RNA-mediated Transcriptional Silencing Research Articles

Control of Chromatin by RNA-mediated Transcriptional Silencing

Control of Chromatin by RNA-mediated Transcriptional Silencing

Long-range control of gene expression via RNA-directed DNA methylation.

RNA-mediated transcriptional silencing, in plants known as RNA-directed DNA methylation (RdDM), is a conserved process where small interfering RNA (siRNA) and long non-coding RNA (lncRNA) help establish repressive chromatin modifications. This process represses transposons and affects the expression of protein-coding genes. We found that in Arabidopsis thaliana AGO4 binding sites are often located distant from genes differentially expressed in ago4. Using Hi-C to compare interactions between genotypes, we show that RdDM-targeted loci have the potential to engage in chromosomal interactions, but these interactions are inhibited in wild-type conditions. In mutants defective in RdDM, the frequency of chromosomal interactions at RdDM targets is increased. This includes increased frequency of interactions between Pol V methylated sites and distal genes that are repressed by RdDM. We propose a model, where RdDM prevents the formation of chromosomal interactions between genes and their distant regulatory elements.

A magnesium-binding nucleotide, a remodeling ATPase, and a wonderful RNA world.

Flipping through the first issue of RNA, two things specifically attracted my eyes: the original edition of The RNA World by Gesteland and Atkins, and a paper by Yi-Tao Yu et al. That classic monograph collected invaluable commentaries and reviews on the whole spectrum of RNA research from the legends and pioneers, on which I often relied for wisdom and insight. That paper by Tim Nilsen's group describing a phosphorothioate substitution study of U6 snRNA in nematode, and along with a similar study in yeast U6 by John Abelson's group, provided early chemical evidence that the bulging uridine (U69 in nematode and U80 in yeast) at the 3′ stem-loop of the U6 snRNA is likely an essential catalytic constituent of the spliceosome. Following on those observations, Shyue-Lee Yean, a postdoc in my lab, spent a good three to four years trying to find answers to a simple question: which comes first, the phosphorothioated U6 block or the Prp2 step? At that time Jean Beggs’s and my groups have shown that Prp2 is an RNA-dependent ATPase that is required for the onset of splicing in the assembled spliceosome. We knew that the ATP hydrolysis by Prp2 induces a conformational rearrangement of the spliceosome that proceeded catalysis. Would phosphorothioated U69/U80 inhibit the spliceosome activation step by Prp2? Shyue-Lee initially synthesized the U6 RNA using polymerase incorporation of the modified nucleotides, but the imprecision of the 3′ ends and low yields forced him to use ligase to stitch together chemically synthesized RNA fragments. Luckily with the help from John Termini's group we not only made the RP stereoisomers but also the SP isomers. The experiments not only answered our initial question—modified U6 does not block the Prp2 step—but also uncovered an unanticipated, magnesium coordinating, and catalytically relevant activity of U80SP. With recent elegant works by the groups of Joe Piccirilli and Jon Staley (in addition to the structural work by the groups of Sam Butcher and Dave Brow) we now know that U80 indeed participates in catalytic metal coordination along with additional nucleotides of U6, and that the spliceosome is indeed a ribozyme. As for Prp2, through the effort of the groups of Soo-Chen Cheng and Reinhard Luhrmann, among others, we now know that the ATP hydrolysis by Prp2 leads to reshuffle several proteins onto or off the spliceosome, explaining the “down-sizing” of the spliceosome by Prp2 that we observed almost two decades ago. A sequence between the branchpoint and the 3′ splice site is critical for productive Prp2 action; however, whether U6 snRNA is involved in this remains unclear. By working on yeast and human splicing factors, I found that not only human factors have more features but also the human cells have more complex responses to splicing perturbations. This is really nothing new; however, it reminds me of the conversation that I had with my musician daughter. She said that different music (classical, jazz, blues, etc.) has different building blocks as well as ways to arrange them; only with an understanding of that structure will a person be able to appreciate the meaning of that music (other than just a feeling). I wonder, although all biological systems have similar or conserved rules, some systems may predominantly use certain types of rules while others rely more on different ones. We have seen some intriguing examples for this in RNA biology—piRNA, RNA-dependent RNA polymerases, RNA-mediated transcriptional silencing, etc. Shall we be surprised if there are more—grand or subtle—to be found? In the midst of organizing and teaching an advanced graduate course on RNA, I found that over two-thirds of the course dealt with topics that were just or not even discovered when RNA was first published. Marvelously, those topics (microRNA, long non-coding RNA, CRISPR RNA, etc.) attract students to the RNA field and create a common language among colleagues from different fields. A legend and recombinant DNA guru in our institution had predicted that the past 50 years of DNA-focused research would give way to RNA-focused research. I am afraid that he is right, again. I would like to thank Tim Nilsen for the opportunity to share these few thoughts with the RNA readers. Although I cannot predict where RNA research is heading, I fully anticipate many new discoveries and excitement regarding RNA will continue in the next 20 years and beyond, so long as we remain curious and keep exploring (with generous supports from funding agencies).

Non-Coding RNAs in Transcriptional Regulation: The review for Current Molecular Biology Reports.

Transcriptional gene silencing guided by small RNAs is a process conserved from protozoa to mammals. Small RNAs loaded into Argonaute family proteins direct repressive histone modifications or DNA cytosine methylation to homologous regions of the genome. Small RNA-mediated transcriptional silencing is required for many biological processes, including repression of transposable elements, maintaining the genome stability/integrity, and epigenetic inheritance of gene expression. Here we will summarize the current knowledge about small RNA biogenesis and mechanisms of transcriptional regulation in plants, Drosophila, C. elegans and mice. Furthermore, a rapidly growing number long non-coding RNAs (lncRNAs) have been implicated as important players in transcription regulation. We will discuss current models for long non-coding RNA-mediated gene regulation.

Specific Tandem Repeats Are Sufficient for Paramutation-Induced Trans-Generational Silencing

Paramutation is a well-studied epigenetic phenomenon in which trans communication between two different alleles leads to meiotically heritable transcriptional silencing of one of the alleles. Paramutation at the b1 locus involves RNA-mediated transcriptional silencing and requires specific tandem repeats that generate siRNAs. This study addressed three important questions: 1) are the tandem repeats sufficient for paramutation, 2) do they need to be in an allelic position to mediate paramutation, and 3) is there an association between the ability to mediate paramutation and repeat DNA methylation levels? Paramutation was achieved using multiple transgenes containing the b1 tandem repeats, including events with tandem repeats of only one half of the repeat unit (413 bp), demonstrating that these sequences are sufficient for paramutation and an allelic position is not required for the repeats to communicate. Furthermore, the transgenic tandem repeats increased the expression of a reporter gene in maize, demonstrating the repeats contain transcriptional regulatory sequences. Transgene-mediated paramutation required the mediator of paramutation1 gene, which is necessary for endogenous paramutation, suggesting endogenous and transgene-mediated paramutation both require an RNA-mediated transcriptional silencing pathway. While all tested repeat transgenes produced small interfering RNAs (siRNAs), not all transgenes induced paramutation suggesting that, as with endogenous alleles, siRNA production is not sufficient for paramutation. The repeat transgene-induced silencing was less efficiently transmitted than silencing induced by the repeats of endogenous b1 alleles, which is always 100% efficient. The variability in the strength of the repeat transgene-induced silencing enabled testing whether the extent of DNA methylation within the repeats correlated with differences in efficiency of paramutation. Transgene-induced paramutation does not require extensive DNA methylation within the transgene. However, increased DNA methylation within the endogenous b1 repeats after transgene-induced paramutation was associated with stronger silencing of the endogenous allele.

Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing

The tRNA gene-mediated (tgm) silencing of RNA polymerase II promoters is dependent on subnuclear clustering of the tRNA genes, but genetic analysis shows that the silencing requires additional mechanisms. We have identified proteins that bind tRNA gene transcription complexes and are required for tgm silencing but not required for gene clustering. One of the proteins, Mod5, is a tRNA modifying enzyme that adds an N6-isopentenyl adenosine modification at position 37 on a small number of tRNAs in the cytoplasm, although a subpopulation of Mod5 is also found in the nucleus. Recent publications have also shown that Mod5 has tumor suppressor characteristics in humans as well as confers drug resistance through prion-like misfolding in yeast. Here, we show that a subpopulation of Mod5 associates with tRNA gene complexes in the nucleolus. This association occurs and is required for tgm silencing regardless of whether the pre-tRNA transcripts are substrates for Mod5 modification. In addition, Mod5 is bound to nuclear pre-tRNA transcripts, although they are not substrates for the A37 modification. Lastly, we show that truncation of the tRNA transcript to remove the normal tRNA structure also alleviates silencing, suggesting that synthesis of intact pre-tRNAs is required for the silencing mechanism. These results are discussed in light of recent results showing that silencing near tRNA genes also requires chromatin modification.

A SWI/SNF Chromatin-Remodeling Complex Acts in Noncoding RNA-Mediated Transcriptional Silencing

SummaryRNA-mediated transcriptional silencing prevents deleterious effects of transposon activity and controls the expression of protein-coding genes. It involves long noncoding RNAs (lncRNAs). In Arabidopsis thaliana, some of those lncRNAs are produced by a specialized RNA Polymerase V (Pol V). The mechanism by which lncRNAs affect chromatin structure and mRNA production remains mostly unknown. Here we identify the SWI/SNF ATP-dependent nucleosome-remodeling complex as a component of the RNA-mediated transcriptional silencing pathway. We found that SWI3B, an essential subunit of the SWI/SNF complex, physically interacts with a lncRNA-binding protein, IDN2. SWI/SNF subunits contribute to lncRNA-mediated transcriptional silencing. Moreover, Pol V mediates stabilization of nucleosomes on silenced regions. We propose that Pol V-produced lncRNAs mediate transcriptional silencing by guiding the SWI/SNF complex and establishing positioned nucleosomes on specific genomic loci. We further propose that guiding ATP-dependent chromatin-remodeling complexes may be a more general function of lncRNAs.

Expanding the action of duplex RNAs into the nucleus: redirecting alternative splicing

Double-stranded RNAs are powerful agents for silencing gene expression in the cytoplasm of mammalian cells. The potential for duplex RNAs to control expression in the nucleus has received less attention. Here, we investigate the ability of small RNAs to redirect splicing. We identify RNAs targeting an aberrant splice site that restore splicing and production of functional protein. RNAs can target sequences within exons or introns and affect the inclusion of exons within SMN2 and dystrophin, genes responsible for spinal muscular atrophy and Duchenne muscular dystrophy, respectively. Duplex RNAs recruit argonaute 2 (AGO2) to pre-mRNA transcripts and altered splicing requires AGO2 expression. AGO2 promotes transcript cleavage in the cytoplasm, but recruitment of AGO2 to pre-mRNAs does not reduce transcript levels, exposing a difference between cytoplasmic and nuclear pathways. Involvement of AGO2 in splicing, a classical nuclear process, reinforces the conclusion from studies of RNA-mediated transcriptional silencing that RNAi pathways can be adapted to function in the mammalian nucleus. These data provide a new strategy for controlling splicing and expand the reach of small RNAs within the nucleus of mammalian cells.

Intended transcriptional silencing with siRNA results in gene repression through sequence-specific off-targeting

Transcriptional gene silencing has been reported with siRNA targeting the promoter region of genes. We tested several siRNAs directed against the human VEGF promoter. Of these, siVFp(-992) exhibited > or =50% suppression of VEGF production in two human cell lines. To determine the specificity of this siRNA-mediated suppression, plasmids were prepared to express a luciferase reporter under the control of VEGF promoters featuring wild-type, mutated, or deleted target sequences. siRNA transfection assays established sequence-specific inhibition of luciferase from the reporter plasmid featuring the wild-type VEGF promoter. However, siVFp(-992) also suppressed the luciferase expression from the plasmids with mutated or deleted target sites, suggesting that silencing was due to a sequence-specific off-target phenomenon, and this was supported by subsequent microarray and bioinformatics analyses. To determine if our concerns regarding the specificity of promoter targeting siRNAs were relevant to other systems where RNA-mediated transcriptional silencing had been previously reported, we tested a published small RNA sequence directed to the HIV(SF2)-LTR promoter. siRNA transfection assays performed in human cells expressing a luciferase reporter gene under the control of the HIV(SF2)-LTR promoter revealed significant suppression whether the target sequence was intact or mutated, or when the entire HIV(SF2)-LTR was replaced by an irrelevant promoter. These data stress the need to examine target specificity when conducting investigations into transcriptional gene regulation with siRNA.

HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double-stranded RNA.

To analyze relationships between RNA signals, DNA methylation and chromatin modifications, we performed a genetic screen to recover Arabidopsis mutants defective in RNA-directed transcriptional silencing and methylation of a nopaline synthase promoter-neomycinphosphotransferase II (NOSpro- NPTII) target gene. Mutants were identified by screening for recovery of kanamycin resistance in the presence of an unlinked silencing complex encoding NOSpro double-stranded RNA. One mutant, rts1 (RNA-mediated transcriptional silencing), displayed moderate recovery of NPTII gene expression and partial loss of methylation in the target NOSpro, predominantly at symmetrical C(N)Gs. The RTS1 gene was isolated by positional cloning and found to encode a putative histone deacetylase, HDA6. The more substantial decrease in methylation of symmetrical compared with asymmetrical cytosines in rts1 mutants suggests that HDA6 is dispensable for RNA-directed de novo methylation, which results in intermediate methylation of cytosines in all sequence contexts, but is necessary for reinforcing primarily C(N)G methylation induced by RNA. Because CG methylation in centromeric and rDNA repeats was not reduced in rts1 mutants, HDA6 might be specialized for the RNA- directed pathway of genome modification.