Judges’ Queries and Presenter’s Replies

  • May 20, 2013 | 04:11 p.m.

    Hello,
    How is your research related to the overall goal of your IGERT project?

  • Icon for: Mallory Freeberg

    Mallory Freeberg

    Presenter
    May 20, 2013 | 09:36 p.m.

    Hi Jerome,
    Thank you for your question! The overall goal of my thesis, of which one chapter is described in my IGERT video and poster, is to elucidate post-transcriptional regulatory mechanisms governing gene regulation in metazoans. To express the information encoded by our genomes, messenger RNAs (mRNAs) are generated as the link between DNA and proteins. Post-transcriptional regulation of this expression can occur at the level of mRNA to either promote its translation to protein or silence it (i.e. no protein is made). mRNA regulation is governed by interactions of mRNAs with specific proteins as well as with small RNA molecules, of which piRNAs are one subclass. Unlike other subclasses of small RNAs, piRNAs are unique in that they are required for silencing specifically transposon RNAs. Other chapters of my thesis include identifying global mRNA:protein interactions in the budding yeast S. cerevisiae; examining the dynamics of mRNA:protein interactions when cells are subjected to external stress; and identifying 3’ untranslated regions of mRNAs (common sites of post-transcriptional regulation) in C. elegans across different tissues and different developmental stages. All of these projects rely heavily on either re-using NGS datasets or generating our own NGS datasets that we then are responsible for sharing with the scientific community.

  • May 21, 2013 | 10:36 a.m.

    Good presentation! What are the likely transcription factors that interact with upstream motif?

  • Icon for: Mallory Freeberg

    Mallory Freeberg

    Presenter
    May 21, 2013 | 11:57 a.m.

    Hi Govindarajan,
    Thank you! A Cell paper was published last fall implicating the Forkhead family of transcription factors (TFs) as recognizing this upstream motif in C. elegans. They showed that knockdown of some proteins in this family moderately affect piRNA levels; however, we have been unable to reproduce their work and suspect that loss of piRNAs in these knockdown animals might be due to defects we observe in sperm and oocyte production. Through an in vitro affinity assay looking for RNAs that interact with all C. elegans TFs, our collaborators have identified another family of TFs that are highly specific for our motif. This family contains sperm- and oocyte-specific factors that could mediate the differential expression of our male- and female-specific piRNAs. Because this search for these TFs is highly competitive, I cannot reveal more information at this time. If you are interested, be on the lookout for publications regarding piRNA TFs in the near future!

  • May 21, 2013 | 11:59 a.m.

    Very exciting work! What are the targets of the piRNAs and do they change in the ageing worm?

  • Icon for: Mallory Freeberg

    Mallory Freeberg

    Presenter
    May 22, 2013 | 03:53 p.m.

    Hi Markus,

    Thank you for your question! So far, little is known about bona fide targets of C. elegans piRNAs beyond what we can predict based on imperfect sequence complementarity. We do know that piRNAs indirectly target the Tc3/mariner class of transposons in worms, as loss of piRNAs leads to upregulation of these transposons. Additionally, recent studies have shown that piRNAs trigger biogenesis of another class of small RNAs, endogenous short interfering RNAs (endo-siRNAs), to elicit gene silencing of a variety of other genes. We observe that predicted gene targets of male piRNAs are depleted of spermatogenesis-related genes, suggesting that there is selective pressure on these piRNAs to avoid targeting genes required for male germline development. Curiously, we see neither depletion nor enrichment of female piRNAs targeting oogenesis-related genes. The next step is to functionally validate these predicted targets of piRNAs to determine the biological relevance of piRNA:target interactions.

    As to your question about aging, the short answer is that we don’t know exactly if or how piRNAs might directly contribute to aging, but there is an indirect link between piRNAs and aging through the germline. C. elegans are hermaphroditic, which means a single worm contains both male and female germline tissue, and develop from embryo to adult stage in about 3 days, living out as adults for a couple of weeks. During embryonic and larval development to adulthood, no piRNAs are detectable. It is only when the animals reach the last larval stage and start to generate germline tissue that piRNAs accumulate. In animals that lack a germline, we actually observed an increased lifespan. Whether or not lack of piRNAs in germline-less animals contribute to this increased lifespan is currently under investigation.

    Thank you for you interest!

  • May 21, 2013 | 02:08 p.m.

    Very interesting work. What are the implications for the larger gender bias in males for abundance of unique piRNAs?

  • Icon for: Mallory Freeberg

    Mallory Freeberg

    Presenter
    May 22, 2013 | 04:35 p.m.

    Thank you for your question! We, too, are interested in explaining the difference between the number of unique male and female piRNAs. We don’t yet have a conclusive explanation, but we think that the answer can be found by exploring the evolution of piRNAs. Although the piRNA upstream motif is conserved in other nematode species, the sequences of the piRNAs themselves are not conserved. This non-conservation of piRNA sequences hints that the sequence itself is not biologically important, but rather having a pool of small RNAs that trigger the degradation of invading RNA sequences or regulate gene expression is important. In the male germline of a hermaphroditic C. elegans adult sperm are produced only during a short time window and then are stored, while in the female germline oocytes are continuously being made over a period of days. Perhaps the limited number of female piRNAs reflects a greater need to avoid accidentally targeting genes necessary for the rapid cellular divisions that produce oocytes.

    It is also worth mentioning that I believe we have not yet identified all of the unique piRNAs in C. elegans, and that continued deep sequencing using modified RNA collection methods that enrich for piRNAs will help elucidate the piRNA pathway more fully. Of all the small RNAs in C. elegans, piRNAs are expressed at the lowest levels and are often drowned out in small RNA sequencing samples by the more abundance microRNAs and short interfering RNAs.

    Thanks for your question! I’m sorry I couldn’t give you a more definite answer at this time.

  • Icon for: Zhaomin Yang

    Zhaomin Yang

    Judge
    May 21, 2013 | 05:02 p.m.

    Interesting presentation! When you say that the biological mechanisms for maintaining fertility are conserved from mammals to bacteria, which mechanisms are you referring to more specifically?

  • Icon for: Mallory Freeberg

    Mallory Freeberg

    Presenter
    May 22, 2013 | 04:09 p.m.

    Thank you for your question! I was mostly referring to general mechanisms by which prokaryotic and eukaryotic organisms successfully reproduce. For example, higher eukaryotic organisms evolved sexual reproduction as a way to promote genetic variation in offspring. Bacteria also promote genetic variation but through conjugation, or horizontal gene transfer, between individual cells. Additionally, other molecular mechanisms independent of piRNA function, for example DNA repair mechanisms, contribute to maintaining genomic integrity and preventing transfer of damaged DNA to an offspring. Finally, many organisms rely on intra- and inter-cellular signaling to determine whether conditions are right for reproduction. Regardless of how “conserved” the exact mechanisms are between simple prokaryotic and complex eukaryotic, the task of promoting reproduction in both genetically and environmentally favorable conditions is a goal of any living organism.

    Thank you for your interest! I hope these examples have helped clarify my point.

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Presentation Discussion

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    Kevin Anderson

    Guest
    May 20, 2013 | 03:12 p.m.

    Do piRNA’s attack transposons after the transposon has separated from the DNA and before it re-inserts? Or, if my question is totally mixed up, could you briefly talk about how transposons relocate and how piRNA affects them?

  • Icon for: Mallory Freeberg

    Mallory Freeberg

    Presenter
    May 21, 2013 | 01:22 p.m.

    Hi Kevin,
    Thanks so much for your question! I regret that I could not get into more detail about transposons in the video. You are absolutely correct that piRNAs attack transposons after they have been copied from the genome and before re-insertion. Briefly, transposons relocate by being transcribed or cut from the genome and using special enzymes encoded by the transposon itself to reinsert back into the genome in another location. piRNAs act by base pairing with the transposon which promotes transposon degradation before it can re-insert. In the spirit of full disclosure, piRNAs actually initiate a more general mechanism called RNA interference, or RNAi which regulates expression of all genes. piRNAs imperfectly base pair with transposons, as well as other harmful or invading RNAs, which acts as a signal to recruit specific proteins to the RNA to drive production of another subclass of small RNAs called endogenous short-interfering RNAs (endo-siRNAs). These endo-siRNAs, which perfectly base pair with the RNA, then recruit additional specific proteins, including nucleases, to destroy the targeted RNA. In this sense, piRNAs act as “sentinels of the genome” to target all harmful RNAs, including transposons, for elimination before they can harm the cell, tissue, or organism.

    I apologize if this is a lot to read. piRNAs are just so cool, especially in worms where they seem to have this general genome surveillance mechanism not yet described in other organisms such as fly, mouse, or human. Please let me know if I can clarify anything else on this point, and thank you for your interest!

  • Icon for: Philomena Chu

    Philomena Chu

    Trainee
    May 23, 2013 | 11:45 a.m.

    Very interesting and important research topic! I think you did a great job explaining how the piRNAs act on transposons in a simple, clear manner.

  • Icon for: Mallory Freeberg

    Mallory Freeberg

    Presenter
    May 23, 2013 | 11:58 a.m.

    Thanks Philomena! I think it’s so interesting how organisms have evolved these defense mechanisms to protect the genome. Because studying these mechanisms is hard in humans (it usually results in sterility which is not something we can easily test!), it’s so important to understand the basic mechanisms in lower organisms. piRNAs have similar function in other organisms, like fly and mouse, but they also have unique functions in these animals that are just as interesting.

    Thanks again for watching my video!

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    Kevin Anderson

    Guest
    May 29, 2013 | 11:09 p.m.

    Thank you for your response! Go Blue!

  • Icon for: Mallory Freeberg

    Mallory Freeberg

    Presenter
    May 30, 2013 | 07:53 a.m.

    You are very welcome! Go Blue! :)

  • Further posting is closed as the competition has ended.

Icon for: Mallory Freeberg

MALLORY FREEBERG

University of Michigan
Years in Grad School: 4

Insights into mechanisms of fertility through study of Caenorhabditis elegans piRNAs

piRNAs are a class of short interfering RNAs (siRNAs) characterized by 21 nt length, 5’ uridine, and no sequence similarity or conservation.1 piRNAs require Piwi protein PRG-1 for accumulation and are involved in germline maintenance and fertility.[2-4] piRNAs suppress harmful genetic elements, including transposable elements, by priming biogenesis of secondary siRNAs that target these elements for degradation.[5-8] piRNAs map primarily to two broad regions of chromosome IV, but their mechanism of biogenesis remains largely unknown. A recently characterized class of germline endogenous siRNAs, 26G RNAs, show distinct expression in either spermatogenic cells or oocytes and embryos.[9,10] We wondered whether piRNAs also show patterns of male or female germline specificity. Although some piRNAs have been detected in both male and female germlines,[2,3] what might distinguish germline-specific patterns of enrichment among all piRNAs is poorly understood. Here, we computationally analyze published sequencing datasets to assess germline specificity of piRNAs. More than 70% of piRNAs are >5-fold enriched in male or female germline. Initial piRNA studies identified a short 8 nt motif (CTGTTTCA) located 46 nt upstream of piRNA loci.[1,2] Strikingly, 80% of male piRNA upstream regions contain the 5 nt core motif GTTTC, compared to less than half for female piRNAs. Additionally, position 1 of the short motif is enriched for C upstream of male piRNAs, which is associated with more robust male piRNA expression; no such pattern is observed for female piRNAs. By generating transgenic C. elegans animals expressing a synthetic piRNA sequence, we show that placing a C-containing short motif upstream of a female piRNA alters its expression pattern to look more like a male piRNA. Furthermore, placing a non-C-containing motif upstream of a male piRNA alters its expression pattern to look more like a female piRNA. These data suggest that the upstream motif orchestrates germline expression patterns of piRNAs in C. elegans.