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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.