|
Short RNAs in Stress and Longevity
Regulatory Pathways
Short RNAs in Stress and Longevity
Regulatory Pathways
Speakers: Frank Slack (Yale University), Ramanjulu Sunkar (Oklahoma State University), Evgeny Nudler (New York University), Anthony Leung (Massachusetts Institute of Technology), Irina Groisman (André Lwoff Institute), Germano Cecere (Columbia University)Presented by the Non-coding RNA Biology Discussion GroupReported by Don Monroe | Posted October 13, 2009 OverviewSince the 1990s, researchers have recognized a growing number of ways in which naturally occurring short RNAs regulate aspects of gene expression, both in normal processes like development and in diseases like cancer. On May 12, 2009, the Academy's Non-coding RNA Biology Discussion Group gathered to explore how these endogenous RNAs affect stress responses and longevity.
Speakers discussed a variety of small RNAs and their roles in determining lifespan, focusing on work in Drosophila, C. elegans, mice, and plants. Topics included recent research on how microRNAs could be used as biomarkers in aging; the role of endogenous small RNAs in plant stress responses; how small RNAs function in heat shock response in eukaryotes; the function of the polymer poly(ADP-ribose) in regulating activity of RNA and RNA-binding proteins in stress granules; and how microRNAs regulate cell senescence. Use the tabs above to find a meeting report and multimedia from this event.
Introduction
Since the 1990s, when scientists discovered that short RNAs can alter gene expression, these segments have become essential tools in biology research. Over the same period, researchers have recognized a growing number of ways in which naturally occurring short RNAs regulate aspects of expression, both in normal processes like development and in diseases like cancer. On May 12, 2009, the Academy's Non-coding RNA Biology Discussion Group gathered to explore how these endogenous RNAs affect stress responses and longevity.
In this context, "longevity" refers not simply to staying alive, but to intrinsic programs that appear to determine lifespan in a wide variety of species. Researchers have explored many facets of these programs, including their close relationships to caloric intake, insulin signaling, reproduction, and stress. Many compelling results come from studies of the roundworm, C. elegans. Frank Slack of Yale University showed that many of the endogenous microRNAs in this creature vary systematically with age, and that some of them appear to be uniquely expressed in older animals. Indeed, manipulating these molecules can decrease, or more significantly increase, the average lifespan of a worm population. Equally important, these molecules could provide biomarkers that flag individuals who will live anomalously long lives.
MicroRNAs are often thought to regulate genetic activity by modulating translation of complementary targets, either directly or by degrading messenger RNA. However, as symposium organizer Alla Grishok of Columbia University has shown in C. elegans, short RNAs can also regulate transcription, presumably by modifying chromatin. Grishok's postdoc Germano Cecere described experiments to identify small interfering RNAs (siRNAs) that use this mechanism endogenously, and the genes that they may regulate. A significant fraction of these genes is involved in longevity and stress response, suggesting that this rapid and reversible regulatory mechanism may underlie many aspects of stress response.
One reason for systematic regulation of longevity may be to protect against the uncontrolled proliferation of cancer cells. At a cellular level, this regulation is expressed as predictable cellular senescence. Irina Groisman of the André Lwoff Institute has explored this phenomenon in mouse embryonic fibroblast cultures. She found that senescence is averted in cells that are lacking the translational regulator CPEB (cytoplasmic polyadenylation element binding protein), whose role she had previously elucidated in the cell cycle. In senescence, the protein may act together with a microRNA to affect the expression of the proto-oncogene Myc.
When the cell is under stress, cytoplasmic structures called stress granules appear, which host both short RNAs and RNA binding proteins. Anthony Leung of the Massachusetts Institute of Technology showed that poly(ADP-ribose) plays a critical role in binding together these diverse components in a dynamic but outwardly stable structure. He further suggested that the polymer may directly regulate some gene expression under stress.
Like many aspects of small-RNA regulation, stress-responsive small RNAs were first identified in plants. Plant researcher Ramanjulu Sunkar of Oklahoma State University described other aspects of RNA regulation that have been found in plants, including a short double-stranded RNA that results from binding between overlapping transcripts from neighboring genes on opposing DNA strands. The agriculturally important stress responses of plants are affected by numerous small RNAs. Sunkar demonstrated that synonymous changes in the RNA sequence of one gene could evade its regulation by microRNA while keeping the protein amino acid sequence intact, resulting in improved stress tolerance.
The best characterized stress response is heat shock, which induces expression of a host of protective proteins. Although the master heat shock factor that triggers this response is well known, Evgeny Nudler of New York University showed that the actual temperature-sensing element is a non-coding RNA he called HSR1, which at 600 nucleotides is much larger than typical regulatory RNAs. HSR1 acts together with the translational elongation factor eEF1A, but this protein can also separately initiate responses to other, non-heat stresses.
The presentations at this symposium covered a wide range of topics, but clearly only scratched the surface. The connections between stress and longevity, and diseases like cancer and Alzheimer's, as well as their regulation by small RNAs, are likely to be the subject of much further research and discussion.
MicroRNAs as Biomarkers for Aging
Speaker:
Frank Slack, Yale University
Highlights
- The reproducible and short lifespan of the nematode C. elegans lets researchers find genetic changes that affect longevity.
- Intrinsic longevity and stress resistance in C. elegans are closely related.
- Some known micro-RNAs, such as lin-4, have a clear effect on longevity.
- Screening identifies many other, previously unknown microRNAs that could play an important role.
- One miRNA is strongly upregulated in older worms, and it may target only a few aging-related genes, among which is eat-3.
MicroRNA mutants and aging
Over the years, many different biological processes have been connected to aging. Oxidative damage, metabolic rate, caloric intake, telomere shortening, and reproduction are all related to the aging process. Frank Slack and his laboratory at Yale University operate on the principle that development and aging are controlled by a developmental program or biological clock. This idea is controversial, in part because evolution ought not to select for post-reproduction longevity. It does select for developmental genes, though, some of which could later affect aging.
Much important aging research has been done in the roundworm, C. elegans, which has a conveniently short lifespan that is highly reproducible at the population level. Mutations of even a single gene can significantly affect lifespan, and at least four processes regulate aging: insulin signaling, caloric restriction, germline signaling, and the clk gene system. The daf-2 gene, for example, codes for the insulin receptor, and mutations in this gene roughly double the lifespan. 
Overexpressing the microRNA lin-4 increases the median lifespan (50% survival) of worms, while disabling it decreases their lifespan.)
In addition to protein-coding genes, some microRNAs also affect longevity. For example, Slack's group showed in 2005 that mutations that disable lin-4 cut lifespan in half. Because many mutations kill animals prematurely, however, longevity researchers "are a little bit suspicious of mutants that cause a short lifespan," Slack noted. So the team also did the opposite, overexpressing the lin-4 miRNA, and found a roughly 25% increase in lifespan.
The expression level of lin-4 can also be stress sensitive, Slack added, and animals engineered to have extra lin-4 are stress resistant. "There's a very tight connection between stress resistance and longevity in C. elegans." This longevity is also linked to insulin signaling, in ways that are still being clarified.
Inspired by these results and by earlier microarray work, Slack and his team undertook a deep-sequencing survey comparing miRNA expression in C. elegans at different ages. In addition to wild-type worms, they looked at worms with a life-extending daf-2 mutation, and at a second population that also had a daf-16 mutation that reverses this life extension. Out of nearly a million sequence reads, about half matched known C. elegans miRNAs. But although the other half matched the genome perfectly, they did not correspond to any functionally-annotated genes, and included almost 30 novel sequences. "Some of them appear only in the daf-2 animals," Slack noted. "Some, we think, are aging-specific."
Changes in gene expression across the lifespan
Postdoc Alex deLencastre generated three cell lines that overexpress the microRNA that is most highly expressed in aging worms compared to young ones. "Each of those independent lines showed a lifespan extension of about two days," Slack noted. "It suggests to us that this miRNA is upregulated in aging and acts to promote normal aging."
Surprisingly, however, the researchers could find "no discernable phenotype during development or during reproduction," Slack stressed. "The gene is not required at all for any earlier stage in development, but is required for aging." Explaining how such a gene can arise may require a subtle evolutionary argument, he said. "We think this might be an aging-specific miRNA."
The function of this miRNA is still unknown. To narrow the possibilities, Slack's team compared its gene targets, as predicted by five different programs. "None of them identified the exact same set of downstream target genes," he noted, but 639 were flagged as targets by more than one program. Interestingly, when the researchers compared those predictions with a set of some 1500 "gerontogenes" associated with aging, only four genes were in both groups.
These genes included two for heat shock proteins, one for one of the 40 C. elegans insulins, and one called eat-3. Only the last one reversed the short lifespan when knocked out. "That suggests that this miRNA might be negatively regulating the expression of eat-3," Slack noted. By disrupting mitochondrial function, he noted, eat-3 mutation may act like caloric restriction, which has long been known to increase lifespan. Further research will be needed to clarify the role this miRNA and the many other microRNAs associated with longevity.
Role of Endogenous Small RNAs in Plant Stress Responses
Speaker:
Ramanjulu Sunkar, Oklahoma State University
Highlights
- Because they cannot move away from stressful conditions, adapting to stress is particularly important for plants.
- The microRNA pathway is only one of several small RNA-based regulatory mechanisms in plants.
- Many short RNAs have a role in plant stress responses, including both microRNAs and natural antisense transcript-derived small interfering RNAs (NATsiRNAs) that require overlapping sense and antisense transcripts.
- Modifying the DNA sequence of a gene that is regulated by a microRNA, while keeping the amino-acid sequence the same, increases stress resistance.
Diverse pathways
The stress response in plants is of great fundamental and agricultural importance. Although researchers have shown that overexpressing some genes can improve stress tolerance, Ramanjulul Sunkar said, "we don't fully understand the entire mechanism of stress tolerance," including how plants initially sense stress. For this reason and others, he said, "it's very hard to translate what we have seen in transgenic plants in the laboratory," and field results are often disappointing.
MicroRNA (miRNA) is well known to be critical in plant development, and many features of RNA regulation were first revealed in plants. But Sunkar noted that "miRNAs in plants not only play an important role in development, but also in stress responses."
miRNAs in plants not only play an important role in development, but also in stress responses.
Endogenous miRNAs are present in both plants and animals; in plants they are more likely to enhance messenger RNA degradation than to affect its translation. But that is only part of the picture, Sunkar noted. "Only 2%-5% of the sequences are miRNAs, and the rest are what we call endogenous siRNAs (small interfering RNAs)," he explained. "There's a lot more variation in plants, relative to animals, with respect to small RNAs." In addition to the miRNA pathway, these molecules can act through the trans-acting (ta-siRNA) pathway, the heterochromatic siRNA pathway, or the natural antisense transcript-derived (NAT-siRNA) pathway.
Sunkar highlighted a double-stranded RNA that is produced through this last pathway, among many that have been shown to be important in stress response. "This siRNA is coming from two overlapping genes, SRO5 and P5CDH," he noted. The double-stranded intermediate requires transcription of both of these genes from opposing strands of DNA. "This is produced specifically under stress conditions," he noted, which is why other researchers had missed it. The stress induces transcription of SRO5, which creates the second strand needed to form the NAT-siRNA, which in turn downregulates the constitutively expressed P5CDH. The product of P5CDH would otherwise break down proteins that might be needed to respond to the stress.
Engineering stress tolerance
Researchers can identify small RNAs that are involved in stress tolerance either by experimentally comparing how stress affects their abundance or by looking for stress related genes in their predicted targets. Both of these approaches point to miR-398. Its expression decreases under stress, and its predicted targets include two copper/zinc superoxide dismutase (CSD) genes, which help prevent accumulation of reactive oxygen species that result from stress. Sunkar and his coworkers found that high levels of miR-398, like those found in leaves, correspond to low expression of these target genes. Similarly, in other tissues, low miRNA levels correspond to high gene expression. Further experiments showed that miRNA levels cause gene expression change through post-transcriptional regulation."The tissue-specific expression of these two CSD genes is regulated by miR-398," he concluded.
Exploiting this understanding, the researchers designed a mutant form of one of the genes. The mutant includes some synonymous base changes in the 5' end that, while not changing the coded protein sequence, destroyed the messenger RNA sequence complementarity with miR-398. "The amino acid sequence is the same as for the wild-type gene," he noted, "except this sequence will not be recognized by the microRNA."
High light stunts wild-type plants (WT), and those that overexpress the normal version of a superoxide dismutase gene (CSD2), but are less harmful to plants with a modified gene that codes for the same protein but whose sequence evades suppression by a micro-RNA.
Transgenic plants that overexpressed this miRNA-resistant gene were more successful under high-light stress than either the wild-type plants or those that overexpressed the normal form that matches the miRNA. "When you use the mutant form," Sunkar said, "you improve the stress resistance." These results support the model that, under normal conditions, the messenger RNA transcripts for the CSD genes rapidly degrade because of the presence of miR-398. Under stress, the disappearance of the miRNA allows transcripts to accumulate and to be translated into stress-detoxifying protein.
Many other small RNAs are involved in the plant stress response. Sunkar and his collaborators have recently developed array technology to probe microRNA levels in stressed plants, including those that respond to stress-related hormones.
RNA Thermosensor and Protein Integrity Sensor Activate the Heat Shock Response in Eukaryotes
Speaker:
Evgeny Nudler, New York University
Highlights
- The ubiquitous heat-shock response is stimulated in mammals by the trimerization of the master regulator HSF1.
- The temperature sensing element that helps trigger the activation of HSF1 is a noncoding RNA, roughly 600 nucleotides long, designated HSR1.
- The sequence for HSR1 is present in many species, but is often absent from reference genomes because of the peculiarities of its gene structure and location.
- Modified versions of HSR1 can render cells more sensitive or more resistant to stress, which could, for example, make cancer cells more susceptible to other drugs.
- The elongation factor eEF1a partners with HSR1 to trigger heat response, but can also directly signal stress associated with protein degradation.
HSR1 and the heat shock response
The heat shock response is the "most general and evolutionarily conserved cell defense mechanism," said Evgeny Nudler of New York University, and involves the expression of a wide range of genes for chaperones and other protective proteins. The "master regulator of all heat shock genes in higher eukaryotes," he said, is the heat-shock factor HSF1, which initiates the response by forming a DNA-binding, phosphorylated trimer.
[Elongation factor] eEF1A and [RNA] HSR1 are necessary and sufficient for [heat-shock factor] HSF activation.
Curiously, when human HSF1 is expressed in Drosophila cells, it initiates the heat shock response at the lower temperature appropriate for flies. "That clearly shows that HSF is not really the sensor by itself," Nudler noted. He and his collaborators identified two other molecules that trigger HSF1. One of these is the translational elongation factor eEF1A, which responds to protein or cytoskeletal damage. The other component is an RNA of approximately 600 nucleotides in length that they designated heat shock RNA 1, or HSR1. This noncoding RNA serves as the actual temperature-sensing element for the heat shock response. Summarizing many experimental results, Nudler said, "eEF1A and HSR1 are necessary and sufficient for HSF1 activation."
Although the researchers have since found homologs of this RNA in many species, including plants, Drosophila, humans, and mice, its sequence is absent from many gene databases. The reason, Nudler said, is that "the original genomes were all made by using bacterial cloning as an intermediate step." Under these conditions, the unusual structure of the HSR1 gene, which is flanked by large inverted repeats, is "extremely unstable," and is lost.
Modifying heat-shock response against cancer
The researchers created an in vitro assay for HSF1 activation, using just the eEF1A and HSR1, to let them explore the mechanism in more detail. For example, by separately using short antisense oligonucleotides that overlap to span the length of the RNA, they identified two regions of the sequence that reduced activation by more than 90%. These regions are presumably critical to a temperature dependent conformation change in the RNA, like those in the riboswitches that Nudler co-discovered. 
The heat shock response requires a temperature-driven conformation change in the heat-shock RNA HSR1 and an abundance of the translational elongation factor eEF1A, which together promote trimerization of the master regulator heat shock factor HSF1.
Modifying the properties of the heat sensor RNA might be useful in the clinic, Nudler said. Activating the heat shock response, for example, might "make neurons or cardiomyocytes more resistant to ischemia," or postpone neurodegenerative protein-folding diseases like Alzheimer's or Parkinson's. He and his colleagues designed modified versions of the RNA that remain in the activating conformation. This molecule, Nudler noted, "could induce a heat shock response without heat shock."
Reducing the heat shock response, by contrast, could sensitize cancer cells to other treatments. As a proof of principle, the researchers generated two breast cancer cell lines that express short-hairpin RNA that is complementary to the critical sequences in HSR1. "When you reduce heat shock response, they become more sensitive" to an agent, Nudler said. "We can target this RNA, making cells more sensitive to conventional chemotherapy." They also used complementary locked nucleic acids to inhibit heat-shock response in vivo, and plan to test them in combination with other cancer treatments in mouse models of cancer.
Nudler noted that the eEF1A can also directly sense non-heat-related stress, since the protein is liberated during translational shutdown and protein cytoskeletal collapse. "eEF1A fits the profile of a general protein-integrity sensor," he noted. "It's most likely the master regulator that can collect information from other sensors, like HSR1."
Translational Regulation of Cell Senescence
Speaker:
Irina Groisman, André Lwoff Institute
Highlights
- The cytoplasmic polyadenylation element binding protein, CPEB, helps regulate the translation of messenger RNA during the cell cycle.
- When CPEB is knocked out, cultured mouse embryonic fibroblasts bypass the usual cellular senescence that helps protect cells from cancer.
- CPEB affects senescence through its effect on Myc, which is mutated in many cancers
- The microRNA miR-15b appears to cooperate with the action of CPEB on Myc, which includes a likely target for this microRNA.
- CPEB localizes in stress granules and near p-bodies.
CPEB and senescence
One way that microRNAs modulate expression is by modifying the rate of translation. Irina Groisman of the André Lwoff Institute has extensively studied a different mechanism of translational regulation, effected by the cytoplasmic polyadenylation element binding protein CPEB. She showed that, at least in some cases, the microRNA miR-15B and CPEB cooperate to control translation.
CPEB recognizes a target sequence, the cytoplasmic polyadenylation element or CPE, in the 3' end of an RNA transcript, and acts through other proteins to modulate the initiation of translation at the opposite, 5' end. This regulation is important during embryonic amphibian and mammalian cell cycles.
Groisman also found that CPEB is necessary for the senescence that normally occurs in mouse embryonic fibroblast (MEF) cells after a few passages in culture. Cells can enter this senescence state after damage or stress. In contrast, CPEB-knockout MEFs bypass senescence, which is relevant to cancer and aging, she notes. "Senescence is a protection stage in cancer transformation. Stem cell senescence is involved in control of longevity."
Senescence is protection stage in cancer transformation. Stem cell senescence is involved in control of longevity.
The protein Ras is an important tool for assessing the cancer potential of cultured cells. "Expression of Ras in immortal cells transforms those cells," into cancer cells, Groisman noted, whereas "expression of Ras in primary cells sends them to premature senescence." Consistent with this difference, the senescence-bypassing CPEB knockout cells become elongated after Ras expression, a sign of cancerous transformation. Adding CPEB to these cells rescued the senescence response, but not if its RNA binding site was mutated. "This experiment suggests that CPEB works as an RNA-binding protein during the senescence phenotype and probably as a translation inhibitor and not activator," she said.
Cooperative action
Groisman showed that CPEB acts on senescence through its effect on Myc, a well-known proto-oncogene. Myc expression is higher in CPEB knockout MEFs, and myc mRNA co-immunoprecipitates with CPEB. Further experiments, she noted, suggest that CPEB is inhibits Myc expression during translation. For example, when the researchers remove the CPE binding site for CPEB from the 3' end of the Myc transcript, "those cells increased expression of the Myc product, and because of that those cells bypass senescence."
The 3' untranslated region of the proto-oncogene Myc has a likely binding site that may allow it to be regulated by the microRNA miR-15b in addition to the cytoplasmic polyadenylation element (CPE) that binds the regulatory protein CPEB.
Under stress conditions, CPEB localized at stress granules. Under normal conditions, they are found near, but not at, p-bodies, Groisman said. "I don't know exactly what this kind of localization means."
When Groisman used immunoprecipitation to find other factors involved in CPEB's regulation of Myc, she found 22-nucleotide RNAs including miR-15b and other miRNAs. In the Myc sequence, she "found a very good site for miR-15b, which is close to the CPE sequence in the Myc 3' UTR. Future work should determine if proximity to the p-bodies is important for translational inhibition by the CPEB. In addition, she said, "I want to verify how localization of CPEB and maybe cooperation with miRNA may be important for the senescence phenotype."
Endogenous RNAi and Stress Response in C. elegans
Speaker:
Germano Cecere, Columbia University
Highlights
- In addition to reducing the stability of messenger RNA and modifying the rate at which it is translated, short RNAs can silence its original transcription from DNA, probably by modifying chromatin.
- Two proteins, ZFP-1, a chromatin factor, and RDE-4, a component of the RNA-processing complex Dicer, are implicated in the cellular regulation of RNA interference.
- Endogenous RNAi repress translation-related genes.
- Endo-siRNAs in C. elegans may mediate response to environmental changes.
RNAi and chromatin modification
Small RNAs are well known to regulate gene expression by affecting the stability of messenger RNA and its translation into protein. But in fission yeast, plants, Drosophila and C. elegans, "it can also act in the nucleus at a transcriptional level, through chromatin modification," notes Germano Cecere of Columbia University.
Cecere's postdoctoral advisor Alla Grishok discovered this transcriptional control in worms as a side-effect of laboratory manipulations. But like other small RNAs that act endogenously, Cecere noted, "It's possible that small RNA molecules can regulate some cellular processes through transcriptional gene silencing."
To try to clarify how transcriptional silencing might occur in C. elegans, the researchers looked for chromatin factors that were in the RNA interference pathway. One such factor, Cecere said, is the zinc finger protein ZFP-1. Among surveys for genes involved in RNA interference, he said, "zfp-1 was found in all of those screens," along with other chromatin-modifying proteins.
The nuclear protein zfp-1, which localizes to chromatin, has been identified in multiple studies as an actor in RNA interference.
To begin to connect siRNAs production and transcriptional regulation, the researchers looked for commonalities between ZFP-1 and another protein designated "RNA-deficient," RDE-4. This protein complexes with the well known protein Dicer to process double-stranded RNA. The researchers chose rde-4, Cecere said, "because we suppose it to be important in the biogenesis of these small RNA molecules."
The researchers compared the gene expression changes for C. elegans mutants that lacked either zfp-1 or rde-4. "We found a very striking, statistically significant enrichment between both the genes [as they] are upregulated and downregulated," Cecere noted. "The expression profile is very similar between the two mutants." Among the upregulated genes are many that are involved in stress response and longevity.
Endogenous siRNA
Having found candidate genes for transcriptional silencing, the team then looked among them for possible RNA targets. To do this, they looked for antisense matches to small interfering RNA (siRNA) sequences, many of unknown function, which had been found in three large surveys. "We wanted to know how the endogenous RNA targets are distributed in the genes upregulated and downregulated in the mutants," Cecere said.
Genes that were upregulated in the mutants were more likely than other genes to contain targets of the endogenous RNA. By contrast, the downregulated genes were not. Most of the genes are involved in protein expression and biosynthesis, Cecere noted. "We started to think that this special profile means that probably this mechanism is at work in stress."
To further explore the stress-response connections, the researchers compared them with the well known yeast stress response expression profiles. He found a significant enrichment in genes that were homologs of those that were downregulated in stressed yeast. This analysis, he said, suggests that "genes repressed upon stress might depend on the endogenous RNAi pathway for their repression."
Genes repressed upon stress might depend on the endogenous RNAi pathway for their repression.
To test whether endogenous siRNA truly regulates part of the stress response, the researchers focused on the translational initiation factor, ifg-1 (eIF4G). "This is one of the genes that are upregulated in both mutants, and that looks to be a siRNA antisense," Cecere noted. In addition, previous studies showed that "knocking down this gene conferred more stress resistance and increased lifespan.
The researchers confirmed that stress increased the corresponding siRNA level, and there was less ifg-1 expression. Moreover, Cecere noted, most of the siRNAs matched an exon that is ultimately spliced into only the longer isoform of two alternate splicings of the protein. As would be expected for siRNA regulation, "upon stress, the long isoform is downregulated," Cecere explained. Further experiments, he said, "suggest that ZFP-1 and RDE-4 work together with endogenous siRNAs in transcriptional regulation of translational factor genes" such as ifg-1. He proposed that this reversible, flexible regulatory mechanism could be very useful for animals that must deal with a frequently changing environment.
Open QuestionsCan miRNAs serve as a biomarker for individual animals that will live a long time?
What mechanisms of RNA-based gene regulations, first identified in plants, will also be discovered in animals?
Can modifications of the activity of heat-shock RNA be an effective therapy, for example making cancer cells or more sensitive or neurodegenerative disease cells less sensitive?
How does the structure of stress granules enter into regulation of gene expression?
How general is the role of poly(ADP-ribose) in post-transcriptional regulation?
In addition to stress responses, what processes are regulated by endogenous siRNA-based transcriptional repression?
What is the significance of molecules located near, but not within, stress granules or p-bodies?
Journal Articles
Frank Slack
Boehm M, Slack F. 2005. A developmental timing microRNA and its target regulate life span in C. elegans. Science 310: 1954-1957.
Boehm M, Slack FJ. 2006. MicroRNA control of lifespan and metabolism. Cell Cycle 5: 837-840.
Friedländer MR, Chen W, Adamidi C, et al. 2008. Discovering microRNAs from deep sequencing data using miRDeep. Nat. Biotechnol. 26: 407-415.
Niwa R, Zhou F, Li C, Slack FJ. 2008. The expression of the Alzheimer's amyloid precursor protein-like gene is regulated by developmental timing microRNAs and their targets in Caenorhabditis elegans. Dev. Biol. 315: 418-425.
Kim SK, Lund J, Kiraly M, et al. 2001. A gene expression map for Caenorhabditis elegans. Science 293:2087-2092.
Ramanjulu Sunkar
Borsani O, Zhu J, Verslues PE, et al. 2005. Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123:1279-1291.
Jagadeeswaran G, Saini A, Sunkar R. 2009. Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis. Planta 229:1009-1014.
Shukla LI, Chinnusamy V, Sunkar R. 2008. The role of microRNAs and other endogenous small RNAs in plant stress responses. Biochim. Biophys. Acta. 1779: 743-748.
Evgeny Nudler
Shamovsky I, Ivannikov M, Kandel ES, et al. 2006. RNA-mediated response to heat shock in mammalian cells. Nature 440: 556-560.
Shamovsky I, Nudler E. 2008. Modular RNA heats up. Mol. Cell. 29: 415-417.
Shamovsky I, Nudler E. 2009. Isolation and characterization of the heat shock RNA 1. Methods Mol. Biol. 540: 265-279.
Shen X, Banga S, Liu Y, et al. 2009. Targeting eEF1A by a Legionella pneumophila effector leads to inhibition of protein synthesis and induction of host stress response. Cell Microbiol. [Epub ahead of print]
Anthony Leung
Leung AK, Sharp PA. 2007. microRNAs: a safeguard against turmoil? Cell 24;130: 581-585.
Leung AK, Calabrese JM, Sharp PA. 2006. Quantitative analysis of Argonaute protein reveals microRNA-dependent localization to stress granules. Proc. Natl. Acad. Sci. USA 103: 18125-18130. Full Text
Schreiber V, Dantzer F, Ame JC, de Murcia G. 2006. Poly(ADP-ribose): novel functions for an old molecule. Nat. Rev. Mol. Cell Biol. 7: 517-528.
Irina Groisman
Groisman I, Huang YS, Mendez R, et al. 2000. CPEB, maskin, and cyclin B1 mRNA at the mitotic apparatus: implications for local translational control of cell division. Cell 103: 435-447.
Groisman I, Jung MY, Sarkissian M, et al. 2002. Translational control of the embryonic cell cycle. Cell 109: 473-483.
Groisman I, Ivshina M, Marin V, et al. 2006. Control of cellular senescence by CPEB. Genes Dev. 20: 2701-2712. Full Text
Germano Cecere
Grishok A. 2005. RNAi mechanisms in Caenorhabditis elegans. FEBS Lett. 579: 5932-5939.
Grishok A, Sinskey JL, Sharp PA. 2005. Transcriptional silencing of a transgene by RNAi in the soma of C. elegans. Genes Dev. 19: 683-696. Full Text
Grishok A, Hoersch S, Sharp PA. 2008. RNA interference and retinoblastoma-related genes are required for repression of endogenous siRNA targets in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 105: 20386-20391. Full Text
Speakers
Frank Slack, PhD
Yale University
e-mail | web site | publications
Frank Slack received his BSc from the University of Cape Town in South Africa, before completing his PhD in molecular biology at Tufts University School of Medicine. He started work on microRNAs as a postdoctoral fellow in Gary Ruvkun's laboratory at Harvard Medical School, where he co-discovered the second known microRNA, let-7. He is currently an associate professor in the Department of Molecular, Cellular, and Developmental Biology at Yale University. The Slack laboratory studies the roles of microRNAs and their targets in development, disease and aging.
Ramanjulu Sunkar, PhD
Oklahoma State University
e-mail | web site | publications
Ramanjulu Sunkar received his BS and MS and degrees in botany, and his PhD in plant stress physiology, from Sri Krishnadevaraya University in Anantapur, India. He obtained his postdoctoral training under Hillel Fromm (Weizmann Institute of Sciences, Israel), Dorothea Bartels (University of Bonn, Germany), and Jian-Kang Zhu (University of California, Riverside). While in Dr. Zhu’s laboratory, Sunkar initiated and developed an independent research program on stress-regulated small RNAs (microRNAs and small-interfering RNAs) in plants.
In 2006, Sunkar joined the Department of Biochemistry and Molecular Biology at Oklahoma State University as an assistant professor and continued to develop this cutting-edge research program on the role of small RNAs in plant stress responses. His laboratory’s research has been funded by the United States Department of Agriculture (USDA), National Science Foundation (NSF), Oklahoma Centre for the Advancement of Science and Technology (OCAST), Oklahoma Tobacco Research Centre (OTRC) and Oklahoma Agricultural Experiment Station (OAES). Currently, he is editing a volume on plant abiotic stress under the series, Methods in Molecular Biology. He served as an ad-hoc reviewer for National Science Foundation (USA), Biotechnology and Biological Sciences Research Council (BBSRC, UK), and other agencies.
Evgeny Nudler, PhD
New York University
e-mail | web site | publications
Evgeny Nudler earned his undergraduate and Master's degrees from the Moscow State University and his PhD in biochemistry in 1995 from the Institute of Molecular Genetics in Moscow, Russia. He joined NYU in 1997 from the Public Health Research Institute of New York City.
Nudler has done pioneering studies in various biological fields. His original work on transcription explained how RNA polymerase moves and recognizes pausing and termination signals in DNA and RNA. His studies on bacterial gene regulation led to the discovery of riboswitches (metabolite-sensing RNA) that control numerous bacterial genes. More recently, his group uncovered key regulators of the heat shock response in eukaryotic cells. He has also made important contributions in the area of nitric oxide biochemistry in both animal and bacterial systems. Nudler has received many prestigious grants and awards including the Searle Scholar Award and NIH Director's Pioneer Award.
Anthony Leung
Massachusetts Institute of Technology
e-mail | web site | publications
Anthony Leung received his MBioch from Exeter College, University of Oxford, UK and his PhD work focused on the dynamic behavior of nucleolar proteome in Angus Lamond’s Laboratory (University of Dundee, UK). He is currently a special fellow of the Leukemia and Lymphoma Society in the laboratory of Phillip Sharp at Massachusetts Institute of Technology. Anthony is interested in how microRNA functions—where it localizes in cells, what proteins it binds to, and what the real targets of microRNAs are. Specifically, he uses deep sequencing technology, quantitative imaging, and proteomics to study how microRNA function changes during cellular stress.
Irina Groisman, PhD
André Lwoff Institute
e-mail | web site | publications
Irina Groisman completed her PhD at Kiev State University in Ukraine, where she studied fidelity of translation in prokaryotes. Her first postdoc was with Hanna Engelberg-Kulka at Hebrew University of Jerusalem, Hadassah Medical School, where she worked on translational regulation of gene expression in prokaryotes. She completed a second postdoc at the University of Massachusetts Medical School in the lab of Joel Richter, where she investigated function of CPEB (cytoplasmic polyadenylation element binding protein) during Xenopus embryonic and somatic mammalian cell cycle. She was later team leader at the Center for Computational Integrative Biology at Massachusetts General Hospital, where she worked on cancer therapy development based on double targeting theory, proposed by Brian Seed. Since the beginning of 2009 Groisman has worked at the André Lwoff Institute, near Paris, France, where she is a group leader investigating the role of CPEB during the mammalian cell cycle. The work may have applications for cancer transformation and aging.
Germano Cecere, PhD
Columbia University
e-mail | web site | publications
Germano Cecere received his BSc from the University of Naples in 2004. Under the supervision of Carlo Cogoni he received his PhD at the University of Rome by contributing to the understanding of the mechanisms of small RNA silencing in the filamentous fungus Neurospora crassa. In 2008 he started a postdoctoral research in Alla Grishok’s lab at Columbia University, where he has begun studying the connection between small RNA silencing and the epigenetic regulation of gene expression in C. elegans.
Don Monroe
Don Monroe is a science writer based in Murray Hill, New Jersey. After getting a PhD in physics from MIT, he spent more than fifteen years doing research in physics and electronics technology at Bell Labs. He writes on physics, technology, and biology.
|
|