You'd Prefer An Argonaute

Targeted 3′ Processing of Antisense Transcripts Triggers Arabidopsis FLC Chromatin Silencing

Fuquan Liu, Sebastian Marquardt, Clare Lister, Szymon Swiezewski, Caroline Dean

Science Express: 3 December 2009.
doi: 10.1126/science.1180278

This week’s punctilious summary and analysis by Igor Ulitsky:

The group of Caroline Dean in Norwich, as well as several other groups, has been extensively studying the regulation of the FLC gene in Arabidopsis. FLC is a key transcription factor in the flowering process, which is very tightly controlled in plants. Their studies have uncovered an extensive regulatory network that converges at the FLC locus, the mechanisms of which include transcriptional regulation, chromatin modification, small RNAs, and now also RNA processing. Two papers on FLC from Dean’s group were published last week – one in Science (Liu et al., the one I’ll focus on) and another in Nature (Swiezewski et al. Nature Vol 462). The Science paper focuses on the so-called “autonomous pathway” of FLC regulation, which promotes flowering by repressing FLC. Previous genetic analyses from the same group have uncovered a number of genes involved in this process, including two RNA-binding proteins, FCA and FPA, the cleavage and polyadenylation specificity factor FY, and an H3K4me2 demethylase, FLD. They have also previously shown (mostly in Liu et al., Molecular Cell 2007) that FCA is physically associated with the chromatin in an intron of FLC, between exons 6 and 7, but did not find clear evidence that FCA binding leads to processing of the FLC gene itself. Instead, it seemed that FCA played a role in regulating the transcription from the FLC locus, through chromatin modification by FLD. In addition, they characterized two anti-sense transcripts in the FLC locus, one short, ending around the physical location of FCA on the chromatin, and one long, ending at the FLC promoter. Interestingly, fca mutants had a higher long form/short form ratio than WT plants.

In this paper, Liu et al. first conducted a genetic screen for suppressors of FCA over-expression (which strongly repressed FLC). They used a fusion of FLC to LUC, and identified several genes that could activate FLC in presence of a strongly activated FCA. These included the known players FY and FLD, but also two additional genes, subunits of the CstF 3’ processing complex, which is highly conserved and essential in many species, including Arabidopsis. Through epistasis analysis, they could show that the CstF subunits indeed function in the FCA pathway. In addition, they show that these mutants have increased transcription of FLC, as evidenced by nascent RNA levels, Pol II binding, and H3K4me3 signatures. Their previous findings now pointed to a role of these subunits in regulating the long form/short form ratio of the antisense transcript. The paper doesn’t convincingly show that the sense FLC transcript is not affected, but it seems that the authors are convinced somehow that only the antisense is affected. Indeed, they find that the CstF mutants fail to process the 3’ of the antisense transcript, which leads in general to higher transcription of the anti-sense, which is similar to the elevation observed in fca mutants, and coincides with an increase in the FLC sense transcript. Overall, in different mutants in the FCA pathway, there is an increase in the abundance of the long form of the anti-sense transcript over the short form. The authors’ model for what happens in the WT strain, in which FCA represses FLC, is as follows: FCA/FY/CstF, through 3’ processing of the antisense transcript, causes shift in long-form/short-form ratio, which leads somehow to recruitment of FLD, which removes the H3K4me2 marks from the body of the FLC gene, leading eventually to down-regulation of both the sense and the anti-sense transcripts. They speculate on what may be the missing link between FCA/FY/CstF and FLD, but there is no clear evidence supporting it.

While the paper focuses on a single gene in Arabidopsis, there are several lines of evidence that this kind of regulation through 3’ processing of an antisense transcript occurs in other regulatory programs. In general, although the paper does not mention it, this kind of mechanism could explain why some regulatory proteins have conserved biding sites in the introns of their targets, as at least some of them may play roles in RNA processing of both the sense and the anti-sense transcipts.

To summarize, this paper, as well as another report on the FLC locus, show how amazingly complicated a relatively straight forward regulatory program (shutting down a target gene) can be. Another global implication that I found in this paper is how useful yet misleading genetic interactions can be. The authors found a genetic link between FCA and FLD, the chromatin modifier at the end of the pathway, in one of their previous studies. However, they could not find a direct physical link between them. As it appears now, FLD appears several pathway steps downstream of FCA, with complex machinery for 3’ processing of the antisense transcript separating them.

The paper was very interesting to read, and it gave a decent introduction to FLC pathway regulation. However, it wasn’t an easy feat to understand it thoroughly. The details of the unfolding of the pathway were not really clear in the first scan, and it was very difficult to understand which results were actually novel, and which have been reported previously. Part of the problem could be the extreme length limitations of Science publications, and lack of simple “textbook” figures in the paper.

RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex

Caryn R. Hale, Peng Zhao, Sara Olson, Michael O. Duff, Brenton R. Graveley, Lance Wells, Rebecca M. Terns and Michael P. Terns

Cell 139 (5): 945-56, 25 November 2009.
doi:10.1016/j.cell.2009.07.040

This week’s ace summary and analysis by Robin Friedman:

The CRISPR (clustered regularly interspaced short palindromic repeats) system is a set of DNA sequences and associated genes involved in prokaryotic immune defense. Since it was discovered that CRISPR loci generate small (usually 25-60 nt) RNAs that often match phage or plasmid sequence, it has been tempting to make an obvious analogy with eukaryotic RNA interference, which can also be used to protect against viruses. However, what little was known about CRISPR mechanism pointed to a DNA-dependent mechanism of invader recognition and defense. One complicating factor is that there are at least nine distinct subtypes of the CRISPR system based on different sets of Cas (CRISPR-associated) genes. In this paper Hale et al. examine a subtype, Cmr, that had not previously been studied.

The authors first showed that in P. furiosis, there are two main species of CRISPR RNA product (termed psiRNAs), 39 and 45 nucleotides long. They purified protein complexes containing these psiRNAs and subjected them to mass spectrometry, finding seven members of the CRISPR-associated Cmr family. Sequencing of the psiRNAs revealed that each species had an 8-nucleotide “psi-tag”, consisting of the 3’ end of the constant repeat sequence, followed by unique guide sequence.

To test the mechanism of the psiRNA-Cmr complex, the authors used several synthetic constructs with sequence similarity to P. furiosis psiRNAs. The complementary RNA sequence was specifically cleaved at two spots, but the sense RNA sequence, unrelated RNA sequences, and a complementary DNA sequence were not cleaved. Truncations of these synthetic complementary RNAs showed that cleavage occurs in the same location, suggesting that the Cmr complex cleaves 14 nucleotides from the 3’ end of the psiRNA. Finally, the authors reconstituted the Cmr complex in vitro with recombinant proteins and synthetic psiRNA and recapitulated the cleavage behavior of the native complex. Only one of the six included Cmr proteins were dispensable for cleavage.

This is one of the simplest Cell papers I have seen. However, its few results are thoroughly proven. There are many questions left to answer about CRISPR function both in this model system and in others, but the scope of this paper is merely to show that the CRISPR system can function through RNA cleavage. This paper finally provides evidence strengthening the appealing analogy between CRISPRs and eukaryotic RNAi, which is sure to stimulate more interest in the system.

Distinct Argonaute-Mediated 22G-RNA Pathways Direct Genome Surveillance in the C. elegans Germline

Weifeng Gu, Masaki Shirayama, Darryl Conte, Jessica Vasale, Pedro J. Batista, Julie M. Claycomb, James J. Moresco, Elaine M. Youngman, Jennifer Keys, Matthew J. Stoltz, Chun-Chieh G. Chen, Daniel A. Chaves, Shenghua Duan, Kristin D. Kasschau, Noah Fahlgren, John R. Yates, Shohei Mitani, James C. Carrington and Craig C. Mello

Molecular Cell 36: 231-244, 1 October 2009.
doi:10.1016/j.molcel.2009.09.020

This week’s comprehensive summary/analysis by Michael Nodine:

Upon screening for mutants defective in RNAi, Craig Mello’s group found that the DICER-RELATED HELICASE-3 (DRH-3) gene was required for germline and soma RNAi. Gu et al. also found that DRH-3 was required for endogenous siRNA (esiRNA) production and/or stability.  When examining the requirement of DRH-3 for esiRNA production more closely, they found that DRH-3 was involved in the production of a specific class of esiRNAs, which they termed the 22G-RNAs due to their length and preference for a 5’ guanosine. 22G-RNAs were found to not have 5’-monophosphates, which are typically found in DICER products, and this observation led the authors to hypothesize that 22G-RNAs are RNA-dependent RNA polymerase (RdRP) products rather than DICER products. They then cloned small RNAs from wild-type and drh-3 samples using a 5’-independent ligation method, and found that 22G-RNAs mapped to ~50% of the protein coding genes annotated in the C. elegans genome.

Interestingly, 22G-RNAs tended to map to the 3’-ends of genes and there was less of a requirement for DRH-3 for 22G-RNAs derived from gene 3’-ends. Since the drh-3 mutants contained point mutations in the conserved helicase domain, this hinted at the possibility that DRH-3 may be part of an RdRP complex and may facilitate its movement along the RNA template by removing inhibitory secondary structures. Consistent with this idea, they found that two RdRPs were redundantly required for 22G-RNA production, and that these two RdRPs along with the tudor domain-containing protein EKL-1 interacted with DRH-3.

They then went on to find that worm-specific Argonautes (WAGOs) were redundantly required for 22G-RNA production. Genes, transposons, pseudogenes and cryptic loci were all found to be targets of 22G-RNAs, and components of the non-mediated decay (NMD) pathway were demonstrated to play a role in the biogenesis of at least a subset of 22G-RNAs. Gu et al. also demonstrated when and where 22G-RNAs function during worm development. WAGO-1 was localized to P-granules, which are localized just outside nuclear pores in the female germline and are thought to play a role in maternal RNA repression and storage. In addition, high-throughput sequencing and developmental northerns suggested that 22G-RNAs are enriched in the female germline and maternally inherited.

Thus, 22G-RNAs are key components of a surveillance pathway, which operates in the female germline and represses protein coding genes, pseudogenes and transposons. Presumably, incorrectly processed protein coding transcripts are targets for 22G-RNA biogenesis/action. However, it remains unknown how aberrant transcripts are recognized. Transcripts lacking poly(A) tails were previously demonstrated to be better substrates for C. elegans RdRPs in vitro, and incorrectly processed transcripts are better substrates for RdRP-dependent RNAi in plants. Fission yeast nucleotidyl transferases have been implicated in the recognition of aberrant transcripts by RdRPs and exosomes, and a homologous nucleotidly transferase, as well as a 3’-5’ exonuclease were found to be required for 22G-RNA production.  Based on these observations, the authors suggest that a nucleotidyl transferase and a 3’-5’ exonuclease, both of which were shown to be required for 22G-RNA production, may function in an exosome-like complex to recognize aberrant transcripts and/or recruit the 22G-RNA RdRP complex. Finally, 22G-RNA pathway components are subcellularly positioned just outside female germline nuclei. Based on their observations, the authors hypothesize that 22G-RNA components may ‘monitor’ the female germline transcriptome and thus function in the surveillance of maternally-inherited RNAs.

qiRNA is a new type of small interfering RNA induced by DNA damage

Heng-Chi Lee, Shwu-Shin Chang, Swati Choudhary, Antti P. Aalto, Mekhala Maiti, Dennis H. Bamford & Yi Liu

Nature 459: 274-277, 14 May 2009.
doi:10.1038/nature08041

This week’s aufschlussreiche summary and analysis by Anna Drinnenberg:

The term “quelling” refers to a posttranscriptional gene silencing phenomenon observed in Neurospora crassa, and was one of the first RNAi pathways to be described (Romano and Macino, 1992). Quelling is triggered by the expression of transgenes, also called “aberrant RNAs,” and results in silencing of both transgenes and cognate endogenous transcripts. It involves the production of double-stranded RNA (dsRNA) by an RNA-dependent RNA polymerase (QDE-1) using the transgenic mRNA as a template. Subsequently, one of two Dicer proteins (DCL-1 and DCL-2) cleaves the dsRNA substrate into small RNA duplexes that get loaded into an Argonaute effector complex containing QDE-2. After cleavage and exonucleolytic digestion of the passenger strand, the other siRNA strand functions as a guide strand in QDE-2 to degrade homologous endogenous transcripts. The physiological role of quelling is thought to be control of transposon expansion in order to preserve genomic integrity.

The authors of this study suggest a new physiological role for components of the quelling pathway in the response to DNA damage. During the process of studying the regulation of QDE-2 they noticed that the expression of QDE-2, at both the mRNA and protein level, is upregulated upon DNA damage caused by adding Histidine, EMS, or Hydroxyurea to the media. Immunoprecipitating QDE-2, they identified a new class of small RNAs ~21nt in length whose abundance is increased in the IP following DNA damage. Interestingly, these small RNAs appear to be shorter than the previously identified siRNAs (~25nt) of the quelling pathway that are produced by the same Dicer proteins (Catalanotto et al., 2004). It will be interesting to determine if the Dicer proteins, that are thought to act redundantly (Catalanotto et al., 2004), can produce small RNAs of different lengths or if the interaction with a cofactor could determine the cleavage interval on the dsRNA substrate.

Most of the small RNAs, which they referred to as “qiRNAs,” are derived from the sense and antisense strands of an rDNA array exceeding the regions that are transcribed into rRNA by Pol1, suggesting that a distinct transcript gives rise to the precursor (aberrant RNA) for the qiRNAs. They noticed that the production of aberrant RNA was not inhibited by thioelutin, a known inhibitor of RNA polymerases. In an attempt to identify the protein that produces the initial qiRNA precursor transcript from the rDNA array, they observed that QDE-1, already known to have RdRP catalytic activity, can also synthesize RNA transcripts using a DNA template. This is a very interesting observation and raises the question if RdRPs in other organisms also have DNA-dependent RNA polymerase activity. Such an activity would make the production of precursor RNAs for small RNAs independent from the canonical transcription pathway for the majority of other cellular RNAs.

In trying to assign a role to the qiRNAs, the authors noticed that the decrease in protein production upon DNA damage is partially blocked in QDE-1 and QDE-3 mutant strains. Moreover, QDE-1 and DCL-1/DCL-2 mutants show increased sensitivity to DNA damage reagents. These observations certainly provide a first hint of function of this pathway. A more detailed follow-up experiment could be a more precise demonstration that the qiRNAs in complex with QDE-2 directly downregulate rRNA transcripts (but this experiment was beyond the scope of this study).

Another recent publication from Cerere and Cogoni (Cecere and Cogoni, 2009) suggests that the small RNAs are involved in copy number control of the rDNA locus, possibly preventing recombination within the array. Changes in the heterochromatic state of the rDNA array could unify both observations: the block in downregulation of the transcripts and the increased recombination rate. Moreover, high-throughput sequencing of the small RNAs as well as RNAseq analysis in Neurospora crassa upon DNA damage might also identify other qiRNA sources and their potential targets.

References:
Catalanotto, C. et al. (2004). Redundancy of the two dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa. Mol Cell Biol 24, 2536-2545.

Cecere, G., and Cogoni, C. (2009). Quelling targets the rDNA locus and functions in rDNA copy number control. BMC Microbiol 9, 44.

Romano, N., and Macino, G. (1992). Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6, 3343-3353.