Guarding the Germ-line Stem Cell Genome

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Guarding the Germ-line Stem Cell Genome

Wednesday, November 3, 2010

The New York Academy of Sciences

Presented By

 

Piwi-interacting RNAs (piRNAs) - a major class of small regulatory RNA - are the most recently discovered class of small non-coding RNAs, and, as their name suggests, they bind to specialized proteins from the Argonaute family, called "Piwi" proteins. Present in both egg- and sperm-producing cells in fruit flies, and in sperm-producing cells in mammals, piRNAs serve to preserve the integrity of the germ-line stem cell genomes. In contrast to miRNAs and siRNAs, little it is still known about how piRNAs are generated and which their mechanisms of action are. This half-day meeting will explore recent research on piRNAs biogenesis and their role in self-renewing divisions of germ-line stem cells, genome integrity, and epigenetic regulation.

Photo credit: Max Planck Institute for Molecular Biomedicine / Kinarm Ko

Agenda

*Presentation times are subject to change.


1:00 PM

Welcome Remarks

1:15 PM

Drosophila Piwi Functions in Hsp90-Mediated Suppression of Phenotypic Variation
Haifan Lin, PhD, Yale University School of Medicine

1:45 PM

piRNA and PIWI Proteins in the Human Ovary
Zev Williams, MD, The Rockefeller University

2:00 PM

Characterizing the Neuronal Function of Aplysia piRNAs
Priya Rajasethupathy, Columbia University

2:15 PM

Protecting the Guardians – Small RNA Methylation by Hen1
Stefan Ameres, PhD, University of Massachusetts Medical School

2:45 PM

Coffee Break

3:15 PM

Distinct 22G-RNA Pathways Direct Genome Surveillance and Chromosome Segregation in the C. elegans
Weifeng Gu, PhD, University of Massachusetts Medical School

3:45 PM

Dissecting the Molecular Function of Piwi Proteins and piRNAs
Nelson Lau, PhD, Brandeis University

4:15 PM

piRNPs: Factors and Targets
Zissimos Mourelatos, MD, University of Pennsylvania, School of Medicine

4:45 PM

Yb body: A Place Where Piwi Meets piRNAs
Haruhiko Siomi, PhD, Keio University School of Medicine

5:15 PM

Networking Reception

Speakers

Organizers

Eric Lai, PhD

Memorial Sloan-Kettering Cancer Center

Dr. Lai received his BA from Harvard University and performed thesis work in the laboratory of Gary Ruvkun, where he learned about developmental gene regulation in C. elegans. He did his PhD at UC San Diego with James Posakony on Drosophila neural patterning and Notch signaling, and continued to study these topics as a postdoc with Gerald Rubin at UC Berkeley. He joined the faculty of Sloan-Kettering Institute in 2005. His laboratory studies diverse topics related to developmental gene control at both transcriptional and post- transcriptional levels.

Marta Murcia, PhD

The New York Academy of Sciences

Speakers

Stefan Ameres, PhD

University of Massachusetts Medical School

Stefan Ameres studied biology at Friedrich-Alexander University Erlangen-Nuremberg, Germany. He did his PhD at the Max F. Perutz Laboratories in Vienna, Austria where he studied the molecular basis for target RNA recognition by small RNAs in the laboratory of Renée Schroeder. Now, Stefan Ameres works as a postdoc with Phillip Zamore at the University of Massachusetts Medical School, where he studies regulatory aspects of RNA silencing pathways in Drosophila and mammals.

Weifeng Gu, PhD

University of Massachusetts Medical School

Dr. Gu received his MD from Peking University Health Science Center (previously known as Beijing Medical University), where he studied human chemokines. He did his PhD at University of Rochester with Eric Phizicky studying tRNA modification. Then he went to UMass Medical school to work with Dr. Craig Mello as a postdoc to study endogenous small RNAs in C. elegans.

Nelson Lau, PhD

Brandeis University

Nelson Lau is an assistant professor of biology at Brandeis University and his laboratory studies gene and genome regulatory processes controlled by small RNAs. Nelson Lau obtained a PhD from MIT with David Bartel, and was a Whitney Foundation Fellow in Robert Kingston’s lab at Mass. General Hospital, where he discovered piRNAs from rats and mice. Nelson Lau is a 2010 Searle Scholar and a recipient of an NIH K99 award.

Haifan Lin, PhD

Yale University School of Medicine

Dr. Lin is Professor of Cell Biology and Director of the Yale Stem Cell Center at the Yale University School of Medicine. Lin’s research has greatly strengthened understanding of the molecular mechanisms that define the unique behavior of stem cells. His early contributions include identification of stem cells in the Drosophila ovary and establishment of these stem cells as an effective model for study. Using this model, Dr. Lin obtained direct evidence for the century-old hypothesis for “asymmetric division” of stem cells, which allows them both to self-renew and to produce differentiated daughter cells. He was also the first to identify and name “niche signaling cells” in the fly model and has been a key player in systematically demonstrating the longstanding “stem cell niche theory” on the essential role of microenvironment signaling in stem cells self-renewal. In the process, Dr. Lin discovered key genes involved in both niche signaling and intracellular regulation, most notably the piwi/argonaute gene family. His work on Piwi proteins lead to the discovery of a group of small RNAs called PIWI-interacting, or piRNAs. The discovery of piRNAs independently by Dr. Lin and others was recognized by Science Magazine as a top scientific breakthrough of 2006. Currently, the Lin lab is exploring the role of these molecules in epigenetic and posttranscriptional regulation of gene expression.

Zissimos Mourelatos, MD

University of Pennsylvania, School of Medicine

Zissimos Mourelatos obtained his M.D. from the Aristotelian University, Greece in 1991. From 1991 to 1995 he was research fellow in the laboratory of Nicholas Gonatas at PENN, studying the cell biology of the Golgi apparatus in neurons and in motor neuron diseases. From 1995 to 1998 he was resident in Anatomic Pathology and clinical fellow in Neuropathology. From 1998 to 2002 he was postdoctoral fellow in the laboratory of Gideon Dreyfuss at PENN. Since 2002 he is a faculty member of the Department of Pathology & Laboratory Medicine at PENN. His laboratory investigates the basic biology of small regulatory RNPs and also how RNA dysregulation contributes to neurodegeneration. He is also a practicing surgical neuropathologist and the director of Neuropathology at PENN.

Priya Rajasethupathy

Columbia University

Priya Rajasethupathy attended Cornell University for her undergraduate studies, and majored in Biological Sciences. She received her BA degree and graduated Suma Cum Laude from Cornell in 2004. She then traveled to India and spent one year as a research student at the National Center for Biological Sciences in Bangalore, India. Upon returning, she joined the MD-PhD program at Columbia University, where she is currently pursuing her PhD in neuroscience with Eric Kandel.

Haruhiko Siomi, PhD

Keio University School of Medicine

Haruhiko Siomi has obtained his Diploma degree (1982) and M.S. degree (1984) in Chemistry ('sugar chemistry') at Gifu University, and his Ph. D. (1988) in virology ('HTLV1') at the Institute for Virus Research in Kyoto University. He joined the Gideon Dreyfuss laboratory in the Department of Biochemistry and Biophysics at the University of Pennsylvania School of Medicine as a HHMI associate in May 1990 and later promoted to Research Assistant Professor ('hnRNPs and FMR1'). In 1999, he became a Professor in the Institute for Genome Research at the University of Tokushima. He then moved to Keio University School of Medicine as Professor in Department of Molecular Biology in 2008. His laboratory investigates various aspects of RNA silencing.

Zev Williams, MD

The Rockefeller University

Zev Williams is a research associate in the laboratory of Dr. Thomas Tuschl at Rockefeller University and a senior fellow in reproductive endocrinology and infertility at Weill-Cornell Medical Center. He received his MD and PhD degrees from the Mount Sinai School of Medicine with Dr. Paul Wassarman where he studied the biosynthesis and assembly of the mammalian egg coat. He continued his studies on the molecular biology of the oocyte through his residency in Obstetrics and Gynecology at the Brigham and Women’s Hospital and Massachusetts General Hospital.

Sponsors

For sponsorship opportunities please contact Cristine Barreto at cbarreto@nyas.org or 212.298.8652.

Promotional Partner

American Society of Gene & Cell Therapy

Abstracts

Drosophila Piwi Functions in Hsp90-Mediated Suppression of Phenotypic Variation

Haifan Lin, PhD, Yale University School of Medicine

Canalization describes an organism’s ability to produce the same phenotype despite genotypic variations and environmental influences. In Drosophila, Hsp90, the Trithorax group proteins, and transposon silencing have been implicated in canalization. Despite this, molecular mechanism underlying canalization remains elusive. Here, we show that the piRNA pathway, but not siRNA or miRNA pathways, is involved in canalization. Furthermore, we isolated a protein complex composed of Hsp90, Piwi, and the Hsp70/Hsp90 Organizing Protein Homolog (Hop), and demonstrated the function of this complex in canalization. Our data indicate that Hsp90 and Hop regulate the piRNA pathway via Piwi to mediate canalization. Moreover, they point to epigenetic silencing of the expression of existing genetic variants and the suppression of transposon-induced new genetic variation as two major mechanisms underlying piRNA pathway-mediated canalization.

piRNA and PIWI Proteins in the Human Ovary

Zev Williams, MD, The Rockefeller University

piRNA and PIWI proteins have been described in germ cells from a broad range of organisms including flies, fish, worms and mice, but have not previously been found in the human ovary. Within the adult mouse testis, expression is limited to immature spermatocytes. We therefore looked at the developmentally equivalent stage in human, specifically ovaries from 18-23 week-old fetuses. RT-PCR and immunohistochemistry confirmed expression of PIWI protein mRNA and protein within fetal, but not adult, oocytes. Deep-sequencing and annotation of total small RNA revealed the presence of piRNA clusters. This research expands the range of expression of piRNA and PIWI proteins to include the human ovary.

Characterizing the Neuronal Function of Aplysia piRNAs

Priya Rajasethupathy, Columbia University

piRNAs are a class of 28-33 nt non-coding RNAs that associate with the piwi protein and have the potential to regulate gene expression at the level of transcription or translation. In both invertebrates and vertebrates, they are understood to be germline-specific, derived from long single-stranded precursors, and to have evolutionary conserved promoters. Some piRNAs emerge from repeat-regions of the genome and function to preserve germline integrity by silencing transposons. The majority of piRNAs, however, are not derived from repeat-regions and their function is unknown. In our studies in the marine mollusk Aplysia, we find an unexpected neuronal expression of piRNAs that stably associate with a neuronally expressed piwi protein. In my talk I will discuss our characterization of the neuronal piRNAs in Aplysia, and more specifically, explore their potential role in the epigenetic control of synaptic plasticity. 

Protecting the Guardians – Small RNA Methylation by Hen1

Stefan Ameres, PhD, University of Massachusetts Medical School

Three small RNA pathways operate in Drosophila: The microRNA- and the RNA interference-pathways regulate diverse aspects of animal development and physiology or defend against foreign genetic elements, like viruses and transposons. The piRNA pathway acts in the germline and ovarian somatic follicle cells to ensure genome stability by targeting transposable elements. In contrast to microRNAs, which mainly interact with their target mRNAs via the so-called ‘seed region' (nucleotides 2 to 7/8 of the small RNA), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs) target transcripts through high or complete complementarity. High complementarity between miRNAs and target mRNAs results in tailing and 3' to 5' exonucleolytic trimming, ultimately decreasing small RNA steady-state levels. The methyltransferase Hen1 methylates the 2' hydroxyl of the 3' terminal ribose of siRNAs and piRNAs. Small RNA methylation protects siRNAs from tailing and trimming. As a consequence, tailing and trimming reinforces sorting miRNAs and siRNAs in flies. I will also discuss our preliminary data on how loss of small RNA methylation affects specifically the piRNA pathway in flies.

Distinct 22G-RNA Pathways Direct Genome Surveillance and Chromosome Segregation in the C. elegans

Weifeng Gu, PhD, University of Massachusetts Medical School

Argonaute-mediated small RNA pathways are highly conserved in Eukaryotes and regulate diverse biological processes. Using deep-sequencing and genetic approaches, we have identified two distinct endogenous small RNA (22G-RNA) pathways in C. elegans, one associated with a group of worm specific AGOs, WAGO1-12, and the other associated with CSR-1. Although the CSR-1 and WAGO Argonauts are equally divergent from the AGO and PIWI clades of Argonautes, they appear to have assumed genome-surveillance functions similar to those linked to PIWI AGOs in vertebrates and insects. Both pathways are dependent on a core complex composed of DRH-3 (Dicer-related helicase), RdRPs (RNA dependent RNA Polymerase), EKL-1 (a Tudor domain protein). Unlike most of the known small RNAs identified in other organisms, the 22G-RNAs are directly synthesized by RdRP, thus bearing a 5' triphosphate Guanidine nucleotide. Surprisingly, DCR-1 plays no role in the biogenesis of these 22G-RNAs. WAGO-1 localizes to germline nuage structures called P granules and silences transposons, pseudogenes, and cryptic loci. WAGO-1 also target thousands of loci that are annotated as genes but by and large have no known function and do not encode recognizable protein domains. In contrast, the CSR-1-interacting 22G-RNAs are antisense to thousands of germline-expressed protein-coding genes, many with a known or essential function. However, CSR-1 does not regulate these targets at the mRNA or protein level. Rather, CSR-1 localizes to chromosomes and is required for proper chromosome segregation. In the absence of CSR-1 or the essential core complex DRH-3/RdRP/EKL-1, chromosomes fail to align at the metaphase plate and kinetochores do not orient to opposing spindle poles. Taken together these findings suggest that AGO pathways target all the transcripts produced in the germline. Transposons, pseudogenes and other non-codoing loci are silenced via the WAGO-1 pathway, while the CSR-1 pathway targets all the functional genes, and in so doing contributes to proper chromosome organization. These findings further broaden our understanding of the biogenesis and varied biological roles of small RNAs and point to their central role in monitoring both gene expression and genome maintenance.

Dissecting the Molecular Function of Piwi Proteins and piRNAs

Nelson Lau, PhD, Brandeis University

Piwi proteins bind an abundant and complex class of small RNAs, piRNAs, that are specifically expressed in animal germ cells. Many piRNAs are homologous and antisense to transposable elements, thus they are postulated to guide Piwi proteins to silence transposon transcripts. However, multiple abundant piRNAs exist that lack homology to transposons, so their regulatory targets and mode of regulation is unclear. The molecular understanding of how piRNAs and Piwi proteins regulate gene and transposon expression is still obscure, and in my talk I will discuss our approaches to examine molecular the regulatory mechanisms the Piwi proteins and piRNAs employ upon possible targets.

piRNPs: Factors and Targets

Zissimos Mourelatos, MD, University of Pennsylvania

Piwi-interacting RNAs (piRNAs) are small RNAs that bind to Piwi family proteins to form piRNPs. Piwi proteins are essential for germline maintenance, specification and differentiation. Piwi proteins contain arginine methylation modifications, which mediate their binding to Tudor domain-containing proteins and the assembly of germ granules. An established function of piRNPs is to suppress transposons in the germline. We will present studies on factors and targets for piRNPs, which uncover a much broader role for piRNPs in the germline, beyond transposon control.

Yb body: A Place Where Piwi Meets piRNAs

Haruhiko Siomi, PhD, Keio University School of Medicine

PIWI-interacting RNAs (piRNAs) function in maintaining the integrity of the genome from invasive transposable DNA elements in germlines. In Drosophila ovarian somatic cells, primary piRNAs are expressed and loaded onto Piwi. Armitage (Armi) sequesters Piwi into Yb bodies; this dynamic event is accomplished by the sequential associations between Armi and Piwi, and then Armi and Yb, the core component of Yb bodies. At Yb bodies, piRNA intermediate-like molecules are loaded onto the Armi-Piwi-Yb complex. Depletion of Zuc causes Piwi to be accumulated at Yb bodies; however, Piwi is barely loaded with mature piRNAs. A Piwi mutant lacking its nuclear localization signal is loaded with mature piRNAs, but hardly represses transposable elements. Thus, Armi, Yb, and Zuc collaboratively control Piwi association with piRNAs and its nuclear function in ovarian somas. These findings suggest a model in which a functional Piwi–piRNA complex is formed and inspected in Yb bodies before its nuclear entry to exert transposon silencing in ovarian somas.

*Additional abstracts coming soon.

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