You'd Prefer An Argonaute

Most “Dark Matter” Transcripts Are Associated With Known Genes

Harm van Bakel, Corey Nislow, Benjamin J. Blencowe, Timothy R. Hughes

PLoS Biology, 8 (5): e1000371, 18 May 2010.
doi:10.1371/journal.pbio.1000371

This week’s illuminating summary and analysis by Igor Ulitsky. It’s Igor’s second contribution to the blog:

The paper is one of at least five interesting RNA-Seq papers that came out in the past month or so (see also Guttman et al. and Trapnell et al. in the May issue of Nature Biotechnology, Kim et al. from Nature 465 (7295): 182-7 and De Santa et al. from the same issue of PLoS Biology). All these studies harness the awesome power of Illumina RNA-Seq to look at (mainly) the murine transcriptome and to try to figure out what it consists of, and how what RNA-Seq tells us differs from what we knew previously. Unfortunately, since the reads in those studies are still <75 long, RNA-Seq still can’t tell us exactly what the transcripts in the cell are, but rather what regions of the genome seem to give rise to RNA (or more precisely – which regions of the genome can we uniquely align reads to). This problem is partially alleviated by paired-end RNA-Seq used in the Nature Biotech papers, but it still has limited power for deciphering transcripts expressed at low levels. The five studies mentioned above tell three different stories about the transcriptome – the two Nature Biotech papers talk about how it is possible to identify thousands of novel exons in known genes, and also to give significantly more accurate exonic structures to some of the previously proposed long non-coding RNAs (lincRNAs). Kim et al. and De Santa et al. talk about a surprising amount of RNA coming from enhancer regions in the mouse genome, RNA whose exact function remains a mystery. The study we’re focusing on – van Bakel et al., tackles a more global question – how much of the polyA+ RNA comes from “known genes”, and how much from everything else – the “dark matter”. This question is naturally of high interest, but addressing it involves a wealth of caveats:

• What are “known genes” (i.e., “non-dark matter”) – protein-coding ones? miRNAs? Coding and non-coding ones with known functions?
• What expression levels can be considered functional? Are all transcripts with relatively low expression levels just noise?
• Are short RNA-Seq reads really informative in terms of the number of different RNA species?
• Are we confident enough in the annotation of the genome with pseudogenes and repeats, both of which can contribute to spurious mappings in intergenic regions?

Despite these caveats, van Bakel et al. do a thorough job of at least trying to answer this question, and do their best to convince the readers that, in fact, very little transcription from the mammalian genome is “dark matter”. When analyzing RNA-Seq data, the majority of the genome does not seem to give rise to detectable polyA+ RNA segments. Then how did ENCODE and related studies report as much as 80% of the genome as being transcribed? The authors begin by showing that tiling arrays are prone to give rise to many false positive calls. They do so by comparing their own tiling array data from human and mouse tissues to published and novel RNA-Seq datasets. Unfortunately, the sets are not completely matched (different labs/starting materials), but the data very convincingly shows that for transcripts with low expression data, the signal from tiling arrays is practically the same as the background – a fertile ground for false positive calls. It is interesting to note that the first part of the paper shows in fact that the data that the authors generated themselves (the tiling arrays) is worse than previously published data (RNA-Seq).

From that point on, the authors focus on RNA-Seq data. They find relatively few completely intergenic stand-alone transcripts that are not captured in some way in the “known genes” databases or at least in existing EST/mRNA collections. This is not very surprising given the effort involved in sequencing ESTs in mouse/human – it could hardly be expected that a lot of polyadenylated transcripts would be abundant in RNA-Seq, but missing from those datasets. It should be kept in mind though, that many of what the authors call “known genes” are in fact non-coding transcripts (based on lack of a long/conserved ORF) with completely unclear function. What about the sequence fragments (seqfrags) that do fall outside of the “known genes” boundaries? About 80% of those reads fall within 10kb of known genes and are likely to represent either unannotated parts of those genes, or transcripts whose biogenesis function is related to the gene adjacent to them, as their expression is generally highly correlated with their neighbors. What about the rest? Are there any interesting RNAs out there in the intergenic space? Well, there are some – the authors identify about 11,000-16,000 seqfrags that are located >10kb away from any known gene and that are significantly different from expected. The novel intergenic transcripts tend to overlap regions of open chromatin –identified using DNAse I hypersensitivity – which suggests that at least some of them could be the enhancer-associated transcripts reported in the parallel studies.

The authors then go on to show that by looking at splice junctions derived from the reads (using the popular TopHat tool) they can reach roughly the same conclusion – most of the spliced polyA+ RNA is already “known” to us. They can still identify about 5,000 novel exons in “known genes”, and those share the general characteristics of the known exons, albeit with lower expression and conservation levels. The imminent problem from this section, and from all the other recent RNA-Seq studies is this: Is anyone keeping track of all those new exons? Updating RefSeq/UCSC/Ensembl? How to update these databases is also an excellent question, as short-read-based studies cannot give us a complete (or close to complete) snapshot of the actual transcript. Anyhow, at this pace, we expect to see many additional papers re-discovering the same set of 5,000 novel exons.

The bottom line?

1. The outback of the genome rarely gives rise to highly expressed and polyA+ transcripts. This does not mean that there is shortage of putative lincRNAs – hundreds of them are already in the “known genes” set, and others may be functional despite low expression levels/proximity to known genes. The jury is still out on the polyA-transcriptome.
2. Annotation of the “canonical genes” in the mouse/human genomes is still not complete and both can be complemented with several thousand additional exons. Let’s hope somebody is keeping track.
3. Many intergenic RNAs are likely to be enhancer-associated (but we still don’t understand why).

This paper (as well as the other recent RNA-Seq studies) was definitely interesting to read, and we can only look forward to what we will learn once long-read RNA-Seq (e.g., Pacific Biosciences) kicks in.

Citation for researchblogging.org:

van Bakel H, Nislow C, Blencowe BJ, & Hughes TR (2010). Most “dark matter” transcripts are associated with known genes. PLoS biology, 8 (5) PMID: 20502517

A dicer-independent miRNA biogenesis pathway that requires Ago catalysis

Sihem Cheloufi, Camila O. Dos Santos, Mark M. W. Chong   &  Gregory J. Hannon

Nature, 465: 584–589, 3 June 2010.
Nature AOP, 27 April 2010.
doi:10.1038/nature09092

This week’s summary and ruminative analysis by Vikram Agarwal. It’s Vikram’s second contribution to the blog:

In this article, Cheloufi and colleagues demonstrate an alternative biogenesis pathway for the maturation of a microRNA. A key question that this study seeks to address is why a member of the Argonaute family of proteins has retained its catalytic activity throughout millions of years of mammalian evolution. Though a handful of examples are known of miRNA-mediated cleavage events in animals, none have been shown to be crucial for target gene regulation and cell viability. The evidence thus suggests the possibility that the catalytically active residues of Argonaute have been conserved for purposes that are distinct from target cleavage.

The authors initially explore the consequences of losing catalytic activity of Argonaute during early mouse embryogenesis. They find that Ago2 is expressed ubiquitously in the early mouse embryo and in nearby placental tissues. Mice with a mutated catalytic residue of Ago2 develop normally through embryogenesis, but exhibit an anemic phenotype and die shortly after birth. Further cell sorting experiments confirm a problem in the maturation of erythrocytes, leading to an accumulation of pro-erythroblasts prior to birth.

During a check of global miRNA expression patterns in wild type and mutant mice, the authors discover that a single miRNA, miR-451, is aberrantly expressed in catalytically deficient mice. This miRNA is already known to have important roles in erythrocyte differentiation. More interestingly, small RNAs mapping to its precursor do not correspond to a canonical pattern one would expect if the miRNA were processed by Dicer. The precursor lacks a detectable miR* fragment, and the 3′ end of the mature miRNA is derived from the loop of the hairpin rather than the stem. The processing of the precursor is biochemically shown to be Drosha, but not Dicer, dependent, when compared to canonical miRNAs such as miR-294 and miR-16. The concluding experiments of the work investigate the role of Argonaute in the processing of pre-miR-451. In vitro purified Ago2 is demonstrated to be sufficient to produce a mature miR-451 cut, although the cleavage product is nearly 8-10 bases longer than expected. Detection of shorter products in in vivo samples, along with untemplated U addition in the 3′ end, implicate the role of an unknown exonuclease in the ultimate maturation of miR-451 into its shorter, functional form.

Overall, the biochemical evidence presented in this study is compelling, and strongly supports a model in which the Piwi domain of Ago2 catalytically cleaves miR-451. A conclusion that is less clear, though presented as an underlying motivation for the study, is that this non-canonical processing pathway explains the retention of Ago2 catalytic activity. An experiment that would provide support to this hypothesis would be to see whether a miR-451 knock-out recapitulates a similar phenotype as a catalytically-deficient Ago2 mutant. At present, it is possible that the target cleavage ability of Argonaute, or a yet unknown mechanism requiring its catalytic potential, could explain the evolutionary pressure for its conservation. Intuitively, it is difficult to comprehend why this non-canonical pathway has been conserved for only a single miRNA out of the hundreds encoded in the genome. The time spans involved during the evolution of the precursor are so vast that there must have been at least some opportunity for small insertions to have converted the miRNA into one that is processed canonically, or produced a novel miRNA convergently with the same seed sequence. Only further work will help elucidate why such strong evolutionary pressure exists to preserve this non-canonical processing mechanism.

Citation for researchblogging.org:

Cheloufi S, Dos Santos CO, Chong MM, & Hannon GJ (2010). A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature PMID: 20424607

Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA

Wenqiang Yu, David Gius, Patrick Onyango, Kristi Muldoon-Jacobs, Judith Karp, Andrew P. Feinberg  &  Hengmi Cui

Nature 451: 202-206, 10 January 2008.
doi:10.1038/nature06468

Cooperation Between Translating Ribosomes and RNA Polymerase in Transcription Elongation

Sergey Proshkin, A. Rachid Rahmouni, Alexander Mironov, Evgeny Nudler

Science, Vol. 328. no. 5977: pp. 504 – 508, 23 April 2010.
DOI: 10.1126/science.1184939

This week’s smart summary and analysis by Pavan Vaidyanathan:

In this paper, the authors propose a mechanism for active physical cooperation between an elongating RNA polymerase (RNAP) and a translating ribosome following the polymerase on the transcribed message.

It has been known for several decades that in prokaryotes the processes of transcription and translation are coupled and occur simultaneously. Ribosomes are loaded on to a message as it is getting transcribed by RNAP. This coupled process serves to maximize efficiency of protein synthesis and allows for rapid changes in gene expression. It is also well established that the coupling of translation to transcription can serve as a means of regulation of RNAP. For instance, if the rate of translation is not as high as the rate of transcription, the increasing distance between the ribosome and RNAP allows for the transcription terminator Rho to bind the message and cause RNAP dissociation from the message. Additionally, numerous amino acid biosynthesis operons are regulated by the mechanism of transcription attenuation in which the rate of translation determines the formation of secondary structure behind RNAP that either allows the continuation of transcription or terminates transcription. In this paper however, the authors propose a novel means of regulation of transcription that depends on a physical interaction between RNAP and the first trailing ribosome behind the polymerase.

The authors first showed that in vivo, the ratio of the rate of transcription (nt/sec) to the rate of translation (aa/sec) is consistently ~3.0 in a variety of environmental conditions and stages of growth. Based on this observation, the authors hypothesize that the rate of transcription in vivo is determined by the rate of translation. To test this, they determined the rate of transcription of a plasmid-derived lacZ message in cells grown in media containing low concentrations of ribosome-binding antibiotic (chloramphenicol). They observed that slowing down the ribosome also slowed down the rate of transcription. They confirmed this by testing the rate of transcription of various messages harboring increasing amounts of rare codons, which are expected to slow down translation. As expected, the rate of transcription was inversely proportional to the percentage of rare codons in the message.

RNAP, like other polymerases, is known to backtrack on a message quite frequently. However, the presence of multiple polymerases on the same message significantly decreased backtracking. The authors hypothesized that the ribosome could improve the efficiency of transcription by serving as a physical block to backtracking and ‘forcing’ the polymerase to go forward. In order to test their model, the authors developed an assay to monitor RNAP backtracking in vivo. The assay employed the single-stranded DNA binding probe, chloroacetaldehyde, to monitor the migration of the transcription bubble. They observed that when a single RNAP molecule was forced to encounter a roadblock (lac repressor bound to DNA), it paused and backtracked. However, when there were two RNAP elongation complexes transcribing the same message, this backtracking was reduced substantially. Similarly, the presence of a ribosome behind RNAP also significantly reduced the incidence of backtracking suggesting that the ribosome could improve transcription by preventing backtracking of the leading elongation complex (EC). Using a similar roadblock system, the authors additionally showed by Northern blot analysis of the transcribed mRNA that the leading EC could had much higher rates of readthrough of the roadblock when it was followed by a second EC or by a ribosome.

The authors conclude that the ribosome directly controls the rate of transcription by preventing RNAP backtracking. Because of this cooperation, the rate of transcription is determined by codon usage and nutrient availability as sensed by the ribosome thus allowing precise regulatory adjustment of transcription to translational needs.

Citation for researchblogging.org:

Proshkin S, Rahmouni AR, Mironov A, & Nudler E (2010). Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Science, 328 (5977), 504-8 PMID: 20413502

DNA Methylation Mediated by a MicroRNA Pathway

Liang Wu, Huanyu Zhou, Qingqing Zhang, Jianguang Zhang, Fangrui Ni, Chang Liu, and Yijun Qi

Molecular Cell, Volume 38: 465-475, 08 April 2010.
doi: 10.1016/j.molcel.2010.03.008

Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP

Markus Hafner, Markus Landthaler, Lukas Burger, Mohsen Khorshid, Jean Hausser, Philipp Berninger, Andrea Rothballer, Manuel Ascano, Jr., Anna-Carina Jungkamp, Mathias Munschauer, Alexander Ulrich, Greg S. Wardle, Scott Dewell, Mihaela Zavolan, and Thomas Tuschl

Cell 141, 129–141, 2 April 2010.
DOI: 10.1016/j.cell.2010.03.009

A Splicing-Independent Function of SF2/ASF in MicroRNA Processing

Han Wu, Shuying Sun, Kang Tu, Yuan Gao, Bin Xie, Adrian R. Krainer, and Jun Zhu

Molecular Cell 38: 67–77, 9 April 2010.
DOI: 10.1016/j.molcel.2010.02.021

Differential regulation of microRNA stability

Sophie Bail, Mavis Swerdel, Hudan Liu, Xinfu Jiao, Loyal A. Goff, Ronald P. Hart and Megerditch Kiledjian

RNA, Advance Online Article, 26 March 2010.
doi: 10.1261/rna.1851510

Caspase-Dependent Conversion of Dicer Ribonuclease into a Death-Promoting Deoxyribonuclease

Akihisa Nakagawa, Yong Shi, Eriko Kage-Nakadai, Shohei Mitani, Ding Xue

Science Express, 11 March 2010.
doi: 10.1126/science.1182374

This week’s methodische summary and analysis–impressively her third contribution to this blog–by Anna Drinnenberg:

In this paper Nakagawa and colleagues describe a new role for Dicer (DCR-1) in the apoptotic pathway of C. elegans. Briefly, the apoptotic pathway can be triggered by many different cellular stimuli, resulting in caspase activation and subsequent fragmentation of nuclear DNA. DNA fragmentation can be separated into two steps: (i) During the first phase a caspase-activated DNA endonuclease catalyzes formation of DNA nicks and breaks (ii) In mammals Caspase 3 activates the Caspase-activated deoxyribonuclease CAD generating 3’ hydroxylated (3’ OH) DNA breaks that can be detected by the TUNEL (TdT-mediated dUTP nick end Labeling) assay. Other endonucleases including EndoG, which translocates from the mitochondria to the nucleus, function at later stages to complete degradation of genomic DNA. Mutants of one or combinations of those endonucleases result in the accumulation of TUNEL-stained nuclei because the resolution of 3’ OH DNA breaks is impaired.

C. elegans has homologues of Caspase3 (CED-3) and EndoG (CPS-6), as well as other endonucleases involved in the second stage of DNA fragmentation, however, a homologue of CAD had not been found in the C. elegans genome. This group therefore aimed to identify the endonuclease that catalyzes the initial formation of 3’ hydroxylated DNA breaks. They performed an RNAi screen in the cps-6 deletion background and selected mutants that showed a decrease in TUNEL signal. DCR-1, a protein so far only known to be involved in RNAi pathways of C. elegans and other species was one hit from the screen.

They further confirmed the TUNEL results using DCR-1 deletion alleles, indicating that DCR-1 acts upstream of CPS-6 and other endonucleases. Moreover, they showed that DCR-1 has pro-apoptotic activity by counting the number of cell corpses during C. elegans embryogenesis in wild-type and dcr-1 deletion strains. Deletion strains of other factors involved in the C. elegans RNAi pathway did not show the same phenotypes indicating that the pro-apoptotic function of DCR-1 is independent of its role in the RNAi pathway. Furthermore, they found that DCR-1 is processed by CED-3, which cleaves its first RNAseIII domain. They named the c-terminal part of truncated form of DCR-1, “tDCR-1”, which lacks most of the full-length protein domains including the Helicase, the PAZ, and even one of the RNaseIII domains.

Studies in vitro incubating tDCR-1 with dsRNA or plasmid DNA showed loss of RNase activity, but a gain of DNase activity. The DNase activity of tDCR-1 appears to be weak, resulting in just a single cut of plasmid DNA instead of complete fragmentation. This would be consistent with a role for tDCR-1 in generating 3’OH DNA nicks, whereas completion of DNA fragmentation is carried out by other endonucleases like CPS-6. It would be therefore interesting to compare the enzymatic activity of tDCR-1 to CAD in the same in vitro assay. They also did some very interesting experiments that convincingly tied the function of the different forms of DCR-1 to either being involved in the apoptotic pathway or the RNAi pathway. For example, they rescued the mutant RNAi phenotype, but not the mutant apoptosis phenotype, of dcr-1 deletion animals by expression of an allele of DCR-1 resistant to CED-3 cleavage. In a complementary experiment, expression of tDCR-1 in the dcr-1 deletion background resulted in rescue of the apoptotic pathway but not the RNAi pathway. Finally, they showed the same acidic amino acids are important for both RNase and DNase activity, demonstrating the similarity of both enzymatic activities.

Nakagawa and collegues performed a very comprehensive study characterizing the role of DCR-1 acting as a functional analog of CAD, indicating that a conserved, caspase-mediated mechanism activates the apoptotic DNA degradation process in both C. elegans and mammals. Further studies could include visualizing the relative abundance of tDCR-1 versus full-length DCR-1 in the nucleus and the cytoplasm. Moreover, studies in other organisms, including those that have a homolog of CAD, could determine if the pro-apoptotic role of DCR-1 is specific to C. elegans or is conserved in other species as well. In conclusion, this study shows the participation of the same protein in two different unrelated pathways, an economical use of the genetic repertoire.

Citation for researchblogging.org:

Nakagawa A, Shi Y, Kage-Nakadai E, Mitani S, & Xue D (2010). Caspase-Dependent Conversion of Dicer Ribonuclease into a Death-Promoting Deoxyribonuclease. Science PMID: 20223951

Alternative splicing produces high levels of noncoding isoforms of bHLH transcription factors during development

Rahul N. Kanadia and Constance L. Cepko

Genes & Development 24 (3): 229-234, 1 February 2010.
doi: 10.1101/gad.1847110

Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins

Brian R. McNaughton, James J. Cronican, David B. Thompson and David R. Liu

PNAS 106 (15): 6111-6116, 14 April 2009.
doi: 10.1073/pnas.0807883106

This week’s shrewd summary/analysis–impressively his third contribution to this blog–by David Weinberg:

In their 2009 PNAS paper, David Liu and colleagues demonstrate that a green fluorescent protein (GFP) variant that has been engineered to have a positively charged surface can penetrate mammalian cells and also chaperone nucleic acids into those same cells. The story began in 2007 when Liu’s lab published their initial characterization of so-called “supercharged” proteins. The motivation was to determine how changing the net charge of a protein can affect its stability. To do this, they began with an extra-stable GFP and changed as many surface-exposed residues as possible to positively charged residues (i.e., lysine and arginine). This resulted in a GFP variant with a net charge of +36 (herein referred to as +GFP) that folded and fluoresced similarly to the original GFP. Amazingly, however, +GFP was highly resistant to aggregation: boiling of the protein eliminated activity, but cooling the boiled protein restored most of the activity. This stabilizing effect of supercharging was not unique to GFP, as similarly supercharged variants of GST (a dimer) and streptavidin (a tetramer) showed similar properties.

So what does all of this have to do with RNA? In 2007, Liu’s lab also noted that +GFP could use its positively charge surface as “molecular Velcro” that can reversibly bind to RNA (tRNA) or DNA (plasmid dsDNA). Of course, +GFP-bound RNA is useless (to a first approximation) in a test tube. Where the PNAS paper begins is with the hypothesis that +GFP might be able to enter cells and thereby escort its bound nucleic acid cargo into the cell as well. The authors begin by conclusively showing that +GFP penetrate a variety of mammalian cells with an efficiency that varies with its charge. To satisfy the cell biologists in the audience, they provide an initial dissection of the mechanism of +GFP uptake using a variety of (mostly chemical) perturbations. From this, they conclude that the mechanism involves energy-dependent endocytosis that is dependent on actin polymerization and sulfated (positively charged) cell surface peptidoglycans but does not require caveolin or clathrin. Now focusing back on RNA, the authors demonstrate that +GFP can bind to siRNAs in vitro – not surprising given their previously published data showing that it can bind to tRNA. Moreover, +GFP-bound Cy3-labeled siRNAs can enter HeLa cells – their FACS data suggest that virtually all cells in the population take up the siRNAs to a similar degree, yielding a quite homogenous population of “transfected” cells. Although Lipofectamine (a standard transfection reagent) can also deliver Cy3-siRNA to HeLa cells, +GFP delivers ~100-fold more siRNA based on fluorescence. More impressively, in 4 other cell lines that are virtually resistant to Lipofectamine-mediated transfection, +GFP delivers huge amounts of Cy3-siRNA without any significant cytotoxicity. Not only do these siRNAs enter the cell, but they can interact with the RNA interference machinery and mediate gene silencing. Further characterization of +GFP-siRNA complexes reveals that +GFP enhances the stability of siRNA in serum and the protein is itself relatively stable in serum. Although less impressive in its efficiency, +GFP (with an HA tag) can also be used to transfect plasmid DNA that gets expressed in the nucleus.

The authors conclude that +GFP may provide an attractive alternative to nucleic acid delivery. Because it uses a general pathway (endocytosis) for delivery, it should in theory work in all cell types. In addition, it is relatively easy to use since it only requires mixing of recombinant +GFP (which can be readily made in E. coli) and the nucleic acid of interest, and adding this to cells. But beyond this immediate application as a transfection reagent, it seems that +GFP and supercharged proteins more generally may become a useful tool for all sorts of biology (which I have no doubt the Liu lab is already exploring).

Citation for researchblogging.org:

McNaughton BR, Cronican JJ, Thompson DB, & Liu DR (2009). Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins. Proceedings of the National Academy of Sciences of the United States of America, 106 (15), 6111-6 PMID: 19307578

Dicer-Independent Primal RNAs Trigger RNAi and Heterochromatin Formation

Mario Halic and Danesh Moazed

Cell 140 (4): 504-516, 19 February 2010.
doi: 10.1016/j.cell.2010.01.019