs in two regions: a major Nrd1/Nab3/Sen1-dependent terminator and a fail-safe terminator governed by the mRNA 3′ end formation machinery. More importantly, data presented here reveal that termination at both sites is followed by polyadenylation of precursors by Pap1 with contribution by Trf4 that is essential for efficient synthesis of mature snoRNAs and for RNA quality control. These findings also ultimately clarify that all Pol II transcripts in yeast, terminated by either mechanism, become polyadenylated by default and only the subsequent mode of the 3′ end processing defines the final form of the RNA.rrp6Δ strain lose their poly(A) tail in the absence of Trf4 or Trf5 (rrp6Δ cells (Out of the two classes of poly(A) polymerases, Pap1 or Trf4/5, the latter option is consistent with observations that sn/snoRNAs polyadenylated in the  or Trf5 . On the 6Δ cells . Our datpap1 mutants point to the activity of Pap1 also at this site. We envisage that Trf4/5 may initiate the process by addition of short oligo(A) tails that are further extended by Pap1 to ensure effective recruitment of exonucleases. This hypothesis would explain how Pap1 recognizes substrates terminated by the Nrd1/Nab3 complex that has no apparent connection with Pap1 but interacts with Trf4. Similarly, both forms of SRG1 CUT, of which the shorter SSRG1 is generated by a Nrd1/Nab3-dependent termination, undergo Pap1-mediated polyadenylation (Addition of poly(A) tails to transcripts terminated at the mRNA-like site by Pap1 is consistent with the well-established function of Pap1 as a component of the CPF in the context of cleavage and polyadenylation machinery. In wild-type cells, only a minority of precursors undergo termination through this site, but they are relatively stable, possibly through heterogeneous nuclear poly(A) RNA-binding proteins, Nab1 and Nab2 . The lennylation . Also, nnylation . This ilNrd1/Nab3, the exosome, and TRAMP interact and function in termination and turnover of several RNAs, including numerous CUTs, snoRNAs, and their intermediates and even some mRNAs . The majIt has been established that poly(A) tail has a double, somehow antagonistic role in RNA stabilization and function and in RNA surveillance. Polyadenylation of snoRNAs, as well as other stable RNAs and CUTs, has been most often attributed to the mechanism of RNA quality control, though it has also been suggested to contribute to snoRNA processing . Our datWhere, then, is the place in our model for the connection between polyadenylation and RNA surveillance? It is still an unsolved riddle how TRAMP/exosome recognizes defective RNA molecules. In the case of hypomodified tRNAs, their abnormal structure contributes to this process . For snoOur model also accounts for the generation of two classes of precursors that are processed with different kinetics. The second terminator may not only act as a fail-safe signal for the escapees but also ensure a pool of precursors, pending the need for further mature molecules. If the required level of snoRNAs is synthesized, and RNP proteins are limiting, these precursors are directed to the discard pathway.rrp6Δ mutant, snoRNAs and poly(A)+ RNAs accumulate in a discrete nucleolar domain distinct from the nucleolar body (NB) or in nucleoplasmic foci in rrp6Δ/rna14-1 and rrp6Δ/rna15-2 strains  total RNA was reverse transcribed with AMV (Promega) using 10 pM ADAPT-oligo(dT)30 or gene-specific primers. cDNA was diluted 10- to 100-fold and used in PCR reactions with ADAPT-oligo(dT)30 and a primer against the mature snoRNA. Resulting bands were excised from agarose gels and sequenced. CR-RT-PCR was carried out on total RNA circularized with T4 RNA ligase (NEB). 5′ end with the TMG cap of snR13 and snR3 was removed prior to circularization by RNaseH treatment in the presence of W274 or W277 oligonucleotides. Circular molecules were amplified by RT-PCR, and products were cloned into pGEM-T Easy (Promega) and sequenced. Oligonucleotides, probes, and primers are listed in General RNA methods are described in the ChIP was performed as described  using Ig


The ability to access, search and analyse secondary structures of a large set of known RNA molecules is very important for deriving improved RNA energy models, for evaluating computational predictions of RNA secondary structures and for a better understanding of RNA folding. Currently there is no database that can easily provide these capabilities for almost all RNA molecules with known secondary structures.In this paper we describe RNA STRAND – the RNA secondary STRucture and statistical ANalysis Database, a curated database containing known secondary structures of any type and organism. Our new database provides a wide collection of known RNA secondary structures drawn from public databases, searchable and downloadable in a common format. Comprehensive statistical information on the secondary structures in our database is provided using the RNA Secondary Structure Analyser, a new tool we have developed to analyse RNA secondary structures. The information thus obtained is valuable for understanding to which extent and with which probability certain structural motifs can appear. We outline several ways in which the data provided in RNA STRAND can facilitate research on RNA structure, including the improvement of RNA energy models and evaluation of secondary structure prediction programs. In order to keep up-to-date with new RNA secondary structure experiments, we offer the necessary tools to add solved RNA secondary structures to our database and invite researchers to contribute to RNA STRAND..RNA STRAND is a carefully assembled database of trusted RNA secondary structures, with easy on-line tools for searching, analyzing and downloading user selected entries, and is publicly available at  The number of solved RNA secondary structures has increased dramatically in the past decade, and several databases are available to search and download specific classes of RNA secondary structures -5. HowevPrevious RNA databases provide secondary structure information, but are specialised in a different direction or follow different goals. The Rfam Database  containsA number of previous databases contain three-dimensional (3D) RNA structures; however, as opposed to proteins, the number of solved RNA 3D structures is much smaller than the number of solved RNA secondary structures.  As such, all these databases do not include molecules whose secondary structures are known but 3D structures are unknown; examples include: the RCSB Protein Data Bank , the NucRNA STRAND spans a more comprehensive range of RNA secondary structures than do previous databases. It currently provides highly accurate secondary structures for 4666 RNA molecules. Since some users of RNA STRAND will likely develop new thermodynamic models, prediction tools or statistical analyses, our data is exclusively determined by carefully conducted comparative sequence analysis , or by eFigure The current release v2.0 of RNA STRAND contains a total of 4666 entries (RNA sequences and secondary structures) of the following provenance:• RCSB Protein Data Bank (PDB) : 1059 en• Comparative RNA Web Site, version 2 : 1056 en• tmRNA database : 726 ent• Sprinzl tRNA Database (September 2007 edition) [• RNase P Database : 454 Rib• SRP Database : 383 ent• Rfam Database, version 8.1 : 313 ent• Nucleic Acid Database (NDB) : 53 entrTable In the future, we intend to regularly check the aforementioned databases for new entries. With our current tools, keeping the database up-to-date will be relatively easy.We created a unique and stable identifier for each entry in the RNA STRAND database. Future releases will keep all previous IDs unchanged.One of the challenging tasks in collecting the RNA STRAND data arose from the fact that the external sources offer data in various formats. We have built tools to convert from all these formats to the CT format, which we use to store all structures internally, and to RNAML, BPSEQ, dot-parentheses and FASTA formats when requested by a user. The format descriptions are accessible on the "Help" page online.i is paired with base at position j, then base at position j is also paired with base at position i.). When performed on our present data, this validation step revealed several inconsistencies in some of the external files, which we brought to the attention of the respective database owners.All external databases we have used in the current version of RNA STRAND, except Rfam, contain highly curated RNA secondary or tertiary structures, therefore we trust the curation methods of these sources. For Rfam we selected a set of reliable structures based on the cited papers, as described in the previous section. Once we converted all the secondary structure external files into the CT format, we checked all files in order to make sure the secondary structures are valid . The main table is MOLECULE, with one row per RNA entry in the database. This table contains as primary key the unique RNA STRAND ID of the entry and further comprises various descriptive fields, including: organism, reference, length, RNA type, external source, external ID, sequence, three levels of abstract shapes using the RNAshapes representation , the metAn additional table TMP_MOLECULE is used to temporarily hold new submissions received via the web interface; for these, we manually check the submission information by checking the cited paper, after which, if the submission is accepted, all further steps required to permanently add the respective RNA(s) to the database are performed automatically.The web interface to RNA STRAND has been created using a set of PHP scripts (version 5.1.2). The main functions of the web interface are searching, browsing, analysis, downloading and uploading.E. coli), external source , length (in bases), the number of molecules in the complex, whether it is a fragment, a sequence pattern using the standard IUPAC nucleic acid codes, an abstract structure or fragment using the RNAshapes representation [The user specifies one or more search criteria in a web-based form. The general criteria include RNA type , organism of origin . Pseudoknots are currently not permitted in the abstract shape representation [Using advanced search criteria, users can search for entries with various structural motifs. For example, when looking for a Y shape with an additional hairpin, one would search for entries that have exactly one multi-loop, three multi-loop branches, three hairpins, one molecule in the complex, and no pseudoknots. This search returns 31 entries, most of which are entation .Support for inspecting large fractions of the database contents is provided via searches with no or very general criteria. For example, it is easy to obtain a list of all RNase P RNA structures contained in the database.Details on individual entries from the result list of any search can be displayed by clicking on an RNA STRAND ID link of the results table. This single entry display comprises general information about the entry, links to the original database entry for this molecule, a secondary structure diagram, details of its secondary structure elements and features, links to other RNA STRAND entries with the same sequence , links to the sequence and secondary structure specification in five formats , and a link to the complete output of the RNA Secondary Structure Analyser.Q1 - 1.3·(Q3 - Q1) or greater than Q1 + 1.3·(Q3 - Q1), where Q1 and Q3 are the first and third quartiles, respectively. Such analyses may guide research pertaining to understanding structural features in naturally occuring RNA molecules, as we outline in the "Utility and discussion" section.In addition to the aforementioned analysis information for individual entries, RNA STRAND also provides histograms or cumulative distribution functions of various molecule characteristics (such as number of pseudoknots per molecule) or structural features (such as number of branches per multi-loop) for all structures in the database or for user-selected subsets, as obtained from the search page. In addition, correlations between various molecule characteristics and molecule length can be obtained. For an unbiased analysis, the user has the option of normalising the data by RNA type (such as tRNA), in which case for each particular RNA type, one data point is obtained by averaging over all the data for molecules of that type. Finally, the user can choose to remove the outliers of the distributions. We use a common definition, according to which a data point is an outlier if, and only if, it is smaller than The set of molecules selected via the search page can be downloaded in one of five supported formats: CT, RNAML, BPSEQ, dot-parentheses and FASTA. Thus, researchers can use specifically selected structures locally.RNA STRAND supports public submission of RNA secondary structures to the database via its web interface. The structure file can be in any of the four supported secondary structure formats  or in the PDB tertiary structure format. Since RNA STRAND is a curated database, newly submitted structures are checked for accuracy and completeness by one of the database administrators before they are added to the database. New additions to the public databases that constitute our external sources will be added to RNA STRAND regularly. This is complemented by the public submission option, which is intended for submission of structures that do not yet belong to any of these databases.RNA STRAND v2.0 contains 4666 RNA molecules or interacting complexes of various types, and an abundance of RNA structural motifs  entries with 100 or more nucleotides contain pseudoknots. We compared the stem length  with the minimal number of base pairs that need to be removed per pseudoknot to render the structure pseudoknot free . Table canonical base pairs, and all other base pairs to be non-canonical.) Figure Our second observation concerns the abundance of non-canonical base pairs and the pairing type of their immediate neighbours. , suggesting that pseudoknots may not follow the same linearity principle.Finally, we found rather strong linear correlations between the number of nucleotides of the RNAs in our database and the number of stems, hairpin loops, bulges, internal loops and multi-loops; the Pearson correlation coefficients are The RNA STRAND database can be used to evaluate the prediction accuracy of energy-based RNA secondary structure prediction software. RNA STRAND v2.0 contains 3704 non-redundant entries containing one molecule that can be used to evaluate software such as CONTRAfold , 403 nonWe have selected 2518 structures out of the 3704 non-redundant entries containing one molecule, after we eliminated the entries with unknown nucleotides and overly large loops.  In addition, we have removed all non-canonical base pairs and the minimum number of base pairs needed to render the structures pseudoknot-free. The resulting structures are used as ground-truth reference structures. We evaluated the sensitivity and positive predictive value (PPV) of CONTRAfold  with varγ between sensitivity and PPV, and thus we report predictions for γ ranging from 2 to 20. When the target of one measure is fixed to the value obtained with "SimFold Turner99", the other is similar as well, showing that on this data set, CONTRAfold 1.1 gives similar average prediction accuracy as "SimFold Turner99". The remaining points of Figure "CONTRAfold 1.1 151Rfam" is the CONTRAfold software version 1.1, as reported by Do et al. . The CONMore importantly, RNA STRAND can facilitate approaches for improving the free energy models underlying energy-based RNA secondary structure prediction software ,7. In thFigure "CONTRAfold 1.1 151Rfam" was trained on a small set of 151 structures from various Rfam families , while "To demonstrate even further the importance of using a large and comprehensive set of known RNA secondary structures for obtaining high-quality free energy parameters, we have used the current version of RNA STRAND v2.0 to obtain a new training set of 2246 structures of average length 246 nucleotides. Using the Maximum Likelihood parameter estimation method described by Andronescu et al. , we haveThese results provide clear evidence for the key role of large and carefully assembled sets of RNA secondary structures, such as provided by RNA STRAND, in the context of determining RNA free energy models. In the future, we are planning to use the RNA STRAND data to train free energy parameters for pseudoknotted structures. Existing energy models for RNA secondary structure prediction methods with pseudoknots are often ad-hoc ,29, and The numerous search criteria supported by the RNA STRAND web interface allow users to select and study molecules with specific structural features. For example, Tyagi and Mathews  studied In recent work on the role of RNA structure in splicing, Rogic et al.  needed tIn addition, RNA STRAND can facilitate the design of optical melting experiments , whose gIn this paper, we presented RNA STRAND, a new database for RNA secondary structure data that provides access to detailed information on known secondary structures as well as statistical analyses of structural aspects of various types of RNAs. We believe that such information will be useful in the context of understanding RNA structure and function; in particular, we expect it to further facilitate the development and evaluation of energy models for secondary structure prediction. Our database is flexible and extensible; it provides a convenient web interface to its major functions and supports searches according to many criteria, including properties of secondary structure elements. The database is publicly accessible and supports the submission of new RNA structures by the research community. We are committed to keeping RNA STRAND up-to-date with new structures that are added to the eight databases of provenance, and we invite submissions of all types of RNA secondary structures, which will help to further expand the database and increase its usefulness.In the future, we intend to add RNA secondary structures obtained from the SHAPE technique ,41, and . The RNA Secondary Structure Analyser, as well as the database tables, are available upon request from the authors.RNA STRAND is publicly available at MA collected the data, implemented conversion and validation scripts, implemented the MySQL database and part of the PHP scripts, performed the statistical analyses and helped to draft the manuscript. VB implemented the vast majority of the PHP scripts and most of the RNA Secondary Structure Analyser. HHH and AC conceived the project, specified the design of the RNA Secondary Structure Analyser, supervised MA's and VB's work, and helped to write the manuscript. All authors checked the accuracy of the database and web interface, read and approved the final manuscript.


Arabidopsis thaliana 24 nt siRNAs requires the Dicer family member DCL3, and clear homologs of DCL3 exist in both flowering and non-flowering plants. However, the absence of a conspicuous 24 nt peak in the total RNA populations of several non-flowering plants has raised the question of whether this class of siRNAs might, in contrast to the ancient 21 nt microRNAs (miRNAs) and 21–22 nt trans-acting siRNAs (tasiRNAs), be an angiosperm-specific innovation. Analysis of non-miRNA, non-tasiRNA hotspots of small RNA production within the genome of the moss Physcomitrella patens revealed multiple loci that consistently produced a mixture of 21–24 nt siRNAs with a peak at 23 nt. These Pp23SR loci were significantly enriched in transposon content, depleted in overlap with annotated genes, and typified by dense concentrations of the 5-methyl cytosine (5 mC) DNA modification. Deep sequencing of small RNAs from two independent Ppdcl3 mutants showed that the P. patens DCL3 homolog is required for the accumulation of 22–24 nt siRNAs, but not 21 nt siRNAs, at Pp23SR loci. The 21 nt component of Pp23SR-derived siRNAs was also unaffected by a mutation in the RNA-dependent RNA polymerase mutant Pprdr6. Transcriptome-wide, Ppdcl3 mutants failed to accumulate 22–24 nt small RNAs from repetitive regions while transcripts from two abundant families of long terminal repeat (LTR) retrotransposon-associated reverse transcriptases were up-regulated. Ppdcl3 mutants also displayed an acceleration of leafy gametophore production, suggesting that repetitive siRNAs may play a role in the development of P. patens. We conclude that intergenic/repeat-derived siRNAs are indeed a broadly conserved, distinct class of small regulatory RNAs within land plants.Endogenous 24 nt short interfering RNAs (siRNAs), derived mostly from intergenic and repetitive genomic regions, constitute a major class of endogenous small RNAs in flowering plants. Accumulation of  P. patens that produced a mix of 21–24 nt siRNAs. Except for their broad mix of sizes, these hotspots were reminiscent of the 24 nt siRNA loci of angiosperms: they tended to associate with decayed transposons, to avoid annotated genes, and to be densely modified with the epigenetic mark 5-methyl cytosine. Deletion of a P. patens Dicer gene abolished production of 22–24 nt siRNAs both from these loci and transcriptome-wide, especially from repetitive regions. We conclude that both microRNAs and intergenic/repeat-associated siRNAs are ancient small RNA regulators in plants, but that the sizes of the siRNAs themselves have drifted over time.Very small RNAs (between ∼21 and ∼30 single-stranded bases) are a ubiquitous component of gene regulation in nearly all eukaryotic organisms. The small RNA repertoire of angiosperms (the flowering plants) is exceptionally diverse and includes conspicuous populations of 21 nt microRNAs, as well a diverse set of 24 nt short, interfering RNAs (siRNAs). The 24 nt siRNAs have well-documented roles in enforcing the silence of parasitic regions of the genome, but are not readily apparent in the small RNA populations of several lineages of ancient, non-flowering plants. We found numerous “hotspots” of small RNA production from the genome of the moss  Most eukaryotes analyzed to date express diverse small silencing RNAs which direct the sequence-specific repression of target RNAs. Small silencing RNAs are bound to Argonaute or Piwi proteins, which modulate target expression by a variety of molecular mechanisms Arabidopsis thaliana, where they are processed by Dicer proteins from long, perfectly double-stranded RNA (dsRNA) precursors. The endogenous dsRNA precursors are most often produced by RNA-dependent RNA polymerases (RDRs). The majority of expressed small RNAs in A. thaliana depend on the activity of two RDR proteins Two major types of small silencing RNAs have been described in plants: MicroRNAs (miRNAs), and short interfering RNAs (siRNAs). miRNAs are ubiquitous regulators of gene expression in animals, plants, and some unicellular eukaryotes. Most plant miRNAs are 21 nts in length and are defined by precise excision from a single-stranded, stem-loop precursor by the action of a Dicer protein. Mature miRNAs often function to repress the expression of an evolved set of protein-coding mRNA targets. miRNAs regulate thousands of mRNAs in animals and have had a profound impact upon the evolution of 3′-untranslated regions A. thaliana specific family members are specialized for distinct endogenous small RNA producing pathways. DCL1 and AGO1 are required for the accumulation and function of most miRNAs trans-acting (tasiRNAs) and are produced after small RNA-mediated cleavage of a primary transcript. tasiRNAs are mostly 21 nts in length, but small amounts of 22 nt tasiRNAs are typically observed as well A. thaliana 24 nt siRNAs correlates with the de novo deposition of repressive DNA and histone modifications Plant Dicers (known as DCLs for “Dicer-Like”), Argonautes (AGOs) and RDRs are all encoded by multi-gene families; in A. thaliana shows two distinct peaks at 21 nts and 24 nts in length DCL3-dependent siRNAs DCL3-dependent 24 nt siRNA pathway might have been a derived feature of angiosperm species. However, when the abundant 21 nt miRNAs and 21–22 nt tasiRNAs are subtracted, the remaining small RNAs from the moss Physcomitrella patens show a broader size distribution of 21–24 nt species DCL3 homolog, these observations prompted us to search for intergenic/repetitive siRNA-producing loci in P. patens. Here, we identified P. patens loci which produced a mix of 21–24 nt siRNAs from primarily intergenic and repetitive regions of the genome, and which were densely populated by the 5-methyl cytosine (5 mC) DNA modification. Using deep sequencing of small RNAs from Ppdcl3 deletion mutants, we observed that PpDCL3 was required for the accumulation of 22–24 nt siRNAs, but not 21 nt siRNAs, from these loci. Loss of PpDCL3-dependent siRNA accumulation correlated with the de-repression of two abundant long terminal repeat (LTR) retrotransposon-associated reverse transcriptase families. Unlike in A. thaliana, P. patens dcl3 mutants also demonstrated developmental abnormalities suggesting that repetitive siRNAs contribute to moss development. These observations demonstrate that a specialized, DCL3-dependent siRNA production system associated with transposons and other non-genic regions of the genome is an ancestral feature of land plants, although the sizes of the relevant siRNAs can differ between lineages.The small RNA population of wild-type P. patens small RNAs P. patens genome by identifying loci corresponding to small RNA production “hotspots”. A previously reported dataset of expressed P. patens small RNAs P. patens MIRNA hairpins or tasiRNA loci. We then ranked genomic loci for their small RNA producing activity based on the number of reads observed for exactly matched small RNAs. To account for the uncertainty of the genomic origins of small RNAs whose sequences matched multiple genomic loci, reads were repeat-normalized by dividing by the number of exact matches between the small RNA and the genome Readily identifiable miRNAs and tasiRNAs account for a minority of expressed P. patens genome clearly fell into two distinct classes, initially discerned based on the lengths of the associated small RNAs: Those which were dominated by RNAs 21 nts in length, and those producing a mixture of 21–24 nt RNAs in a strikingly consistent ratio  and Pp23SR  loci, respectively. Most loci (89 out of 100) had at least one corresponding small RNA which uniquely mapped to the genome , while the Pp21SR loci were mostly between 100 and 1,000 nts in length , thus leading to the erroneous conclusion that they were derived from a long dsRNA precursor instead of a single-stranded, stem-loop precursor. Many of the NA dyads . Thus, tP. patens genome was contained within inverted repeats (Pp21SR loci and 85% of the Pp23SR loci) had at least some overlap with one or more inverted repeats. However, the percentage of nucleotides which overlapped with inverted repeats for most of the Pp23SR loci was close to the genome-wide value of 6.2% , and other elements  as judged by values obtained using randomized cohort loci  had a single integration of the knock-out construct only at the targeted locus (data not shown); these two lines were used for all further studies.We hypothesized that the bination . Four inbination . In all y RT-PCR . DNA bloPpdcl3 mutants produced gametophores faster than the wild type, which reminded us of the previously reported Pprdr6 phenotype Ppdcl3, Pprdr6, and the wild type in detail. Similar sizes of protonemata were inoculated onto minimal media with or without ammonium supplementation and monitored for gametophore number over a fortnight. Regardless of ammonium availability, the rate of gametophore production in Ppdcl3 mutants was accelerated relative to wild-type  were also densely methylated indicating that 5 mC deposition at small RNA loci is not strictly limited to Pp23SR loci  were designed and used for RT-PCR. Transcripts for PpRT3 and PpRT6 were not detectable in wild type but significantly accumulated in Ppdcl3-5 and Ppdcl3-10  and AtDCL3 (which produces 24 nt siRNAs) by directing the accumulation of 22–24 nt siRNAs. However, AtDCL2-dependent siRNA loci do not coincide with AtDCL3-dependent siRNA loci and, unlike PpDCL3, AtDCL2 makes only a very small overall contribution to the overall siRNA population AtDCL3 is clearly not the most closely related plant Dicer to AtDCL2DCL2 arose from an ancestral DCL3 gene.PpDCL3 ablated 22–24 nt siRNA production from Pp23SR loci but had no effect upon 21 nt siRNAs. Thus, we hypothesize that a second DCL also acts in conjunction with PpDCL3 to produce Pp23SR-derived siRNAs. Besides PpDCL3, the P. patens genome contains one DCL4 homolog and two DCL1 homologs AtDCL4 is required for chiefly 21 nt tasiRNA, secondary siRNA, and viral siRNA accumulation, we suspect that its moss homolog produces the residual 21 nt RNAs at the Pp23SR loci. Interestingly, these residual 21 nt siRNAs are not dependent upon PpRDR6, whose A. thaliana homolog is closely associated with DCL4 function. Instead, PpRDR6 partially contributed to 22–24 nt small RNA accumulation in Pp23SR loci. This functional redundancy is not exactly the same as that previously reported at A. thaliana 24 nt siRNA loci: In A. thaliana, the products of other DCLs only become apparent upon loss of DCL3 function P. patens they are present even in the wild-type.Deletion of Pp23SR loci assayed had dense 5 mC modifications in all contexts. This further solidifies the connection of the Pp23SR loci to the 24 nt siRNA loci of A. thaliana. It is possible that, as in A. thalianaPp23SR loci can direct de novo cytosine methylation in the asymmetric CHH context. However, complete removal of PpDCL3 function and the consequent loss of all 22–24 nt siRNAs at Pp23SR loci did not substantially affect the maintenance of wild-type 5 mC patterns in most instances examined. It may be that the residual 21 nt siRNAs which persist at Pp23SR loci in the Ppdcl3 background are sufficient to maintain 5 mC deposition, buffering the effect of the Ppdcl3 mutation. The fact that the two transposon-associated Pp21SR loci, which are naturally devoid of PpDCL3-dependent 22–24 nt siRNAs, also possessed dense 5 mC modifications in all contexts is consistent with this idea. Alternatively, it is possible that PpDCL3-dependent siRNAs are critical for the establishment, but not the maintenance, of 5 mC patterns at the Pp23SR loci and perhaps elsewhere. Indeed, components of the 24 nt siRNA pathway are required at the A. thaliana SDC locus to initiate 5 mC deposition in all contexts, but not to maintain these patterns once established dcl3 mutation does not substantially affect the maintenance of 5 mC deposition at several other A. thaliana loci A. thaliana loci where 5 mC density is affected in dcl3 mutants , while maintenance of 5 mC density is only weakly diminished at the MEA-ISR and 5S rDNA loci in A. thaliana dcl3 mutants Pp23SR loci in the Ppdcl3 mutants is consistent with previous observations from A. thaliana. A third possibility is that the 5 mC and siRNAs associated with Pp23SR loci reflect independent processes which act in parallel to maintain the silence of the affected genomic regions.All Atdcl3 mutants lose most 24 nt repetitive siRNA accumulation Ppdcl3 plants, which lacked 22–24 nt repetitive siRNAs, displayed an accelerated production of gametophores relative to the wild type. While this developmental acceleration is less severe than that of Pprdr6 plants, several lines of evidence suggest that PpDCL3 and PpRDR6 might have partially overlapping roles: Both mutants have similar developmental abnormalities, TAS3 tasiRNA accumulation is slightly decreased in Ppdcl3 mutants, and Pprdr6 mutants slightly impact the production of repetitive 22–24 nt siRNAs. The Ppdcl3 developmental phenotype is unlikely to be the result of stochastic epialleles because it is identical in multiple, independently-derived mutants and does not require several generations to become manifest. One possibility is that slight tasiRNA defects in Ppdcl3 are responsible for the developmental phenotype. Alternatively, a PpDCL3-dependent small RNA, such as a miRNA with unusual biogenesis requirements, could directly regulate genes critical for gametophore development. It is also possible that the repetitive siRNAs themselves, or the suppression they seem to confer on certain repetitive elements, plays a role in moss development.Pp23SR “hotspots” was drastically reduced in Ppdcl3 mutants. However, neither the maintenance of 5 mC DNA modifications nor the transcriptional suppression of these hotspots were substantially altered in Ppdcl3 mutants. However, transcript accumulation of two highly abundant LTR retrotransposon-associated RT families was strongly enhanced by deletion of PpDCL3, despite the persistence of PpDCL3-independent 21 nt siRNAs. Thus, PpDCL3 is necessary for the suppression of at least some highly repetitive elements, most likely as a direct result of 22–24 nt siRNAs. It is important to emphasize that the transcriptional reactivation of RT domains observed in Ppdcl3 mutants does not necessarily correlate with re-activation of transposition-competent elements – we have not directly tested for transposition. However, the data do suggest that the repetitive 22–24 nt siRNAs may indeed play important roles in repressing transposition and defending genome integrity. We conclude that AtDCL3-derived, repetitive siRNAs are, like miRNAs and tasiRNAs, a distinct class of specialized small RNAs with ancient origins within the land plants.Production of 22–24 nt siRNAs from the most abundant P. patens expressed small RNAs from PpTAS3a–d loci (accessions BK005825–BK005828). Draft assembly version 1.1 of the P. patens genome was used throughout P. patens miRNAs used in the analysis of the new P. patens small RNAs were from miRBase version 11.0. Newly generated small RNA datasets were deposited with NCBI GEO (GSE12468).P. patens draft genome assembly version 1.1 using NCBI blastall version 2.2.17 (non-default parameters: -p blastn -e 0.1 -F F -W 6 -m 8 -v 100000 -a 2) and filtered to retain only exact matches. The repeat-normalized magnitude of each small RNA's expression from a given locus was calculated by dividing the number of times a given small RNA was sequenced by the total number of exact matches in the genome Small RNAs from GSE5103 were mapped to the Standalone CENSOR  were utilized for amplification of 5′ upstream (970 bp) and 3′ downstream (1035 bp) fragments of PpDCL3 ORF using the P. patens genomic DNA as a template; the resulting fragments were separately cloned into the pCR 4-TOPO TA cloning vector . The fragments were released by HindIII/XhoI and BglII/MluI restriction enzymes, respectively, and then ligated into the pUQ vector containing a hygromycin resistance cassette  which was originally constructed on the basis of pUC19. The resulting disruption construct was digested with HindIII and MluI restriction enzymes to be linearized and precipitated. Polyethylene glycol-mediated protoplast transformation was performed as previously described 5′-GCGTTTGAATTTGGTTCCACCACC-3′/5′-AGATAGCTGGGCAATGGAATCCGA-3′) and BF/BR primer sets (5′-GGGTTTCGCTCATGTGTTGAGCAT-3′/5′-GCGAGCATTGTGCAAGTTTCCGTA-3′) were used for the analysis of 5′ and 3′ integration events, respectively. RT-PCR analyses were performed to identify the removal of Ppdcl3 transcript with two sets of primers  and the accumulation of hpt transcript with a primer set . RNA was isolated from 7 d-old protonemal tissue with Trizol reagent  and reverse transcribed using a SuperscriptIII reverse transcriptase  following the manufacturer's instructions. Thermocycling conditions were as follows: initial denaturation at 95°C for 5 min, followed by 35 cycles of 30 sec at 95°C, 1 min at 55°C, 30 sec at 72°C, and terminated by a 10 min-final extension at 72°C. Ppubiquitin primers . Cultures were grown as described above. Small RNA-enriched fractions were prepared by precipitating high molecular weight RNAs in the presence of 0.5 M NaCl and 10% (m/v) Polyethylene glycol  and recovering the supernatant. Pre-adenylated 3′ adapters  were added using T4 RNA ligase without exogenous ATP; the wild-type library used linker 1 (5′-AppCTGTAGGCACCATCAATddC-3′), both Ppdcl3 libraries used linker 2 (5′-AppCACTCGGGCACCAAGGAddC-3′) and the Pprdr6-19 library used linker 3 (5′-AppTTTAACCGCGAATTCCAGddC-3′). Ligated products were gel purified and then ligated to a 5′ adapter composed of RNA (5′-GUUCAGAGUUCUACAGUCCGACGAUC-3′) using T4 RNA ligase with ATP. After gel purification, samples were reverse transcribed using oligos appropriate to the specific 3′ linker . cDNA libraries were then amplified using a constant 5′ oligo (5′-AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3′) and 3′ oligos specific for each of the linkers . Amplified libraries were then gel-purified and two samples were mixed containing equal amounts of the wild-type, a Ppdcl3, and the Pprdr6-19 library. Mixed samples were then sequenced using an Illumina genome analyzer by Fasteris, Inc. . A detailed protocol is available upon request.Total RNA was extracted using Tri-Reagent  per the manufacturer's instructions from 10-day old protonemal cultures of the wild-type, P. patens version 1.1 draft genome assembly using oligomap Raw FASTQ files were first processed to identify 3′ linker sequences as follows: The 5 nt sequence representing the first five bases of one of the 3′ linkers was searched for within an 8 nt window of the read (positions 19 through 26). If found, all linker bases were removed and the resulting small RNA read was assigned to the appropriate sample based upon the identity of the linker. If no 5 nt matches to any of the linkers was found, or if more than one was found, the read was discarded. Reads which exactly matched the sense strand of one or more rRNAs  were then discarded. The remaining reads were mapped to the P. patens protonemal tissues using a Nucleon Phytopure Plant DNA extraction kit  following the manufacturer's instructions. Bisulfite conversion of 500 ng of genomic DNA was performed using an EZ DNA Methylation kit  following the manufacturer's protocol. Regions analyzed were amplified from 2 µl of bisulfite converted DNA using sets of specific primers  was treated with 20 units of McrBC  for 4 h at 37°C. Amplification was performed using sets of specific primers (Genomic DNA was isolated from seven day-old  primers . The pri primers .PpRT3 and PpRT6) or 50 cycles  of 30 sec at 95°C, 1 min at 50°C, 30 sec at 72°C, and terminated by a 10 min-final extension at 72°C. The same primers as in the McrBC assay were used for amplification of small RNA loci, whereas sets of specific primers were used for PpRT3 and PpRT6 , followed by a DNase treatment  following the manufacturer's protocol except that the length of incubation was 20 minutes. 500 ng of the RNAs were converted to cDNA using Superscript III reverse transcriptase  primed with random hexamers  following the manufacturer's instruction. Thermocycling conditions were as follows: initial denaturation at 95°C for 5 min, followed by 35 (for nd PpRT6 .32P-ATP using T4 polynucleotide kinase. Blot hybridization was carried out in 0.05 M sodium phosphate (pH 7.2), 1 mM EDTA, 6×SSC, 1×Denhardt's, 5% SDS. Blots were washed 2–3 times with 2×SSC, 0.2% SDS and one time with 1×SSC, 0.1% SDS. Blots were hybridized and washed at temperatures 10°C below the Tm of the oligonucleotide. The sequences of the oligonucleotides used for the detection of small RNAs are listed in Total RNA was separated in a 12% denaturing polyacrylamide gel containing 8.3 M urea in TBE buffer, and electroblotted onto nylon membranes for 1 h at 400 mA. Radiolabeled probes were generated by end-labeling of DNA oligonucleotides complementary to miRNA, tasiRNA sequences and the U6 snRNA control with γFigure S1P. patens PpDCL3. A) Schematic of homologous recombination scheme. Labeled arrows indicate oligos used for PCR and RT-PCR analyses. Solid rectangles indicate exons, lines indicate introns. CaMV: Caulifolwer Mosaic Virus, hptII: hygromycin phosphotransferase II gene. Not to scale. (B) PCR of genomic DNA using the indicated primer pairs. (C) RT-PCR analyses of gene expression using the indicated primer pairs.Targeted deletion of (0.59 MB PDF)Click here for additional data file.Figure S2Pp23SR loci by length and 5′ nucleotide. Each graph represents the Pp23SR matched portion of the indicated small RNA library. A, U, G, and C refer to the identity of the 5′ nucleotide. NRPM: Normalized reads per million.Distribution of small RNAs from (0.19 MB PDF)Click here for additional data file.Figure S3Distribution of all genome-mapped small RNAs by length and 5′ nucleotide. (A) Each graph represents the indicated small RNA library, counted by abundance (number of reads). A, U, G, and C refer to the identity of the 5′ nucleotide. (B) As in A, except tallied by uniquely obtained sequences regardless of abundance.(0.26 MB PDF)Click here for additional data file.Figure S4PpActin primer was used a control.RT-PCR on the selected small RNA producing loci. (1.23 MB PDF)Click here for additional data file.Table S1Physcomitrella patens.Novel miRNAs from (0.06 MB XLS)Click here for additional data file.Table S2Pp21SR loci.(0.04 MB XLS)Click here for additional data file.Table S3Pp23SR loci.(0.05 MB XLS)Click here for additional data file.Table S4Primer sets used for amplification of small RNA loci.(0.07 MB DOC)Click here for additional data file.Table S5Bisulfite sequencing data.(0.11 MB DOC)Click here for additional data file.


Human APOBEC3 proteins are editing enzymes that can interfere with the replication of exogenous retroviruses such as human immunodeficiency virus (HIV), hepadnaviruses such as hepatitis B virus (HBV), and with the retrotransposition of endogenous retroelements such as long-interspersed nuclear elements (LINE) and Alu. Here, we show that APOBEC3G, but not other APOBEC3 family members, binds 7SL RNA, the common ancestor of Alu RNAs that is specifically recruited into HIV virions. Our data further indicate that APOBEC3G recognizes 7SL RNA and Alu RNA by its common structure, the Alu domain, suggesting a mechanism for APOBEC3G- mediated inhibition of Alu retrotransposition. However, we also demonstrate that APOBEC3F and APOBEC3G are normally recruited into and inhibit the infectivity of ΔVif HIV1 virions when 7SLRNA is prevented from accessing particles by RNA interference against SRP14 or by over expression of SRP19, both components of the signal recognition particle. We thus conclude that 7SL RNA is not an essential mediator of the virion packaging of these antiviral cytidine deaminases. APOBEC proteins are members of a family of polynucleotide cytidine deaminases (CDA) that play important roles in antiviral defence. Human APOBEC3G and 3F can block the replication of a wide array of exogenous retroelements, including retroviruses such as human immunodeficiency virus (HIV) and murine leukaemia virus (MLV) ,2, and hAPOBEC family members can also act on endogenous substrates, notably retroelements. APOBEC3A, 3G and 3F can block the propagation of endogenous retroviruses such as intracisternal-A particles (IAP) ,16 or MuThe Alu family of repetitive sequences is one of the most successful groups of mobile genetic elements, having multiplied to more than one million copies in the human genome in some 65 million years of primate evolution ,23. IntePlasmids pCMV4-HA expressing the HA-tagged form of APOBEC3G and APOBEC3A were a kind gift from M. Malim . Human APOBEC3B and APOBEC3F cDNAs were amplified from activated peripheral blood lymphocytes. We used primers cem196: 5'-agattagcttggctgaacatgaatccacagatcag-3' and cem197: 5'-ttacttctagagtttccctgattctggagaatgg-3' for APOBEC3B and primers cem157: 5'-agattaagcttccaaggatgaagcctcacttcag-3' and cem156: 5'-ttacttctagactcgagaatctcctgcagcttgc-3' for APOBEC3F. These cDNAs were then introduced in the HindIII and XbaI sites of the pCMV4-HA plasmid, replacing the human APOBEC3G cDNA. The resulting proteins correspond to the NP_004891 and NP_660341 NCBI entries, respectively. The cDNAs for APOBEC3C, APOBEC2, APOBEC1 and AID come from B. Matija Peterlin and Yong-Hui Zheng  and were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. All cDNAs for APOBEC family members were inserted into the same expression vector pCMV4-HA. Single-aminoacid substitutions were made on APOBEC3G coding sequence using Quickchange site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions. Plasmids for GAG expression and NC deletion construct, Zwt-p6, were kindly provided by P. Bieniasz . Plasmids for Alu retrotransposition, were a kind gift from T. Heidmann (Alu-Sb1: pAlu pA+ neoTet) and from J. Moran (L1-RP: pJM101 L1-RP Δ neo). Plasmids for SRP19 and SRP19 Δ 6 were kindly provided by Xiao-Fang Yu . For 7SL RNA-APOBEC3G binding competition experiments, sequences encoding the 7SL Alu and S-stem domains were cloned in pLVCTH  downstrSecondary structures of 7SL RNA and Alu-Sb1 were predicted using the Sfold software at the Sfold Web server (Wadsworth Center).HA-tagged APOBEC proteins and derivatives were expressed into Hela cells. In some cases competitors were co-transfected at the indicated ratios. 48 h later, total homogenate was obtained from confluent 10-cm plates using 500 μl per plate of high stringency RIPA lysis buffer  complemented with 1× Protease Inhibitor Cocktail Set I  and Prime RNase Inhibitor at 1000 units/ml (Eppendorf). Beads-immobilized anti-HA antibody (Roche) was used to immunoprecipitate HA-tagged proteins (50 μl beads + 200 μl homogenate), before extensive washes with low stringency lysis buffer and final elution with 100 μl of RNase-free distilled water.6 copies of a lentiviral genomic plasmid  . Two sets of specific primers  were used to amplify 7SL RNA by SYBR-Green Real-Time PCR (Applied Biosystems). As an internal standard, 10 were in5 HeLa cells using JetPei (Polyplus). The day after they were plated on 10 cm dishes, and selected once they had reached confluence in 2 mg/ml G418 (Invitrogen). After 60 h medium was changed and G418 concentration was reduced to 0,5 mg/ml. After 72 h colonies were fixed and coloured .pAlu pA+ neoTet (1 μg), pJM101 L1-RP Δneo (1 μg), and cytidine deaminases-expressing plasmids (1 μg) were co-transfected into 10Vif-defective HIV-1 particles were produced by transient transfection of 293T cells with Fugene (Roche) in presence or absence of antiviral cytidine deaminases with or without HIV-1 Vif. In some cases (as in figure APOBEC3G-specific immunofluorescence was performed as previously described (25).293T cells were seeded at 60% confluency in 6 cm plates in triplicates and transfected with pSR14 and pSR-Luc, which produces shRNA against SRP14 subunit and Luciferase respectively, using calcium phosphate mediated transfection. Puromycin dihydrochloride  was used to select the transfected cells 24 hours post-transfection. After 24 hours more, cells were washed with TBS  and replaced with fresh medium containing puromycin at 0.5 μg/ml. At 120 hours after transfection the cells expressing shRNA were further transfected with lentiviral plasmids together with APOBEC3G and APOBEC3F. Cells and virions were collected at 144 hours after transfection when the SRP14 and 7SL RNA are significantly downregulated. Cells were grown in the presence of cycloheximide (Sigma) during the whole procedure at 5 μg/ml concentration to improve efficiency of targeting into ER and thereby improving the viral titer.6 293T cells were seeded into 15 cm plates in duplicates approximately 24 h before transfection with HIV-1 ΔVif and SRP19myc or SRP19 Δ6myc plasmids at ratios of 4:1 and 2:1 (SRP19:HIV-1), in presence and absence of A3G and A3F. Virus was collected 36 h post medium change. Virus supernatant was cleared of cellular debris by centrifugation at 3000 rpm for 15 min in Heraeus megafuge centrifuge and filtration through a 0.2-μm pore size membrane (Millipore). Virus particles were then concentrated without sucrose cushion by ultracentrifugation at 196,000 × g for 2 h at 16°C in a Beckmann Coulter optima L-80 XP ultracentrifuge. Viral pellets were suspended in lysis buffer [ and RNase inhibitor (Promega)]. Cellular and viral RNAs were extracted using RNeasy Micro Kit  and QiAMP Viral RNA mini kit , respectively.7 × 10Normalized quantities of samples from both cells and virus were suspended in 5× sample buffer and 20× reducing agent (Fermantas) denatured for 5' at 95°C and resolved on a Tris-glycine SDS-Polyacrylamide gel followed by western blot. HA-tagged proteins (A3G and A3F) were detected using peroxydase-conjugated rat monoclonal antibody . Proliferating cell nuclear antigen (PCNA) was used as a protein loading control and was detected using a mouse monoclonal antibody  followed by a secondary sheep anti-mouse antibody conjugated to horseradish peroxidase. Myc tagged proteins (SRP19 and SRP19Δ6) were detected using C-myc rabbit polyclonal antibody (SC-789) from Santacruz followed by secondary donkey anti-rabbit antibody conjugated to horseradish peroxidase. p24 (Capsid) of virus was detected using anti p24 antibody  followed by secondary sheep anti-mouse antibody conjugated to horseradish peroxidase.The Alu and 7SL RNAs are closely related and share a common secondary structure: the Alu domain Fig. . AccordiTo investigate a hypothetical role for Alu-related RNAs in APOBEC3G HIV-1 virion incorporation, we turned to a library of point mutants of the cytidine deaminase. We identified a series of single amino acid mutants with either partial  or complete  HIV packaging defect, which correlated with an inability to block the infectivity of Δ Vif HIV-1 (not illustrated). Amongst the three mutants that completely failed to be incorporated in HIV-1 virions, W127L stood out as exhibiting the same stead-stated levels of expression and cytoplasmic localization as wild type, and moreover was fully sensitive to Vif-induced degradation Fig. . AccordiSeveral studies have pointed to the role of some cellular RNA(s) as a bridge between NC and APOBEC3G, important for the virion packaging of the CDA -14,35,36These results were consistent with a model in which 7SL RNA mediates the recruitment of APOBEC3G into HIV-1 virions, although they provided only correlative evidence. To probe the issue more directly, we examined the incorporation of the cytidine deaminase into virions produced from cells in which the SRP RNA was downregulated. First we tried to downregulate 7SL RNA by transfection of small interfering RNAs targeted against its sequence. However, these attempts were unsuccessful (data not shown). We thus turned to an indirect approach. For this, we took advantage of the fact that downregulation of SRP14 (a protein constituent of the SRP) by RNA interference leads to destabilization of the signal recognition particle and degradation of 7SL RNA Fig. . It was To confirm these data, we used a second approach. SRP19 is a key component in the formation of the SRP complex, demonstrated to be the first SRP protein to bind the 7SL RNA during assembly of the complex , and to W127L), that was stably expressed, W127L properly localized, and still sensitive to Vif-induced degradation, but defective for 7SL RNA and Alu RNA binding. This mutant also failed to inhibit Alu retrotransposition. These data support a model whereby APOBEC3G blocks Alu retrotransposition by recognizing the Alu domain common to all Alu-related RNAs, rather than the trans-acting LINE-encoded Orf2 protein. This is consistent with recent evidence suggesting that APOBEC3G does not interact with any of the polypeptides encoded by LINE-1 [In this work we characterize the binding of APOBEC3G to 7SL RNA, the nucleic acid scaffold of the signal recognition particle (SRP). The SRP contains, in addition to 7SL RNA (or SRP RNA), at least six polypeptides, named according to their apparent molecular mass . This multimolecular complex recognizes the signal peptide present at the N-terminal end of secreted and transmembrane proteins, as it emerges from ribosomes translating the corresponding mRNAs. These mRNAs are then targeted by the SRP to the endoplasmic reticulum (ER) membrane, where translation of the new ER-translocated protein continues. Assembly of the SRP proceeds through the recognition of the 7SL RNA Alu domain by the SRP9-SRP14 heterodimer , the othy LINE-1  and, whey LINE-1 . The bin7SL RNA is the most abundant RNA found in retroviral particles. For each molecule of viral genomic RNA, there are 7 molecules of 7SL RNA in an HIV-1 virion, and levels of 7SL RNA in particles are ten-thousand fold higher than those of actin messenger RNA, even though the latter is much more abundant in producer cells . 7SL RNAFinally, in an effort to elucidate the importance of 7SL RNA for APOBEC3G and 3F incorporation into HIV1 virions, we first produced virus from cells in which 7SL RNA was downregulated by RNA interference against SRP14. These particles lacked 7SL RNA but still incorporated other small cellular RNA species such as 5S and Y3, and did not exhibit significant changes in APOBEC3G and APOBEC3F levels. As the decreased infectivity of these virions somewhat hampered the interpretation of our data, we used a second approach by overexpressing SRP19, in order to sequester 7SL RNA away from assembling retroviral particles as recently described ,40. The The binding of APOBEC3G to 7SL RNA through its Alu domain suggests a mechanism for APOBEC-mediated inhibition of Alu retrotransposition. However, biochemical and functional analyses of ΔVif HIV1 particles devoid of 7SL RNA indicate that this RNA species is not essential for the virion recruitment of the antiviral cytidine deaminase.DB, SP and DT conceived the overall design of the study, BM performed the initial analysis of the A3G mutants, AL and KS helped with and supervised, respectively, the 7SL RNA binding studies, DB, SP and DT wrote the paper. All authors read and approved the final manuscript.


Schizosaccharomyces pombe have revealed the structure of centromeric heterochromatin is affected in RNAi pathway mutants. It has also been shown in fission yeast that the heterochromatin barrier is traversed by RNA Pol II and that the passage of RNA Pol II through heterochromatin is important for heterochromatin structure. Thus, an intricate interaction between the RNAi machinery and RNA Pol II affects heterochromatin structure. However, the role of the RNAi machinery and RNA Pol II on the metazoan heterochromatin landscape is not known. This study analyses the interaction of the small RNA machinery and RNA Pol II on Drosophila heterochromatin structure.Heterochromatin is the tightly packaged dynamic region of the eukaryotic chromosome that plays a vital role in cellular processes such as mitosis and meiotic recombination. Recent experiments in dcr-2, ago1, ago2, piwi, Lip [D], aub and hls). Immunofluorescence analysis of polytene chromosomes from trans-heterozygotes of RNA Pol II and different mutations of the small RNA pathways show decreased H3K9me2 and mislocalization of Heterochromatin protein-1. A genetic analysis performed on these mutants showed a strong suppression of white-mottled4h position effect variegation. This was further corroborated by a western blot analysis and chromatin immunoprecipitation, which showed decreased H3K9me2 in trans-heterozygote mutants compared to wild type or single heterozygotes. Co-immunoprecipitation performed using Drosophila embryo extracts showed the RNA Pol II largest subunit interacting with Dcr-2 and dAGO1. Co-localization performed on polytene chromosomes showed RNA Pol II and dAGO1 overlapping at some sites.The results in this paper show genetic and biochemical interaction between RNA Pol II (largest and second largest subunit) and small RNA silencing machinery components  and the small RNA silencing machinery in  The metazoan chromosome consists of two distinct functional compartments based mainly on their transcriptional competence and higher order chromatin packaging. Heterochromatin is tightly packed and has a paucity of actively transcribed genes. It plays a vital role in biological functions such as determining the distribution of meiotic recombination, telomere maintenance and sister chromatid cohesion -3. The mSchizosaccharomyces pombe and mouse cells  stock has a pericentric inversion between the white gene and the centric heterochromatin. This arrangement results in a variegated eye pattern. Many genes acting as chromatin modifiers suppress or enhance the position-effect variegation (PEV) effect. We used mutations in the second largest subunit of RNA Pol II 140. The mutant alleles used were RNA Pol II 140 (A5) and RNA Pol II 140 (wimp). The A5 allele is a null mutant with a five amino acid deletion while wimp is an antimorph , piwi[2], hls [616], hls [125], aub [QC42 ], aub [P3a] and RNA Pol II alleles  and piwi[1], as well as RNA Pol II 140 (A5) and hls [125] on DX1 homozygous flies. In each case the trans-heterozygotes reversed the silencing of mini-white to a much greater extent when compared to either single heterozygotes or control flies with no mutations. The exact molecular mechanism of DX1 silencing has not been elucidated but is believed that pairing sensitive silencing might be one of the contributing factors. The experiments performed with RNA Pol II mutants suggest that there is also involvement of a transcriptional silencing component.The suppression of PEV is a reflection of the changes in the chromatin structure of heterochromatin. The effect of RNA Pol II and RNA silencing machinery trans-heterozygote mutants was not confined to the heterochromatin environment of the chromocentre. This was shown in an experiment employing transgenic flies that have seven tandem copies of 1 Figure , that ar fashion . We testDrosophila, H3K9me2 modification is concentrated at the centric heterochromatin. H3K9me2 is also interspersed along the euchromatin arms where it is accumulated on transposable elements /+; (2)RNA Pol II 140(A5)/+; hls [E61]6/+; (3)RNA Pol II140 (A5)/+; piwi[1]/+; and (4)RNA Pol II 140(A5)/+; Lip [D]/+ [Lip is synonymous with Dmp68 [The strong suppression of PEV in trans-heterozygotes of RNA Pol II and RNA silencing machinery components led us to investigate the heterochromatin structure at the chromocentre of polytene chromosomes. In elements . We reasip [D]/+  /+ and RNA silencing machinery mutants alone showed very modest to no change in H3K9me2 levels compared to the wild type. However, trans-heterozygotes for the combined mutants showed a strong reduction in H3K9me2 levels compared with wild type and single heterozygotes . The tubulin locus (in euchromatin) did not show any enrichment of H3K9me2 nor any significant difference in the amount of H3K9me2 between the control (In(1)w [m4h];+/+), single heterozygous mutants and the double heterozygotes of RNA Pol II 140 and dcr-2(L811fsX). However, at the white (which lies in the vicinity of centromeric heterochromatin due to the inversion) locus, the double heterozygotes of RNA Pol II 140 (A5)/+; dcr-2 (L811fsX) showed significant reduction (about fourfold) of H3K9me2 compared to the control as well as to single heterozygote mutants w [m4h] genetic background indicates the importance of H3K9me2 in suppressing the white locus. The ChIP results are consistent with the PEV analysis and Western blot results.We next performed chromatin immunoprecipitation (ChIP) using H3K9me2 antibodies on adult flies. The combination of RNA Pol II and dcr-2 was selected because it gave the strongest suppression of PEV in the w mh backgroRNA Pol II215 W81) is located on the X-chromosome, a PEV analysis of male flies was not possible and the fact that a translocation balancer chromosome between the X chromosome and the second chromosome was not available precluded any immunofluoresence analysis on larval polytene nuclei. The mutant allele used was W81, which has a truncated carboxyl terminal domain (CTD) due to the presence of a premature stop codon.In addition to the second largest subunit of RNA Pol II, we also studied the effect of the RNA Pol II's largest subunit mutation on H3K9me2 levels in the adult carcass /+; dcr-2(L811fsX) showed a significant reduction of H3K9me2 in Western blot analysis compared to the wild type as well as RNA Pol II215(W81)/+ alone. The reduction of H3K9me2 with two different subunits of RNA Pol II, in combination with dicer-2 mutations, provides further evidence of a role of RNA pol II in heterochromatin formation in conjunction with RNA silencing genes.Trans-heterozygotes of Drosophila is characterized by strong deposition of heterochromatin protein-1 (HP1). With a reduction of H3K9me2, HP1 is deposited at various low affinity-binding sites along the chromosome arms  heterochromatin environment. When trans-heterozygotes of RNA Pol II140 (A5)/+; ago1(k04845)/+ were introduced into the In(1)w [m4h] background, there was moderate suppression of PEV  CTD antibody  and dAGO1 /+; TAF-1/+ and the single heterozygote TAF-1/+ shows no effect on suppression of PEV  is the major component of the transcription initiation complex in eukaryotes. Trans-heterozygotes of white RNAi. The trans-heterozygotes of (1) RNA Pol II(A5)/+; piwi[1]/+; (2) RNA Pol II (A5)/+; hls [125]/+; and (3) RNA Pol II (A5)/+; dcr-2(L811fsX)/+ had no effect on w-IR RNAi /+ heterozygotes in which the dose of RNA Pol II is halved, the effect is more pronounced on the heterochromatin structure , but the w-IR RNAi pathway is unaffected under these circumstances. To obtain further insight into the role of RNA Pol II in silencing, we tested for an effect on TGS silencing involving the interaction between Alcohol dehydrogenase-white hybrid transgenes /+ also had no effect on relieving the silencing of Adh-w by w-Adh transgenes.In order to test whether the double heterozygous combinations affect post-transcriptional functions of the RNA silencing machinery, selected genotypes were examined for an effect on ; dcr-2L8fsX/+ hadansgenes . In thesckground ,32. When)/+; hls 5/+; and Drosophila, S. pombe and mouse defects in the RNAi machinery lead to a compromised heterochromatin structure and aberrant regulation of transposable elements. Recent studies in S. pombe, Arabidopsis and mouse have further elucidated the mechanism by which small RNAs arising from heterochromatic repeat transcription modify histone methylation in the heterochromatin. Plants have an exclusive RNA Pol V dedicated for heterochromatin maintenance . In contrast, dcr-1, which has a key role in miRNA metabolism, had no role in determination of heterochromatin structure in our studies. The interaction of RNA Pol II with other proteins in the RNA silencing pathway displayed a similar level of effect on heterochromatin structure.We observed genetic and biochemical interaction between the second largest subunit of RNA Pol II and RNA silencing genes. The genetic interaction had a chromatin component in terms of suppressing PEV and reduction of H3K9me2 levels. The interaction between RNA Pol II and Dicer-2 was the strongest in terms of suppressing PEV of piwi and aub in suppressing PEV and H3K9me2 reduction in combination with RNA Pol II. PIWI and Aub associate with a unique class of rasiRNAs called piRNAs that are found predominantly in the germline cells and are independent of the Dicer processing machinery  PEV suppression and H3K9me2 levels; and (2) miRNAs have a central bulge making it difficult to explain how specificity is achieved when miRNAs base pairs imperfectly with nascent centromeric transcripts/centromeric DNA. The involvement of AGO1 probably suggests that small RNAs arising out of cleavage of nascent heterochromatic transcripts have some affinity for AGO1.Recent experiments in plants reveal the presence of a RNA Pol V complex which shares some subunits of RNA Pol II and participates in RNA silencing . BecauseS. pombe. The interaction between RNA Pol II and Dicer-2 in Drosophila reflects a similar mechanism. The exact role of the RNA Pol II second largest subunit mutations on small RNA synthesis from the centromere is not known but it is possible that it disrupts the biochemical interaction between the CTD of the largest subunit and Dicer-2. The heterochromatin specific histone modifications are dependent on both the RNA pol II complex as well as small RNA silencing machinery components.Our experiments suggest that RNA Pol II transcription through centromeric heterochromatin results in a nascent transcript that could fold into a stem loop structure by virtue of the presence of many direct and inverted repeats present in the heterochromatin. This acts as a substrate for Dicer-2 generating endo-siRNAs, which guide chromatin modifications at the heterochromatin. The interaction of Dicer with RDRC has been shown to be crucial for centromeric heterochromatin structure in All flies were grown at 25°C on standard food medium. The genotypes were:y w; RNA Pol II 140(A5)/TM3, SbRNA PolI 215(W81)/FM7aru[1] h[1] th[1] st[1]cu[1]RpII140 [wimp] sr[1[ e [s] ca[1]/TM3, Sb[1]y w; ago2(414)y w; eyFLP; FRT 42 D dcr-2(G173E)/CyOy w; eyFLP FRT 42 D dcr-2(L811fsX)/CyOy w; eyFLP FRT 82B dcr-1(Q1147X)/TM3, Sercn1 P(ry t 7.2) ago-1(04845)/CyOru st hls [E616] e ca/TM3, Sb ery cv c sbd hls [DE8]/TM2, Ubx ry ew1; hls [125] e/TM3, Sb eaub [QC42]/CyOw1; aub Df(P3a)/CyOy w; piwi[1]/CyOy w; piwi[2]/CyOy w; DX1/CyOIn(1)w [m4h]; SM6a/Gla; TM3, Sb/Ser. The single heterozygotes of RNA Pol II 140 in the w [m4h] background were then mated with RNA silencing mutants in a similar background. The F2 males were then sorted into different single and double heterozygous groups and analysed for PEV.The male flies of each of the genotypes mentioned above were crossed to female piwi[1] on DX1 silencing, the DX1(lac w) transgene array was recombined with Sco and balanced over CyO to produce DX1 Sco/CyO flies. The males of DX1 Sco/CyO were then crossed to female piwi[1]/CyO. In the next generation, female non-curly DX1 Sco/piwi[1] females were selected, which then were crossed to male w; Gla/SM6a; TM3, Ser/Sb. In the next generation non-Sco flies DX1 piwi[1]/CyO were obtained.In order to analyse the effect of Fly heads from 10 animals were homogenized in methanol containing 0.1% hydrochloric acid (HCl). The absorbance of the supernatant was measured at 480 nm after the centrifugation of the fly head homogenate. Three independent experiments were performed in each case.RNA Pol II 140 and dcr-2, piwi and hls, we crossed them into a TCyO Tb background. In the next step, males of RNA Pol II 140 and females of piwi[1], dcr-2(G173E), hls [125]  CyO Tb) were crossed and non-Tb third instar larvae were selected for further analysis. Three to four pairs of salivary glands each from control and trans-heterozygotes were dissected in 0.7% NaCl. The glands were then fixed for about a minute in 3.7% formaldehyde in phosphate buffered saline . The glands were then kept in a solution of 45% acetic acid and 3.7% formaldehyde for about 2 min and then squashed. The slide was placed on dry-ice for 20 min, the cover slip removed and then washed twice in PBS for 10 min each and blocked for 30 min in a solution of PBS containing bovine serum albumin (BSA). The following antibodies were used at 1:100 dilutions: Sxl , HP1 (from Dr S Elgin) and H3K9me2 (Upstate). The primary antibodies were incubated overnight at 4°C. On the following day, the slides were washed twice in PBS and blocked in PBS-BSA solution. The slides were then blocked with 5% goat serum for 30 min at 37°C. The secondary antibodies (1:100 goat anti rabbit-conjugated with fluorescein isothiocyanate and 1:200 goat anti-mouse conjugated with Texas red) were then applied to the slide for about 1 h at 37°C. The slides were washed twice in PBS and visualized using fluorescence microscopy after application of 4', 6-diamidino-2-phenylindole. The images were adjusted using Photoshop CS3 version software.To analyse trans-heterozygotes of The gently squashed polytene spreads  were preAdult flies (aged 12-15) were dissected to remove the ovaries. The carcasses were then homogenized in HEPES buffer containing protease inhibitor cocktail (Pierce). The homogenate was then acidified with HCl to a final concentration of 0.2 N HCl and kept on ice for 1 h. The homogenate was then centrifuged at 11,000 g for 15 min and the supernatant was then neutralized with NaOH. The histone enriched protein lysate was then boiled with Laemmli sodium dodecyl sulphate sample buffer and loaded onto the gel. The Western blot analysis was performed by standard procedures . The antWild type embryos at 6-18 h were homogenized in total lysis buffer containing 1 M Tris pH 8, 150 mM NaCl, 10 mM EDTA, 10% glycerol and protease inhibitor cocktail (Pierce). The homogenate was then kept for 15 min on ice. After centrifugation the lysate (about 500 μg) was applied to the activated amino link resin beads. The beads had RNA Pol II monoclonal antibodies 8WG16 (50 μg from Covance) covalently linked to the beads. The lysate was incubated with the beads with gentle mixing at 4°C overnight. The interacting proteins were eluted and analysed by western blots. Native mouse serum was used as a negative control. The antibodies used were Dicer-2 and Ago-1 rabbit polyclonal antibodies.Salivary glands from wild type larvae were squashed and fixed in formaldehyde and acetic acid solutions as described above. AGO1 antibody (Abcam) was diluted 1:4 in PBT (PBS+Triton X-100). In order to test for the specificity of the antibody the specific peptide bound by the AGO1 antibodies was used at 1:50 dilution, mixed with AGO1 and incubated at room temp for 30 min with occasional gentle shaking. Sxl antibody  was used as an internal control. The nuclei were observed by standard methods as described above.Drosophila animals (adults) for 10 min at room temperature. After blocking the reaction with glycine, and after three washes (5 min each at 4°C with buffer A1), subsequent steps were performed as described in  PEV compared with control and single heterozygote mutants. All the male flies were of same age (4 days after eclosion).Click here for fileImmunofluorescence analysis of polytene chromosomes in RNA Pol II and small RNA pathway trans-heterozygote mutants. H3K9me2 modification is strongly reduced in trans-heterozygotes compared with the control. The FITC (green) channel shows H3K9me2 antibody signal while the Texas red shows Sxl antibody signal. Representative images from five different experiments (approx 50 pairs of nuclei) have been examined. The genotypes of each polytene nucleus has been indicated.Click here for fileWestern blot analysis of heterochromatin protein-1 (HP1) in small RNA and RNA Pol II trans-heterozygote mutants. Western blot analysis with HP1 and tubulin (loading control) antibodies on adult carcasses of the indicated genotypes. No significant upregulation of HP-1 was observed in mutants compared with wild type. The standard error bars were calculated from three different experiments.Click here for fileWestern blot analysis to check the specificity of the dcr-2 antibody. The western blot analysis performed on third instar larvae shows the absence of the specific band at ~200 kDa in dcr-2 (L811fsX).Click here for fileAmino acid sequence of RNA Pol II second largest subunit and dAgo-2. The consensus heterochromatin protein-1 binding pentapeptide sequence (PxVxV) is highlighted in bold letters.Click here for filedcr-1(Q1147X) and RNA Pol II 140(A5) in w [m4h] backgroundEye pigment analysis of . Trans-heterozygotes of dcr-1(Q1147X) and RNA Pol II 140(A5) didn't affect position-effect variegation. Three independent replicas were performed. Standard error is shown. The genotypes of male flies are indicated.Click here for fileRole of miRNA machinery in heterochromatin formation. Immunofluorescence analysis of polytene chromosomes using H3K9me2 (FITC) and Sxl (Tx red) antibodies on the noted genotypes.Click here for fileCo-localization of AGO1 and RNA Pol II (8WG16) on polytene chromosomes. The arrows indicate sites of co-localization between AGO1 and RNA Pol II. Canton S wild type third instar larvae were used.Click here for fileAnalysis of AGO1 localization. The upper panels show AGO1 and Sex lethal proteins on female polytene chromosomes. The lower panel is the control experiment demonstrating the specificity of the antibody using AGO1 specific blocking peptide. Sxl is the internal control and it is unaffected by AGO1 blocking peptide.Click here for fileTaf-1 (TATA Box Associated factor 1) on position-effect variegation using In(1)w [m4h] male fliesEffect of . All male flies were of the same age (4 days after eclosion).Click here for fileEffect of RNA Pol II and RNA silencing machinery on white-IR post transcriptional gene silencing (PTGS). Trans-heterozygotes of RNA Pol II 140 and small RNA mutants don't affect w-IR PTGS. The genotypes of male flies are indicated.Click here for fileAdh-white (1 copy) and white-Adh (2 copies) female fliesEffect of RNA Pol II 140(A5) and small RNA silencing machinery (hls [125]) on . All flies were observed 2 h after eclosion.Click here for file


Earlier this year plant scientists met in Santa Fe, New Mexico at the Keystone Symposium "RNA Silencing Mechanisms in Plants". Sessions included small RNA biogenesis and signalling, development and stress responses, small RNA-directed DNA methylation, and interaction with pathogens. This report highlights some of the prominent and recurring themes at the meeting and emerging arenas of future research. Many of the seminal discoveries in RNA silencing were made in plant systems and, due in large part to excellent genetics, plant science continues to contribute greatly to our understanding of small RNA biology. At the Keystone Symposium "RNA Silencing Mechanisms in Plants" in Santa Fe, New Mexico, over 150 plant scientists met to share their latest research. Many of the topics discussed will undoubtedly increase understanding of RNA silencing in diverse systems.ARF3 repression and restricts ARF3 activity to the abaxial domain[ARF3 expression establishes a discreet domain of ARF3 accumulation, helping define leaf polarity.Movement of small RNAs over both long and short distances was a major topic at the meeting. The systemic aspect of RNA silencing is well documented ,2, yet tArabidopsis leaves that constitutively expressed GFP and detected patches of silencing spreading from the bombarded cells [IR71. This mutant lacks siRNAs from a portion of the inverted repeat, yet when a wild-type scion was grafted onto mutant rootstock, siRNAs were detected in roots from the entire inverted repeat, demonstrating that endogenous siRNAs or an siRNA precursor transcript from this locus move from shoot to root [To investigate whether cell-to-cell mobility is a general feature of small RNAs, Olivier Voinnet  bombarded GFP siRNAs into ed cells . To rule to root .Arabidopsis grafts to demonstrate that small RNAs are capable of long distance travel. Using deep sequencing, long-distance movement of siRNAs from thousands of genomic loci was identified [dcl2 dcl3 dcl4 triple mutant rootstocks eliminated the possibility that longer transcripts might transit before being cleaved into siRNAs in the distal tissue. These analyses indicate that siRNAs from some loci are mobile, while other siRNAs are not, and that movement of siRNAs might be responsible for many of the siRNAs that accumulate in root tissue. The Voinnet and Baulcombe groups demonstrate that mobile siRNAs are functionally indistinguishable from locally synthesized siRNAs in that they target DNA methylation in the cells that receive the signal [David Baulcombe  also described analysis of entified . Graftine signal ,6.Brassica napus phloem sap under different nutrient conditions. Several new sRNAs were identified that change abundance in phloem sap independently from the levels in leaves or roots, and it appears clear that movement of many sRNAs is controlled in response to nutrient conditions. For at least one molecule (miR395a), movement of the mature miRNA seems to enhance expression of the precursor in distal tissue [There is evidence that the plant vasculature system is a highway of small RNA molecules. Julia Kehr  addressed the biological relevance of this transport route by deep sequencing the small RNA population in l tissue . These rIR71 is transcribed in response to stress. Small RNAs from IR71 cause DNA methylation in cis and are able to silence reporter transgenes in trans [IR71 or other inverted repeats.A silenced state might be transmitted from parent to progeny ,8 in expin trans , indicatArabidopsis is released after a shift from low temperature to elevated temperature. Silencing was re-established when plants were returned to normal growth conditions, but specific genomic regions remained de-repressed. Further analysis indicates that one such genomic region contains a Copia-like retrotransposon now known as Onsen (Japanese for "hot spring"). Although Onsen remained transcriptionally active after heat treatment, no new insertions of the element were detected in wild-type plants, either somatically or in the germline. Transposition of Onsen was detected in the nrpd1 (RNA Pol IV) mutant, but only in the generation following the heat stress of seedlings. Paszkowski speculates that a variety of transposable elements in the genome respond to distinct environmental stimuli and under appropriate conditions could transpose to increase genetic diversity in progeny.Jerzy Paszkowski  also described how the environment might affect silencing by suppression rather than activation of silencing-related RNAs. He presented evidence that transcriptional gene silencing in Onsen by Pol IV-dependent siRNAs and a delay in the timing of transposition would buffer the genome against transient environmental fluctuations. A similar process, described by Janne Lempe , buffers genetic and environmental variation in order to maintain robust phenotypes. This variation is unmasked in the absence of functional HSP90 [nrpe1 and nrpd2/e2. Notably nrpe1 (RNA Pol V) mutants of Arabidopsis show significantly increased phenotypic variation and uncover cryptic genetic variation.Post-transcriptional repression of transposable elements such as Onsen. These tantalizing results indicate that RNA silencing and transgenerational effects will be a fertile field for future research in both plants and animals.Another condition known to trigger genetic and epigenetic alterations in the genome is the blending of variant genomes through interspecific hybridization. David Baulcombe is also investigating how small RNA populations change during hybridization between cultivated and wild varieties of tomato. Analysis of small RNA populations in a series of introgression lines indicates that hybridization activates small RNA production from specific genomic regions. This alteration in epigenetic state is reflected in altered gene expression, creating transgressive  expression states in introgressed lines. Surprisingly, siRNA activation is not immediate upon hybridization, indicating that critical epigenetic control might occur in the germline, similar to the retrotransposition of Arabidopsis by NRPD1 and NRPE1, respectively) and are specialized for siRNA production and transcriptional gene silencing [Another recurring theme at this meeting was the complex interactions between different polymerases. Through duplications of Pol II, plants have evolved two additional DNA-dependent RNA polymerases (Pol IV and Pol V). Pol IV and Pol V contain distinct largest subunits  described a weak allele in the second largest subunit of Pol II, NRPB2, which indicates that Pol II might transcribe a subset of loci regulated by Pol IV [If Pol IV is limited to "paused transcription" templates y Pol IV . In addiRmr7, causes almost complete lack of 24 nt siRNAs but does not cause the developmental phenotypes observed when all Pol IV activity is lost through mutation of the largest Pol IV subunit, Rmr6 [rmr6 mutants, maize plants with mutations in Rmr1, encoding a protein with similarity to SWI/SNF nucleosome remodeling enzymes, lack 24 nt siRNAs. However transposable elements are reactivated only in rmr6 plants, indicating that repression of transposable elements is due to exclusion of Pol II from the chromatin by Pol IV rather than siRNA production and RNA-directed DNA methylation [Polymerase functions are even more intricate in maize, where there are three genes for the second largest subunit of Pol IV. Jay Hollick  described how these paralogues have subfunctionalized. Loss of one gene, it, Rmr6 . The comhylation . Our undNicotiana benthamiana - an important aspect of defence against this virus [Arabidopsis RDR1 and RDR6 are required for potent defence against cucumber mosaic virus (CMV) [During replication viruses produce double-stranded RNA that is recognized and cleaved by host Dicers. These primary siRNAs also combat the virus by "slicing" single-stranded viral transcripts, thereby triggering secondary siRNA formation via host RNA-dependent RNA polymerases (RDRs). RDR activity is important in excluding potato virus X from the meristem of is virus . Shou-Weus (CMV) . During us (CMV) , it remavia RNA silencing was presented by Mathew Lewsey . He demonstrated that the CMV 2b protein repressed responses to the phytohormone jasmonic acid (JA) [Early evidence that viruses might directly manipulate host plant processes cid (JA) . JA doescid (JA) . Therefocid (JA) .Although many mRNAs are cleaved by microRNAs, only a subset of these transcripts attract RDR activity and produce endogenous secondary siRNAs (ta-siRNAs) . Presentcis and trans but are distinct from siRNAs because they do not require RDR2 for synthesis [Arabidopsis. Like their rice counterparts, Arabidopsis lmiRNAs are produced from a single strand of an RNA hairpin, require DCL3 and AGO4, and target DNA methylation to homologous DNA. But unlike rice lmiRNAs, Arabidopsis lmiRNAs also require RDR2 and Pol IV, which suggests complex biogenesis of these small RNAs in different species.Our understanding of small RNAs in plants has progressed rapidly since the advent of next-generation deep sequencing technologies and novel categories of small RNAs continue to be uncovered. Yijun Qi  described 24 nt miRNAs in rice. These long miRNAs (lmiRNAs) associate with AGO4-clade Argonaute proteins and trigger asymmetric DNA methylation in ynthesis . HailingDRD701 (OsRDR6) and OsDCL4, presumably for production of a double-stranded precursor, but they do not require OsDCL3a, the Dicer responsible for cleavage of non-phased 24 nt siRNAs. The increased availability of plant genome sequences and the decreasing price of small RNA deep sequencing will undoubtedly sustain the discovery of novel small RNA classes for some time to come.Xiaofeng Cao  also described a recently discovered category of small RNA in rice - phased 24 nt siRNAs. In addition to hundreds of loci producing ta-siRNA-like phased 21 nt siRNAs, rice contains many loci producing 24 nt phased siRNAs . These "The Keystone Symposium "RNA Silencing Mechanisms in Plants" was a stimulating meeting with many excellent scientific presentations. It is clear that plant systems are poised to unravel many important questions in RNA silencing, including systemic movement and the transgenerational impact of small RNA, interaction between endogenous and virus-derived silencing systems, and mechanics of secondary siRNA production. Solving these riddles and applying plant models to other systems will ensure RNA silencing in plants remains a fertile field.The authors declare that they have no competing interests.