Tag Archives: Drosophila

The Transposon/piRNA/Chromatin Nexus
















Uploading piRNAs to the Cloud.












Opening up the RNA-chromatin network

In eukaryotic nuclei, DNA is coiled around histone proteins to form nucleosomes. The pattern by which nucleosomes are compacted into higher-order structures determines the accessibility of chromatin and hence it’s transcriptional activity. Many different factors, including the linker histone H1, histone modifications, chromatin remodelling enzymes and non-histone proteins play important roles in structuring chromatin. Various classes of RNA have also been implicated in regulating the higher-order structure of chromatin. Among many examples; Argonaute associated small silencing RNAs are known to sometimes exert their inhibitory effects by directing histone modifications or DNA methylation and lncRNAs have been shown to serve as cis-acting scaffolds coordinating the action of histone-modifying enzymes. It’s been known for decades that RNA makes up a proportion of chromatin, but exactly what types of RNAs and what their roles are is not yet clear. A new paper in Molecular Cell (Schubert et al.) sets out to answer these questions, characterising chromatin-associated RNAs in Drosophila and finding an important role for RNA in regulating chromatin compaction and accessibility.

Schubert et al. found that RNAs were involved in maintaining the accessibility of chromatin using an assay in which chromatin is digested by a nuclease (DNase). This digestion creates a ladder of DNA of different sizes on a gel, ranging from single nucleosomal fragments to far larger pieces. The extent of digestion is dependent on the level of chromatin condensation; the more compacted the nucleosomal structure, the more refractory it will be to DNase digestion. The researchers found that incubation of chromatin with an RNase prior to DNase treatment resulted in more compacted chromatin; DNase digestion was less efficient. Using different RNases and inhibitors they discovered that the RNA population involved was single-stranded and synthesised by RNA polymerase II.

Similar results were found when the authors used density-gradient centrifugation to isolate chromatin. They found a fraction of RNA associated with chromatin (caRNA) that when digested, resulted in chromatin becoming more compacted and shifting to higher density fractions. Interestingly, this RNA-dependent chromatin condensation effect is reversible; when the compacted chromatin was extracted and incubated with fresh cellular extracts, it reopens, again migrating in lighter fractions, re-associated with RNA. This caRNA-dependent chromatin accessibility is also dependent on chromatin-associated proteins, as it could not be rescued under denaturing conditions.

Using mass spectrometry, the authors identified 59 proteins that had lower affinity for chromatin after removal of RNA. One of these was the highly abundant chromatin decondensation factor 31 (Df31). A study that I have long intended to write about (Filion et al. 2010) used the binding of Df31 and 52 other chromatin associated proteins to determine that rather than dividing Drosophila chromatin into two types: transcriptionally repressed heterochromatin and active euchromatin, we should instead think in terms of five different classes which they colour coded. Green, blue and black chromatin are broadly transcriptionally repressed, whilst red and yellow are euchromatic and more transcriptionally active. Df31 is found bound to these red and yellow types of more open chromatin. Schubert et al found that Df31 chromatin binding is stabilised by caRNAs. Df31 binds histone H3 in the absence of RNA, but its affinity is substantially enhanced by the addition of RNA. RNAi knockdown of Df31 causes a fraction of genomic DNA to be more compacted.

Deep sequencing of the pool of caRNAs revealed that they were enriched for non-coding RNAs, especially a class termed small nucleolar RNAs (snoRNAs). snoRNAs are known to guide the modification of bases in ribosomal, transfer and messenger RNAs. They have also been implicated in RNA editing and splicing. Schubert et al. found that 30 of the 186 snoRNAs expressed in Drosophila embryos were found associated with chromatin. Using fluorescent in situ hybridisation, they showed that two of the most highly enriched snoRNAs could be visualised binding to the interbands of Drosophila polytene chromosomes (ie. euchromatin). These two snoRNAs were able to ‘rescue’ compacted chromatin in the density-gradient experiments, and were shown to directly interact with Df31.

The authors also found that extracts of human cells could rescue RNase treated compacted chromatin, and that snoRNAs are found associated with chromatin in human cells.

Schubert et al have therefore characterised a novel conserved role for snoRNAs mediating the accessibility of higher-order chromatin structures. As none of the protein components known to complex with snoRNAs in snoRNPs were identified in the mass spectrometry experiments, it appears that snoRNAs form distinct ribonucleoprotein complexes to mediate this chromatin associated role. Df31 is one such important interactor, linking chromatin-associated snoRNAs and histone proteins within nucleosomes to maintain accessibility of red and yellow chromatin. However, the knockdown of Df31 resulted in far more limited and localised chromatin compaction than RNase treatment, suggesting that the role of caRNAs goes beyond the maintenance of open euchromatin, but also to regulating heterochromatic accessibility. Seeing as 58 other chromatin proteins, as well as many other snoRNAs and other caRNAs, were implicated in RNA-associated chromatin accessibility functions, this initial study has just revealed the tip of an iceberg. Just how this RNA-chromatin network functions to maintain accessibility requires a lot of work. As with many of the best studies, the light shone serves to partially illuminate the scale of our ignorance.

Schubert T, Pusch MC, Diermeier S, Benes V, Kremmer E, Imhof A, & Längst G (2012). Df31 Protein and snoRNAs Maintain Accessible Higher-Order Structures of Chromatin. Molecular cell PMID: 23022379

Filion GJ, van Bemmel JG, Braunschweig U, Talhout W, Kind J, Ward LD, Brugman W, de Castro IJ, Kerkhoven RM, Bussemaker HJ, & van Steensel B (2010). Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell, 143 (2), 212-24 PMID: 20888037

Upstream ORFs and Regulation of Translation

Protein expression can be rapidly and responsively regulated at the level of translation. Translational regulation commonly involves trans-acting factors such as miRNA complexes or RNA-binding proteins, specifically binding cis-regulatory sequences. A study from last year – Medenbach et al. – has demonstrated an interesting mechanism in which translation of a transcript is repressed by the initiation of translation at a short upstream open reading frame (uORF).

Sexual organisms need a mechanism to balance the levels of transcription from sex chromosomes between sexes. In mammals this ‘dosage compensation’ is achieved by inactivating one of the X chromosomes in females; Drosophila instead hypertranscribes the single X chromosomes of male flies. In females, hypertranscription is prevented by the translational silencing of male-specific lethal (msl)-2 mRNA by a key sex determination factor, the RNA-binding protein Sex-lethal (SXL).

SXL exerts translational control on msl-2 transcripts by two distinct mechanisms. Binding in the 3’UTR, in conjunction with a co-repressor, it blocks the recruitment of the ribosomal pre-initiation complex to the 5’UTR. Any pre-initiation complexes that escape this control are then challenged by a failsafe mechanism; SXL bound to the 5’UTR causes destabilisation of the small ribosomal subunit upstream of the SXL-binding site.

Medenbach et al. analysed the mechanisms of this 5’UTR SXL-mediated translational control, using constructs in which altered msl-2 5’UTRs drove expression of a reporter gene in a cell-free system. The msl-2 5’UTR contains three small upstream ORFs. Mutations preventing initiation in the first two of these uORFs did not impair translational control, but when the third uAUG was mutated the reporter construct was translationally de-repressed. This effect was dependent on a SXL-binding site slightly downstream of the uORF, mutation of which abrogated the derepression. The repression was dependent on the presence of SXL. By itself the uORF reduced translation of the downstream reading frame two fold, but in the presence of SXL, downstream translation was repressed more than 14 fold.

The uORF encodes a di-peptide (Methionine-Threonine). By swapping the threonine-encoding codon and the subsequent stop codon, and by deleting the stop codon, the researchers showed that translational repression required uORF initiation and not translational elongation or termination.

Medenbach et al. went on to investigate just how widespread this mechanism of translational control may be. Computationally scanning the Drosophila transcriptome, they found that 58% of 5’UTRs contain one or more upstream initiation codons, whilst 4.3% contain putative SXL binding sites. 268 mRNAs (1.3%) contain both a uORF and a SXL-binding motif at the appropriate distance from one another to make them candidates for a similar mode of SXL-mediated translational repression. They then tested 12 of these candidate 5’UTRs in their reporter assay, and found that 6 of them did indeed mediate SXL-dependent translational repression. They also demonstrated that the regulatory cassette consisting of the uORF, the intervening 21nt, and the SXL-binding site was capable of mediating repression when inserted into the 5’UTR of an unrelated gene.

uORFs have previously been shown to regulate translation of the main reading fame in other contexts. The authors reference two examples using different mechanisms; a system involving termination and reinitiation from 4uORFs in a budding yeast transcript that doesn’t require any trans-acing factors; and another fungal mechanism in which ribosomal stalling at the termination codon of an uORF causes the mRNA to be degraded. In contrast, the translation regulation system in the msl-2 5’UTR utilises a binary module requiring only translational initiation at the uORF and binding of SXL. How exactly this works with respect to the ribosomal pre-initiation complex is not yet clear. Recognition of an initiation codon triggers a conformational change in this complex from a scanning ‘open’ structure to a closed conformation, followed by subunit joining to make a complete translationally competent ribosome. The authors found that SXL interacts with two subunits of the elF3 initiation factor, but weren’t able to prove the importance of these interactions for translational repression.

Medenbach et al’s computational scan of the Drosophila genome showed that the use of this binary module is an important mechanism by which SXL regulates the expression of many proteins. Similar uORF modules may be utilised by other RNA binding proteins, but it’s also possible that other uORFs function in quite different ways, as suggested by the examples from fungi. What’s beyond doubt, seeing as more than half of mammalian and Drosophila genes contain them is that uORFs are an important factor in translational regulation.

Medenbach J, Seiler M, & Hentze MW (2011). Translational control via protein-regulated upstream open reading frames. Cell, 145 (6), 902-13 PMID: 21663794

Amyloid-like oligomers and long-term memory.

A new paper in Cell, shows that the formation of amyloid-like oligomers of a RNA-binding protein in synaptic membranes is necessary for the persistence of memories in Drosophila.

If memory is the maintenance of learning associated changes in synaptic efficacy and number, and these synaptic modifications are associated with changes in protein composition, how do they persist for years when individual protein molecules are turned over in days?

Cytoplasmic polyadenylation element-binding proteins (CPEBs) are a family of RNA-binding proteins that have been shown to regulate activity-dependent protein synthesis at synapses. A Drosophila CPEB, Orb2, has been shown to be required for the long-term persistence of memory (Keleman et al.). Previous studies on a CPEB from the sea slug Aplysia, had shown that it had prion-like properties, in that it could exist in two conformational states: a monomeric form and a self-perpetuating multimeric form with amyloid-like properties. Synaptic stimulation leads to the conversion of Aplysia CPEB into the multimeric form, and the formation of a self-sustaining synaptic mark. Blocking the activity of multimeric ApCPEB inhibited the long-term facilitation of a synaptic response (Si et al.).  Based on these Aplysia studies, Majumdar et al. hypothesised that Orb2’s role in Drosophila long-term memory could also be mediated by activity-dependent formation of amyloid-like oligomers.

Purifying Orb2 from Drosophila head extracts, Majumdar et al. show that it either exists in a monomeric form, or in oligomers consisting of between two and six Orb2 molecules. Orb2 oligomers are resistant to denaturation by heat or detergents, showing that they have amyloid-like characteristics. The oligomeric form of Orb2 appears to be localised to synaptic membranes, whilst the monomeric form is mainly found in cell bodies. Stimulation of neurons involved in memory formation increased the level of oligomeric Orb2.

Majumdar et al. go on to show that there are two different isoforms of Orb2 involved in the formation of Orb2 oligomers, Orb2A and Orb2B. Although the Orb2 oligomers found in vivo were mainly composed of Orb2B, the researchers found Orb2 oligomerisation requires Orb2A. Both isoforms contain a glutamine rich unstructured prion-like domain that acts as a substrate for oligomerisation, but a short 8 amino acid N-terminal domain of Orb2A catalyses the oligomerisation reaction. Orb2A appears to regulate the formation of amyloid-like Orb2 oligomers.

The previous study (Keleman et al.) that showed that Orb2 was involved in long-term memory stabilisation had used a technique that reduced the Orb2 concentration. By generating flies that carried a single point mutation in the Orb2A N-terminal domain, specifically impairing Orb2 oligomerisation, Majumdar et al. went on to show Orb2 oligomerisation is necessary for long-term memory persistence.

This work is exciting on two different fronts. It shows a mechanism for the generation of stable synaptic marks by the formation of amyloid-like oligomers. This could be a more widespread phenomenon underlying memory formation and persistence. More broadly, this study yields insight into potential physiological roles for amyloid formation and prion-like mechanisms.

Insoluble amyloid-like aggregations are a common pathological characteristic in human neurodegenerative conditions such as Alzheimer’s disease and Parkinson’s disease. In the case of prion diseases such as Creutzfeld-Jacob disease, amyloidogenic forms of Prion-Protein propagate the disease by seeding the conformational changes to the non-pathogenic form. The authors of the paper do not call the Orb2 oligomers prions, as the mechanisms underlying oligomerisation are not understood. However, in the case of ApCPEB, multimerisation was shown to be self-sustaining, and hence prion-like.

Is there a link between the preponderance of amyloid associated neurodegenerative diseases and the role of amyloid-like oligomers in memory? It is probably unwise to extrapolate too far from these results. However, one could postulate that if this is a widespread mechanism of memory formation, amyloid associated diseases could be more likely to affect the brain. Likewise, if amyloid-like protein multimerisation has normal physiological roles in the brain, it could be a more permissive environment for amyloid formation than other tissues.

Majumdar, A., Cesario, W., White-Grindley, E., Jiang, H., Ren, F., Khan, M., Li, L., Choi, E., Kannan, K., Guo, F., Unruh, J., Slaughter, B., & Si, K. (2012). Critical Role of Amyloid-like Oligomers of Drosophila Orb2 in the Persistence of Memory Cell, 148 (3), 515-529 DOI: 10.1016/j.cell.2012.01.004

Si, K., Choi, Y., White-Grindley, E., Majumdar, A., & Kandel, E. (2010). Aplysia CPEB Can Form Prion-like Multimers in Sensory Neurons that Contribute to Long-Term Facilitation Cell, 140 (3), 421-435 DOI: 10.1016/j.cell.2010.01.008

Keleman, K., Krüttner, S., Alenius, M., & Dickson, B. (2007). Function of the Drosophila CPEB protein Orb2 in long-term courtship memory Nature Neuroscience, 10 (12), 1587-1593 DOI: 10.1038/nn1996

small silencing RNAs. I: Piwi-interacting RNAs.



The piRNA system in Drosophila



The piRNA pathway in somatic follicle cells


The germline piRNA pathway and ping-pong amplification.







of further interest: piRNAs in the brain: epigenetics and memory