CONTENT

1. Introduction
2. Programs
     2-1. RNA-protein interactions
     2-2. RNA chemistry
     2-3. RNA regulation in protozoa
     2-4. RNA silencing
     2-5. RNA transport and localization
     2-6. 3’ End processing
     2-7. RNA in disease
     2-8. RNA decay
     2-9. Mentor-Mentee luncheon – Finding a post-doctorial position
3. Concluding remarks

1. Introduction

The nineteenth annual meeting of the RNA society was held in Quebec, a beautiful historic city in Canada. Approximately a thousand researchers in the field of RNA gathered in this year. In addition, two satellite meetings were held right before and after the RNA meeting: (1) Fourth Annual Summer Symposium on Cellular Dynamics of Macromolecular Complexes that focuses on Structure and Dynamics of RNA interactions and (2) 1st International Symposium on Stress-Associated RNA Granules in Human Disease and Viral Infection for who studies how RNA granule affects human diseases.  In this report, I will be focusing on the RNA society meeting.

There were sixteen sessions and five workshops depending on the area in the field of RNA biology. We had approximately two hundred talks and five hundred posters presented at the meeting. Two poster sessions were held in the evening (9:00PM to 11:00PM). Moreover, there were two additional career programs, mentor-mentee luncheon and time management workshop. Since many of the works have not been published yet, details of the research will not be described to keep it confidential. First day of the conference comprised welcoming reception, dinner party, and two keynote addresses by Phillip Zamore at UMass medical school and Robert Schneider at NYU school of medicine. For the following days, sessions started at 8:30AM and normally ended at 7:00. Poster sessions were held after dinner. Entire schedule of the meeting is available at the link below.

2. Programs

Oral presenters were given 10 minute for their presentation and 2 minute for questions except for the keynote speakers.

2-1. RNA-protein interactions

Feng Qiao laboratory from University of California at Irvine made the first presentation. They investigat-ed the interaction between TLC1, the RNA subunit of telomerase, and Ets2p, one of the proteins in the te-lomerase RNP. By using phosphorothioate footprinting, they found important elements of TLC1 in the interac-tion with Ets2p.

The second presentation was done by Julian J.-L. Chen laboratory from Arizona State University, and they showed how telomerase precisely define where it pauses DNA synthesis with high fidelity identifying se-quence determinants in telomerase RNA.

Christiane Branlant group studies the eukaryotic Box C/D RNPs that are involved in rRNA processing and UsnRNP biogenesis. Utilizing structural and genetic analyses, they identified a new player in C/D RNP assembly, Hit1p/TRIP3, which stabilizes the scaffolding protein of C/D RNP assembly.

John Woolford group showed importance of expansion segments of 25S rRNA in Saccharomyces cere-visiae utilizing rDNA mutagenesis system to manipulate the expansion segment. They found that lose of the expansion segment leads growth defect, suggesting it is important for ribosome biogenesis. Study of the pre-cise functions of the expansion segment is under active investigation.

Arlen Johnson group studies the RNA-RNA and RNA-protein interaction in ribosome biogenesis, and they demonstrated that A RNA helicase, Ecm16, is responsible for the release of U3 snoRNA during pre-40S maturation at the talk.

Elisabeth Tran group from Purdue University made the sixth presentation. They studied Dbp2, RNA-directed helicase, that is important for mRNP assembly, and they presented a model how Dbp2 is recruited to nascent RNA and promotes RNA-binding protein assembly during transcription.

2-2. RNA chemistry

Joseph Piccirilli group in University of Chicago aims to study RNA imaging techniques. To investigate the mechanism how a RNA aptamer called Spinach fluoresces upon fluorophore binding, they crystallized antibody-bound Spinach with and without fluorophore bound. They analyzed the significance of the G-quadruplex motif of Spinach for fluorophore binding, and this study would provide a base for engineering new RNA aptamers for various purposes.

Peter Unrau group developed a better RNA visualization system called RNA mango. They made a 39-nt G quadruplex type RNA aptamer that fluoresces when it binds thiazole orange dyes. This system solves prob-lems that the previous RNA visualization systems have such as high intrinsic background and weak dye-aptamer interaction.

Kevin Weeks laboratory studies the structure of the dengue virus RNA. They tried to characterize the secondary structure of the dengue virus RNA using SHAPE (selective 20-hydroxyl acylation analyzed by primer extension) chemical probing technology. They combined the result of SHAPE with bioinformatics to predict the secondary structure of RNA in the virus or in the absence of the virus, and the results identified the putative protein binding regions of the viral RNA.

2-3. RNA regulation in protozoa

Christine Clayton group studied how trypanosomes regulate mRNA splicing and degradation. They meas-ured the rates of trans splicing and mRNA decay of trypanosome and found that there are two categories of mRNAs depending on the degradation rate. One group is mRNAs with short half-lives that are initially degraded fast and reach a slower phase. Another group involves long-lived mRNAs that are degraded at slow rate in the beginning but show rapid destruction later. These seem to depend on whether mRNAs are dead-enylated or not before degradation. Also, they identified the proteins that interact with mRNAs by doing high-throughput screening and found over 200 proteins that increase or decrease the expression of mRNA. Since many novel proteins identified, this would help other researchers to study the function of RNA binding proteins that are previously unknown.

Dror Eliaz group from Bar-Ilan University investigated the regulation of trans-splicing of Trypanosoma brucei mRNA under ER-stress. T. brucei is a human pathogen that causes sleeping sickness. They identified PK3, a serine threonine kinase, as a key regulator of trans-splicing under ER stress in which trans-splicing is inhibited and cells reach programmed cell death eventually. In addition, they found that spliced leader RNA is secreted from the parasite, which suggests the presence of quorum sensing of the parasite.

Ken Stuart group at Seattle Biomed presented a novel method to study the functional architecture of editosomes in Trypanosoma brucei. They used chemical cross-linking/mass-spectrometry (XL/MS) and random mutagenesis to determine the protein-protein interaction of editosomes and found 33 editosome protein pairs. In addition, they mapped essential residues in editosome proteins using these approaches. Interestingly, they found that essentiality of some residues changes depending on the life cycle of the parasite suggesting that editosomes functions differently at distinct life cycle stages.

2-4. RNA silencing

Sarah A. Woodson at Johns Hopkins University revealed the structure of bacterial Hfq protein with rpoS mRNA. Hfq is a RNA binding protein that modulates small non-coding RNA(sRNA)-mediated translational regulation. They identified RNA motifs on rpoS mRNA that Hfq protein binds. In addition, they provided struc-tural model in which the rpoS-Hfq complex is shown and proposed that Hfq alternates its interaction with rpoS mRNA moving between closed and open status.

Joan Steitz laboratory at Yale University studied a non-canonical microRNA biogenesis. They found that some microRNAs are generated by RNA polymerase II dependent manner, and they have 5’-m7G-cap. Its gen-eration is microprocessor-independent, but its cytoplasmic processing is Dicer dependent as canonical mi-croRNAs are. In addition, 5’-capped strand is not efficiently incorporated into Argonaute. This allows us to design microRNA therapeutics minimizing the off-target effects because it can be designed to generate only one functional microRNA strand.

Gunter Meister group at University of Regensburg investigates the function of argonaute (Ago) proteins in miRNA-mediated gene silencing. Not all ago proteins have endonuclease activities, and they engineered these Ago proteins to turn them into cleavage-competent Ago proteins. They determine the important domains in Ago proteins that confer the cleavage function, which allows us to predict whether other Ago protein has endonuclease activity or not.

Martin Simard laboratory at St-Patrick Research Group in Basic Oncology studies the degradation miRNA. They previously found decapping enzyme DCS1 forms a degradation complex with XRN1 in C. elegans. Ongoing study aims to better understand miRNA degradation by this modulator protein. They identified DIP-1 (DCS-1 interacting protein) that is involved in developmental defects associated with mRNA misregulation. They found miRNAs and target mRNAs that are affected by dip-1 mutant, suggesting it is an important post-translational modifier in miRNA degradation.

Samuel Pfaff at the Salt Institute for Biological Studies investigates RNA processing that contributes to motor neuron identity. They identified a miRNA that is specifically enriched in motor neurons. Interestingly, this miRNA is generated by novel motor neuron-specific alternative splicing and polyadenylation mechanism. In addition, these alternative splicing and polyadenylation are located in the same intron where the miRNA is, suggesting the interactions among RNA processing machineries. Furthermore, they found that knockout of this miRNA affects survival of mice after birth due to yet unknown causes. Ongoing study aims to characterize the function of this miRNA.

2-5. RNA transport and localization

Mikko J. Frilander group at University of Helsinki studies how mRNA export from nucleus to cytoplasm is regulated. They found a novel mechanism of mRNA retention in the nucleus, and the minor spliceosome is involved in the process. They also found that U11 binding to the 3’-UTR is required for the nuclear retention, indicating that mRNA export is regulated by U11 snRNP.

Karla Neugebauer laboratory at MPI-CBG investigates the role of SR proteins in mRNA export. The SR proteins function in pre-mRNA splicing. They found all of SR proteins shuttle between nucleus and cytoplasm with different shuttling capacities. In addition, they found shuttling patterns of the SR proteins are different in cell types suggesting cell-type specific functions of the SR proteins. Next, they aimed to identify target mRNAs of individual SR proteins by microarrays and RNA-seq. They found that shuttling capacities correlate with the expression level of target mRNAs, and particular splice-isoforms are related to high shuttling SR proteins. This suggests that the SR proteins with different shuttling capacities regulate the export of specific mRNA isoforms.

Xianying Cui group at University of Toronto studies ER localization of mRNAs. They determine the frac-tion of mRNAs that are targeted and anchored to the ER, and they also found the cis-element that is required for mRNA anchoring. They focus on mRNAs that encode tail-anchored ER-resident proteins. These mRNAs are initially thought to be free-floating in the cytoplasm, but they showed that it is actually targeted to ER. They also found that initial targeting of these mRNAs is dependent on translation. These data suggest an alternative pathway by which tail-anchor proteins are localized to the ER by targeting mRNAs to ER membrane.

Dierk Niessing group at Helmholtz Zentrum Munchen studies the formation of motor-dependent transport particles that function in mRNA localization. They particularly focused on ASH1 mRNA in yeast that is an example for a nuclear transit of mRNPs. Loc1p is known to be required for efficient ASH1 mRNA localiza-tion, but there is limited information regarding the mRNA assembly. Thus, they aimed to determine how Loc1p-containing pre-mRNA assembles. They showed the stable mRNP complex of Loc1p with other proteins and important factors for motor activation. In addition, they did a single-particle motility assay to show move-ment of the reconstituted complex in vitro. This is the first time mRNA assembly was showed from the begin-ning at the molecular level.

2-6. 3’ End processing

3’ processing of mammalian pre-mRNA is governed by the cleavage and polyadenylation specificity fac-tor (CPSF). CPSF is a multi-protein complex that binds to the polyadenylation signal of mRNA at the 3’UTR, cleaves it, and add a poly(A) tail. It is thought to have at least six subunits, but the actual composition is not clear because the complex was not reconstituted in vitro. Elmar Wahle laboratory at Martin-Luther-University Halle-Wittenberg showed an in vitro reconstitution of CPSF that is active on AAUAAA-specific RNA binding and polyadenylation. They found the subunit that is responsible for the recognition of RNA sequences. In addition, they found the subunits that may be required for the cleavage reaction.

Yongsheng Shi group in University of California at Irvine studies the alternative polyadenylation of mRNA. It is important post-transcriptional gene regulation, but its regulation and functions still remain to be elusive. They showed that one of the mRNA 3’ processing factor, Fip1 as a regulator of embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. In addition, they identified the factors important for the Fip1-mediated alternative polyadenylation. Together, it suggests a novel post-transcriptional regulation of stem cell-renewal and pluripotency.

Mammalian/mechanistic target of rapamycin (mTOR) is known to regulate translation that is important for cell proliferation and growth. Jeongsik Yong group at University of Minnesota is expanding its function from translation to post-transcriptional gene regulation. They found that mTOR regulates alternative cleavage and polyadenylation. In addition, mTOR reprograms factors that are involved in the alternative cleavage and polyadenylation process, and it alters the length of 3’UTR. They showed that this process is independent of translation.

2-7. RNA in disease

Atze Das group in University of Amsterdam identified miRNA from human immunodeficiency virus type 1 (HIV-1). They found that HIV-1 transacting responsive (TAR) RNA element is a source of miRNAs.  Its func-tion is still unclear, but it appears that HIV-1 uses this element to prevent the cleavage of its own RNA genome.

Jeffrey Kieft laboratory at University of Clorado Denver School of Medicine studies the structure of the Flaviviral RNA. They especially focus on the conserved Xrn1-resistant RNA structures (xrRNAs) of the Flavivural RNA. By x-ran crystallography, they found that it has a unique three-dimensional topology that 5’-end of the RNA pass through a ring structure of the same RNA strand, and this confers the Xrn1-resistance to the 5’-end of the RNA. Interestingly, this Xrn1-resistant 3’UTR is important for the virulence because of yet unknown function.

Alexei Korennykh group at Princeton University studies the regulation of RNA sequence recognition by kinase-linked receptors RNase L and Ire1. Ire1 is a unfolded protein response protein which cleaves out an unconventional intron from target mRNA. RNase L is a homologue of Ire1, and it cleaves viral and self-RNA to promote the interferon response. By providing structures and mechanisms of RNA sequence recognition by these receptors, they would like to provide a platform to design small molecules to regulate the interferon response.

Gene Yeo laboratory at University of California San Diego generated a human iPSC models to study my-otrophic lateral sclerosis and frontotemporal dementia  (ALS/FTD). They hypothesize that accumulation of RNA foci due to an expanded GGGGCC repeat in the C9ORF72 locus causes the changes in gene expression which leads to motor neuron degeneration in ALS. They generated stable iPSC line expressing the GGGGCC repeats, and it results in nuclear RNA foci. This allows to compare cells with or without the repeat expansion, and they eventually were able to separate patient cells to fractions and analyze the changes in gene expression. They found specific gene expression patterns, and it would provide a foundation for developing disease biomarkers.

Eran Hornstein group studies microRNA dysregulation in amyotrophic lateral sclerosis (ALS). They showed that the expression of microRNAs are downregulated in human ALS motoneurons, and decreased Dicer activity appears to be the primary reason for the downregulation.  They suggested that the regulation of microRNA biogenesis is important for the neurodegeneration, and Dicer activity is post-translationally regulated.

Jocelyn Cote laboratory at University Ottawa investigates spinal muscular atrophy (SMA), which is a leading cause of infant deaths. They found that CARM1 expression increases in SMA, and more interestingly they found CARM1 as a novel regulator of nonsense-mediated mRNA decay (NMD). Coimmunoprecipitation identified UPF1 and UPF2 as interacting proteins of CARM1, suggesting that CARM1 acts in the early steps of NMD. They identified the NMD targets regulated by CARM1, and those targets are are also misregulated in SMA. This suggests the importance of CARM1 in SMA.

2-8. RNA decay

Elena Conti group in Max Planck Institute presented a crystal structure of a yeast Pan2-Pan3 core com-plex that is important for deadenylation of mRNA before its degradation. They recapitulate the catalytic activity of yeast Pan2-Pan3 complex in vitro. In addition, they showed that one Pan2 and two Pan3 subunits make a complex in the x-ray crystallography, and the N-terminal domain of Pan2 is important for the interaction with the Pan3 homodimer.

Bertrand Seraphin group focuses on the other side of mRNA decay pathways especially in the fate of 5’ m7G cap during the mRNA decay. After deadenylation mRNA can be degraded from the 3’-end by the RNA exosome or the 5’-Cap is removed by decapping enzymes followed by the Xrn1-mediated degradation from its 5’-end. m7GDP (di-)nucleotides are the by-products of the Xrn1-mediated decay, and they suggested that it is converted to m7GTP followed by DcpS-mediated hydrolysis to m7GMP. Finally m7GMP is converted into yet unknown by-products that do not contain the methyl group. In addition, they showed that the biochemical reaction of DcpS is evolutionarily conserved among eukaryotes.

Jeff coller laboratory in Case Western Reserve University investigated the determinant of mRNA half-life in yeast. They found that mRNA half-life is varies depending on the codon used, which suggests that transla-tional elongation rate is an important factor for determining the mRNA half-life. They also suggested that co-don optimization affects the decapping of mRNA not the deadenylation.

Herve Le Hir group studies how the Upf1 protein mediates nonsense-mediated mRNA decay. They uti-lized magnetic tweezers to analyze the activity of a single Upf1 protein and found that it translocate onto RNA with high processivity. Also, the translocase activity of Upf1 is regulated by its interaction with Upf2. This study is the first time to show eukaryotic RNA helicase translocase onto RNA substrate. Furthermore, they suggested that Upf1 is able to remodel RNA-protein complex.

Nobuyoshi Akimitsu group utilized 5’-bromo-uridine (BrU) Immunoprecipitation chase-deep sequencing analysis to identify the RNA species regulated by Upf1-dependent RNA degradation pathway. They identified over 200 transcripts that are directly regulated by Upf1, and the result suggests that over 600 transcripts are regulated by Upf1 directly and indirectly. They also identified a degradative element in RNA transcripts for Upf1-mediated degradation.

2-9. Mentor-Mentee luncheon – Finding a post-doctorial position

This was an informal gathering in that several graduate students and post docs have lunch with a cou-ple academic and industry mentors to answer questions about career. I chose a table of “finding a post doctorial position”. We had six students and three young academic mentors. We asked many questions how to find the post doc position after our graduation.

Here, I summarize their suggestions. First, look for the position at least a year before you graduate. If you get your position early, you will have enough time to apply for fellowship, which is critical for having your position stable, and it will help you get a job eventually. Second of all, think about what you want to do in a new laboratory. PIs do not like if you just list the techniques you have in your CV. They would like to see how much you really want to do research in their laboratories. Thus, it is important to think about what the laboratory is doing and come up with some ideas about the projects you would like to commit. Then, you write them down in your cover letter. Lastly, you should be excited what you are going to do. This needs strong commitment, and it is going to be what you would do for your lifetime. Thus, find a topic that makes you get excited.

3. Concluding remarks

Apparently, the RNA society has grown up rapidly for nineteen years. Dozens of different fields have emerged, and those involve subjects from basic science to translational research. Especially, the CRISPR system is notable since it comprised an entire session even though it was not covered on this report. In addition, many other topics could not discussed in this report due to the page limit. Topics discussed were randomly selected. Next, meeting of the RNA society will be held at University of Wisconsin, Madison.