Poster Session 5: RNA Modification & Condensation 21:00 - 22:00 Wednesday, 27th May, 2020 Poster

53 Chemo-enzymatic synthesis of site-specifically modified and photo-caged

Bozana Knezic1,2, Sara Keyhani1,2, Alexey Sudakov1,2, Alexander Heckel1, Harald Schwalbe1,2 1Goethe University Frankfurt, Frankfurt, Germany. 2Center for Biomolecular Magnetic Resonance (BMRZ), Frankfurt, Germany

Abstract

Posttranscriptional editing of RNAs occurs in all types of cells, often for regulatory purposes. Editing includes splicing and ligation reactions affecting larger parts of the RNA. Other types of modifications are the deamination or methylation of single affecting smaller RNA regions. Such reactions happen, for instance, with A (to I) and C (to U). These modifications impact the sequence and may alter the secondary structure of the RNA. Furthermore, they can lead to a different amino acid sequence and can ultimately affect the translation efficiency and diversify the cellular phenotype. In addition, there is evidence that posttranscriptional modifications affect liquid-liquid phase separation (LLPS) and as a consequence could participate in the support or even prevention of various diseases. In order to conduct experiments designed to investigate the impact of posttranscriptional editing, preparation of site-specifically modified RNAs is absolutely essential. For this purpose, solid-phase synthesis is the favored method up to this day. However, the technique is limited in terms of length of the desired RNA construct to a maximum of approximately 50 nt, if pure samples in sufficient quantities are required. To overcome this limitation, this project considers a chemo-enzymatic RNA synthesis approach[1]. This method was developed in our labs and allows the site-specific incorporation of modified nucleotides into a target RNA of virtually unlimited length. We were able to incorporate particular modified nucleotides into different long RNAs. This includes and other naturally occurring nucleotides, fluorinated nucleotides and nucleotides tagged with photolabile-protecting groups. The intention of this study is to develop a library of modified nucleotides, which are compatible with the before-mentioned synthesis approach. This approach could significantly support other methods and projects involved in RNA sequencing and modification recognition. In this case, a given site- specifically modified RNA could serve as a bench-mark for newly designed sequencing methods.

[1] S. Keyhani, T. Goldau, A. Blümler, A. Heckel, H. Schwalbe, Angew. Chemie Int. Ed. 2018, 57, 12017–12021.

Presenting author email [email protected]

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RNA Modification & Editing 76 m6A restricts axonal growth in Drosophila through modulation of Fragile X mental retardation target selection

Alessia Soldano1, Lina Worpenberg2, Chiara Paolantoni2, Sara Longhi1, Miriam Mulorz3, Tina Lence3, Hans- Hermann Wessels4, Giuseppe Aiello1, Michela Notarangelo1, FX Reymond Sutandy3, Marion Scheibe3, Rhagu R. Edupuganti5, Anke Busch6, Martin M. Möckel7, Michiel Vermeulen5, Falk Butter3, Uwe Ohler4, Christoph Dieterich8, Alessandro Quattrone1, Jean-Yves Roignant 2 1Centre for Integrative Biology, University of Trento, Trento, Italy. 2Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland. 3Institute of Molecular Biology, Mainz, Germany. 4Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany. 5Radboud Institute for Molecular Life Sciences, Oncode Institute, Nijmegen, Netherlands. 6Bioinformatics Core Facility, IMB, Mainz, Germany. 7Protein Production Core Facility, IMB, Mainz, Germany. 8Klaus Tschira Institute for Integrative Computational Cardiology and Department of Internal Medicine III, University Hospital Heidelberg; German Center for Cardiovascular Research (DZHK), Heidelberg, Germany

Abstract

The abundant mRNA modification N6-methyladenosine (m6A) regulates a variety of physiological processes through modulation of RNA metabolism. m6A is particularly enriched in the nervous system of several species and its dysregulation has been associated with neurodevelopmental defects as well as neural dysfunctions. In Drosophila, the loss of m6A alters fly behavior but the underlying mechanism and the role of m6A during nervous system development has remained elusive. Here we found that impairment of the m6A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions, as well as, in the adult mushroom bodies. We identify Ythdf as the main m6A reader in the nervous system required for limiting axonal growth. Mechanistically, we show that Ythdf interacts directly with Fragile X mental retardation protein (Fmr1) to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6A pathway controls development of the nervous system by modulating Fmr1 target selection.

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RNA Modification & Editing 209 Programmable RNA base editing with a single engineered protein

Wenjiang Han1,2, Wendi Huang1,2, Tong Wei1,2, Yanwen Ye1,2, Miaowei Mao1, Zefeng Wang1 1CAS-MPG Partner Institute for Computational Biology, Chinese Academy of Sciences, Shanghai, China. 22.University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China

Abstract

Programmable base editing of RNA enables re-writing on specific sites. Current tools for specific RNA editing dependent on the assembly or recruitment of the guide RNA into an RNA/protein complex, which may cause delivery barrier, low editing efficiency, and high immunogenicity. Here, we report a new set of tools, RNA editing with individual RNA-binding enzyme (REWIRE), to perform precise base editing with a single engineered protein. The REWIRE system contains a human-originated programmable RNA-binding domain (PUF domain) to specifically recognize its target and ADAR or Apobec3A deaminase domain to achieve A-to-I or C-to- U editing. Using this system, we achieved up to 80% editing efficiency in A-to-I editing and 60% efficiency in C- to-U editing, with little non-specific editing in the targeted region and a low level of off-target effect globally. We were able to apply the REWIRE system to rescue the disease-associated base mutations and to modify mitochondrial RNAs. The RNA binding module of this system was further optimized to increase the editing specificity and minimize off-target effects. As a single-component base editing system originated form human , REWIRE presents a precise and efficient RNA-editing platform with potential in basic research and therapy.

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RNA Modification & Editing 226 C/D Box RNAs protect tRNA Met(e) from stress-induced clivage

Patrice Vitali, Tamàs Kiss CBI-CNRS; Université Paul Sabatier, Toulouse, France

Abstract

Site-specific 2'-O-ribose methylation of mammalian rRNAs and RNA polymerase II-synthesized spliceosomal small nuclear RNAs (snRNAs) is mediated by small nucleolar and small Cajal body (CB)-specific box C/D ribonucleoprotein particles (RNPs) in the nucleolus and the nucleoplasmic CBs, respectively. Here, we demonstrate that 2'-O-methylation of the C34 wobble of human elongator tRNAMet(CAT) is achieved by collaboration of a nucleolar and a CB-specific box C/D RNP carrying the SNORD97 and SCARNA97 box C/D 2'-O-methylation guide RNAs. Methylation of C34 prevents site-specific cleavage of tRNAMet(CAT) by the stress-induced endoribonuclease angiogenin, implicating box C/D guide RNPs in controlling stress-responsive production of putative regulatory tRNA fragments.

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RNA Modification & Editing 274 -wide analysis of ADAR3 binding and the role of ADAR3 in increasing MAVS activity in response to double-stranded RNA

Reshma Raghava Kurup, Emilie Oakes, Aidan Manning, Pranathi Vadlamani, Heather Hundley Indiana University, Bloomington, IN, USA

Abstract

RNA editing contributes to transcriptome diversity by influencing regulatory processes such as RNA splicing, stability, localization and translational efficiency. -to-inosine (A-to-I) editing is one of the most abundant types of RNA editing in humans and is carried out by ADARs. In humans, there are three ADAR family members, ADAR1, ADAR2 and ADAR3. ADAR3 is less studied due to brain-specific expression and lack of known editing activity. Recently our lab demonstrated that ADAR3 is elevated in glioblastomas compared to adjacent normal tissue and binding of ADAR3 to one neuronal transcript, GRIA2, leads to inhibition of editing by ADAR2. However, the role of ADAR3 in the regulation of editing and in glioblastoma is not well understood. A transcriptome-wide analysis was performed to determine RNA targets of ADAR3 and the effects of ADAR3 on RNA editing in the U87 glioblastoma cell line. Using RIP-seq analysis, 3316 ADAR3 bound targets were identified. I found that ADAR3 expression resulted in differential editing and gene expression transcriptome- wide. Around 80% of sites have reduced editing and the majority of differential edited sites are located in 3’UTRs. In this study, I showed that ADAR3 act as a negative regulator of ADAR1-mediated editing. Previous studies have shown that loss of ADAR1 editing activity leads to activation of the double-stranded RNA (dsRNA) immune response pathways. The dsRNA sensors activate MAVS, leading to expression of type I IFNs and inflammatory cytokines thus playing an important role in the antiviral response and autoimmunity. Upon exogenous dsRNA treatment, ADAR3 expressing cells showed elevated expression of immune response downstream of MAVS. I also demonstrated that ADAR3 binds to the MAVS transcript and reduces ADAR1- mediated editing of the MAVS 3’UTR. Even though ADAR3 did not affect MAVS expression at the mRNA level, MAVS protein level is increased in ADAR3 expressing cells. My data suggest that ADAR3 has a unique role in the regulation of MAVS expression independent of ADAR1 and presumably RNA editing. The results from this study will provide a better understanding of ADAR3 mediated gene regulation during normal development as well as in diseases.

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RNA Modification & Editing 288 An uncharacterized human synthase modifies the HCV RNA genome and is important for viral replication

Erin Borchardt1, Wenyu Lin2, Raymond Chung2, Brett Lindenbach1, Wendy Gilbert1 1Yale University, New Haven, CT, USA. 2Massachusetts General Hospital, Boston, MA, USA

Abstract

Diverse chemical modifications found in nature decorate viral RNA genomes and are poised to impact the viral life cycle. Pseudouridine, which is the most abundant chemical modification in human cells, rigidifies the RNA backbone and affects RNA-RNA and RNA-protein interactions in various contexts. Recent work from our lab and others has revealed a complex pseudouridine landscape that includes many modified sites in messenger RNAs as well as all classes of non-coding RNA. Here, we report the first characterization of site-specific viral RNA pseudouridylation. Using high-throughput pseudouridine profiling, we discovered 43 high-confidence distributed across the hepatitis C (HCV) genome and hundreds of novel pseudouridines throughout cellular RNAs in HCV-infected cells. We identify multiple host pseudouridine synthases that directly modify viral genomic RNA and show that one of them, PUS7L, is required for normal viral replication in cultured hepatocellular carcinoma cells. Further, we define specific RNA sequences common to most PUS7L targets, demonstrating a unique sequence motif for a previously uncharacterized human PUS protein. Work is ongoing to interrogate specific PUS7L-dependent pseudouridines for various roles in HCV infection including recoding of viral proteins and innate immune evasion. Together, our results reveal PUS7L as an active pseudouridine synthase in human cells and establish pseudouridine as a pro-viral RNA modification for HCV.

Presenting author email [email protected]

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RNA Modification & Editing 296 RNA modifications as biomarkers for RNA virus infection in cell lines and Aedes mosquitoes

Rachel Netzband1, Will McIntyre2,1, Pheonah Badu1, Gaston Bonenfant1, Sean Bialosuknia1,3, Alexander Ciota1,3, Daniele Fabris2,1, Cara Pager1 1University at Albany, Albany, NY, USA. 2University of Connecticut, Mansfield, NY, USA. 3Wadsworth Center, New York State Department of Health, Albany, 12203, USA

Abstract

Zika virus (ZIKV) received global attention during an outbreak in the Americas in 2016-2018, where virus was associated with neurological and developmental defects in infected individuals. The virus is primarily transmitted via the Aedes species mosquitoes. Currently there are no antivirals or vaccines. It is critical to understand the molecular biology and pathogenesis of ZIKV. Our lab is interested in the mechanisms by which ZIKV interacts with and subverts the cellular . To pursue this, we use , a highly sensitive and unbiased analytical approach. Recently we showed that the distribution of post-transcriptional modifications (PTMs) on RNA in liver and kidney cells changed upon ZIKV infection. Moreover, we demonstrated that the viral genomic RNA was heavily modified, well beyond the characterized N6-methyladenosine modification (m6A). ZIKV cycles between human and mosquito hosts, thus the goal of this research was to investigate the PTM profiles in a mosquito cell line and compare to ZIKV infection in living mosquitoes. Specifically, Aedes aegypti mosquitoes were infected by providing an infectious bloodmeal (n=48) and isolating those individuals that fed on blood. As a control we had 31 blood-fed uninfected mosquitoes. RNA from whole mosquitoes was harvested 14 days post-infection by homogenization and TRIzol extraction. Using phosphodiesterase and nuclease P1 we digested RNA into mononucleotides, and then analyzed PTMs using mass spectrometry. Between mock- and ZIKV-infected mammalian cells we observe distinct changes in the PTM landscape. Similarly in mosquitoes we find changes in the PTM profiles, both different between mock- and ZIKV-infected mosquitoes and between mammalian cells and mosquitoes. In mammalian cells, we observed the appearance of two variants of dimethylcytosine only during ZIKV infection. Excitingly, we found that whole mosquitoes have other PTM biomarkers, such as dimethylguanosine, inosine, and methyladenosine that may be used to predict the infection status of an individual mosquito. Our research opens up the opportunity for a sensitive and unbiased detection method of infected mosquitoes in the field.

Presenting author email [email protected]

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RNA Modification & Editing 322 Elucidating the modification status of tRNAs in response to environmental and metabolic stress

Mariana Mandler, Christina Fitzsimmons, Kayla McDonald, Pedro Batista NCI, Bethesda, MD, USA

Abstract

RNA modifications extend the functionality of transcripts. While over 170 RNA modifications have been identified so far, determining the function and location of many of these has been limited by available reagents and technologies. 4-Thiouridine (s4U) is a naturally occurring RNA modification found in bacteria and tRNAs, and functions as a near-UV sensor and a regulator of cell growth, in response to which levels of specific tRNAs have been reported to change. Here, we have adapted a chemical approach to capture and identify s4U-containing RNAs in a high-throughput manner using Next-Generation Sequencing. With this method we can observe enrichment for tRNAs known to be modified, amid potential depletion of tRNAs that lack this modification. This method allowed us to ask several questions regarding whether and how s4U modifications are influenced by environmental conditions. Bacterial cells were cultured in different types of media and RNA was collected from cells at different stages of growth. While the enrichment of specific tRNAs did not vary significantly between different stages of growth in the same media, we identified a few tRNAs, such as threonine (ACU), where s4U levels did respond to growth stages. Enrichment of these s4U-containing tRNAs varied mostly between bacteria grown in different types of media. These results suggest that nutrients provided by the extracellular environment, known to impact metabolic pathways, can influence the levels of s4U-modification in tRNAs within the cell. Altogether, a high-throughput approach to map and quantify s4U- containing tRNAs has elucidated how environmental nutrients can impact the levels of tRNA modifications in bacteria and can be applied to identify s4U modified RNAs in any sample.

Presenting author email [email protected]

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RNA Modification & Editing 328 Adaptation of enhanced crosslinking and immunoprecipitation (eCLIP) for the high-throughput, high-resolution mapping of N6-methyladenosine modifications

Sergei Manakov1, Ines Rabano1, Alexander Shishkin1, Kylie Shen1, Heather Foster1, Peter Chu1, Eric Van Nostrand1,2, Gene Yeo1,2 1Eclipse Bioinnovations, San Diego, CA, USA. 2University of California, San Diego, San Diego, CA, USA

Abstract

N6-methyladenosine (m6A) is the most prevalent epitranscriptomic RNA modification found in and has been shown to regulate many stages of RNA biology including splicing, secondary/tertiary structure, localization, stability, and translation. Adenosine modifications are actively added, recognized, and removed by a series of “writer”, “reader”, and “eraser” proteins, making m6A a dynamic and reversible system for regulating RNAs. Further, there are now clear links between m6A writer, reader, and eraser proteins as well as m6A state of specific mRNAs to cancer pathogenesis, indicating that both global and specific dysregulation of m6A can have targeted effects that lead to disease. Thus, there is a need for methods to robustly profile m6A- modification states in both cell lines as well as tissues, and in normal as well as diseased or modified states. Advancements in high-throughput sequencing have led to the development of methods to map m6A transcriptome-wide, but there remains a need for improved methods that are scalable across conditions and treatments and with low abundance samples, such as blood or patient tissue. To address this need, we adapted enhanced crosslinking and immunoprecipitation (eCLIP) to create an optimized protocol for profiling m6A modification sites with single nucleotide resolution and 20-50-fold less starting RNA material (m6A-eCLIP). Using m6A-eCLIP, we recover traditional m6A motif enrichment and positioning enrichment at stop codons, validating the accuracy of the approach. Further, the improved handling and efficiency enabled us to profile m6A status across several different experimental conditions, showing that m6A-eCLIP can be a robust method that is widely applicable for epitranscriptomic profiling.

Presenting author email [email protected]

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RNA Modification & Editing 351 Random mutagenesis identifies a novel essential domain in editosome RNase III proteins that are required for editing and differ between life cycle stages in

Jason Carnes, Suzanne McDermott, Ken Stuart Seattle Children's Research Institute, Seattle, WA, USA

Abstract

Multiprotein editosomes catalyze gRNA-specified insertion and deletion of to create functional mitochondrial mRNAs in Trypanosoma brucei. Three functionally distinct editosomes are distinguished by their single KREN1, KREN2, or KREN3 RNase III endonuclease and, respectively, KREPB8, KREPB7, and KREPB6 partner proteins. These endonucleases perform the first catalytic step of editing, cleaving mRNA in diverse mRNA/gRNA heteroduplex substrates. We identified divergent and likely non-catalytic RNase III domains in KREPB4, KREPB5, KREPB6, KREPB7, KREPB8, KREPB9, and KREPB10 editosome proteins. Because known RNase III endonuclease functional domains are dimeric, the editing endonucleases may form heterodimers with one or more of these divergent RNase III proteins. We show here using conditional null cell lines that KREPB6, KREPB7, and KREPB8 are essential in both procyclic form (PF) and bloodstream (BF) cells. Loss of these proteins results in growth defects and loss of editing in vivo, as does mutation of their RNase III domain that is predicted to prevent dimerization. Similarly, mutation of the U1-like zinc finger domain results in growth defects. Furthermore, random mutagenesis experiments reveal a novel domain shared among KREPB6, KREPB7, and KREPB8 that is essential for function. Loss of KREPB6, KREPB7, or KREPB8 also dramatically reduces cognate endonuclease abundance, as do many but not all studied mutations, indicating that KREPB6, KREPB7, and KREPB8 stabilize their respective endonucleases. The phenotypic consequences of repression are more severe in BF than in PF, indicating differences in endonuclease function between developmental stages that could impact regulation of editing. These results suggest that KREPB6, KREPB7, and KREPB8 form heterodimers with their respective endonucleases to perform mRNA cleavage. We also present a model wherein editosome proteins with divergent RNase III domains function in substrate selection via enzyme- pseudoenzyme interactions.

Presenting author email [email protected]

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RNA Modification & Editing 381 Chemical Approaches to Illuminate the Epitranscriptome

Ralph Kleiner Princeton University, Princeton, NJ, USA

Abstract

The properties of eukaryotic messenger RNA can be modulated by dynamic chemical modifications that occur post-transcriptionally (known as the “epitranscriptome”). These include N6-methyladenosine (m6A), which regulates mRNA turnover, translation, nuclear export, and splicing, as well as other modifications on the nucleobases. A major challenge is to identify the functional consequences of these marks and elucidate the molecular mechanisms by which they affect gene expression programs in cells. To address this gap in our knowledge, we have developed and applied chemical biology strategies to characterize to study the effects of mRNA modifications on cellular processes. First, we describe a chemical proteomics approach relying upon photocrosslinkable diazirine-containing synthetic RNA oligonucleotide probes and quantitative proteomics to profile readers of N6-methyladenosine (m6A) and N1-methyladenosine (m1A). In addition to identifying known m6A binders, we find that YTH-domain proteins bind specifically to m1A. Investigation of YTHDF2, indicates that this protein controls the stability of m1A-modified transcripts. Further, we find that m6A disrupts RNA binding by the stress granule proteins G3BP1/2, UPS10, CAPRIN1, and RBM42, providing a link between mRNA modifications and the integrated stress response. Second, we describe a strategy for the metabolic incorporation of non-canonical nucleotides into cellular RNA. We have applied protein engineering to - cytidine kinase 2, an enzyme in the pyrimidine nucleotide salvage, to alter its substrate specificity. Remarkably, introduction of this mutant enzyme into mammalian cells enables the incorporation of bulky C5- modified pyrimidine nucleosides into RNA. We have used this approach for the metabolic incorporation of novel azide-containing biorthogonal nucleotides for visualizing RNA synthesis and turnover, as well as diazirine- containing nucleosides for protein-RNA photocrosslinking. We anticipate that our approach will serve as a general strategy for modifying RNA in living cells with diverse modified bases for probing RNA biology. Taken together, our work should improve our understanding of fundamental RNA regulatory mechanisms and provide powerful and general strategies for interrogating the function of mRNA modifications.

Presenting author email [email protected]

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RNA Modification & Editing 395 Physiological implications of the non-canonical NAD+ cap removal by DXO1 in

Aleksandra Kwaśnik1, Michał Krzysztoń2, Joanna Kufel1 1Institute of Genetics and Biotechnology, University of Warsaw, Warsaw, mazovian, Poland. 2Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, mazovian, Poland

Abstract

DXO1 from Arabidopsis thaliana is a member of DXO protein family that participates in the removal of non- canonical RNA NAD+ cap (deNADding) and in the quality control of a canonical m7G cap synthesis (5’QC). These functions are executed by the phosphodiesterase PD-(D/E)XK active site that hydrolyzes various RNA 5’- end structures and further proceeds with the exoribonucleolytic degradation of the RNA body. Our previous work showed that DXO1 is unique among its eukaryotic homologs. Architecture of DXO1 catalytic site allows for a very efficient NAD+ cap removal, but 5’-3’ exoribonuclease activity is diminished and 5’QC properties are completely inhibited, unless the plant-specific N-terminal extension (NTE) is deleted from the DXO1 sequence. Unexpectedly, strong deNADding activity of DXO1 appears unrelated to the most exemplified morphological and molecular phenotypes of dxo1 mutant plants that include altered RNA metabolism and chloroplast function. Instead, all implications of DXO1 deficiency that we observed before were solely dependent on NTE.

Presence of a strong deNADding activity of DXO1 in vitro prompted us to search more thoroughly for its physiological function, but we expected that only a limited subset of RNA molecules would be direct targets of DXO1 enzymatic activity. We set out to identify them with a transcriptomic approach on dxo1 plants and transgenic lines expressing various levels of wild-type, catalytically mutated and N-terminally truncated DXO1 in a dxo1 background. We also included fry1 plants that show constitutive accumulation of a chloroplast stress signal, 3’-phosphoadenosine-5’-phosphate (PAP), which is the inhibitor of DXO1 and three plant XRN 5’-3’ exoribonucleases. To reduce the cost and time required to generate transcriptomic data from a large set of RNA samples, we applied BRB-seq (bulk RNA barcoding and sequencing) that employs early multiplexing allowing for the parallel preparation of multiple 3’ cDNA libraries. Comparison of the obtained datasets allowed us to select potential targets of DXO1 enzymatic activity that were further analyzed in a transcript-specific manner. Moreover, our results compared with the publicly available NAD+-capped RNA sequencing data provide an input to the understanding of the non-canonical RNA capping in Arabidopsis.

Presenting author email [email protected]

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RNA Modification & Editing 405 Poly(UG)-tailed RNAs in Genome Protection and Epigenetic Inheritance

Aditi Shukla1, Jenny Yan1, Anne Dodson1, Dan Pagano1, Marvin Wickens2, Scott Kennedy1 1Harvard Medical School, Boston, MA, USA. 2University of Wisconsin, Madison, WI, USA

Abstract

Mobile genetic elements threaten genome integrity in all organisms. MUT-2/RDE-3 is a ribonucleotidyltransferase (rNT) required for transposon silencing and RNA interference (RNAi) in the model organism C. elegans. When tethered to RNAs in heterologous expression systems, RDE-3 can add long stretches of alternating non-templated uridine (U) and guanosine (G) ribonucleotides to the 3’ termini of these RNAs (polyUG or pUG tails). Here, we show that, in its natural context in C. elegans, RDE-3 adds pUG tails to transposon RNAs, as well as to targets of experimental and endogenous RNAi in the C. elegans germline and soma. pUG tails with more than 12 perfectly alternating 3’ U and G nucleotides convert otherwise inert RNA fragments into potent and specific agents of gene silencing. Injection of pUG RNAs into C. elegans germlines induced small interfering (si)RNA expression, suggesting that pUG tails may mark RNAs as templates for RdRP. Indeed, MS/MS analysis of proteins binding to pUG RNAs, as well as directed coIP analyses, show that RdRP is a pUG RNA-binding protein. Together, our data show that pUG tails promote gene silencing by recruiting RNA- dependent RNA Polymerases (RdRPs) to pUG RNAs so that pUG RNAs can be used as templates for siRNA production. In germ cells, pUG RNAs localize to a phase separated condensate termed the Mutator foci, whose assembly we show is necessary for coordinating the pUG RNA pathway. Finally, we show that iterative cycles of pUG RNA-templated siRNA synthesis and siRNA-directed mRNA pUGylation mediate transgenerational epigenetic inheritance (TEI) in the C. elegans germline. Together, our data lead us to speculate that one function of RNA pUGylation is to convert otherwise inert RNA fragments into RNA-based inoculates, which allow parents to immunize their progeny against the damaging effects of transposons. Incidentally, our MS/MS analysis also identified TDP-1, the C. elegans ortholog of the mammalian UG-binding protein TDP-43, as a highly specific pUG tail-binding protein. Progress, if any, towards understanding the function for TDP-43 in pUG RNA biology will also be reported.

Presenting author email [email protected]

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RNA Modification & Editing 421 Investigating the cytoplasmic regulation of the SAM synthetase MAT2A mRNA

Juliana Flaherty1, Anna Scarborough1, Ashwini Kumar2, Chao Xing2, Nicholas Conrad1 1University of Texas Southwestern Medical Center, Department of Microbiology, Dallas, TX, USA. 2University of Texas Southwestern Medical Center, McDermott Center Bioinformatics lab, Dallas, TX, USA

Abstract

S-adenosyl methionine (SAM) is the methyl donor for nearly all cellular methylation events, so cells tightly regulate intracellular SAM levels. In most human cell types, SAM is synthesized by the SAM synthetase encoded by the methionine adenosyltransferase 2A (MAT2A) gene. To control SAM levels, cells regulate MAT2A mRNA accumulation by at least two mechanisms mediated by 6 regulatory hairpins (hp) in the MAT2A 3 ´ UTR. We proposed that the MAT2A transcript is regulated by SAM-dependent detention of the last intron. Upon SAM starvation, the dwell-time of the hp methyltransferase METTL16 on hp1 increases, resulting in splicing induction. In the cytoplasm, MAT2A mRNA is subject to further regulation. We and others have suggested that METTL16-mediated methylation of hp 2-6 decreases cytoplasmic stability of the MAT2A mRNA. However, the mechanisms controlling MAT2A mRNA stability remain elusive. To identify proteins involved in regulating cytoplasmic stability of MAT2A mRNA, we developed a cell line that expresses a reporter construct consisting of GFP fused to the final 2 of MAT2A. The construct lacks the detained intron, so we can monitor splicing-independent regulation by the MAT2A 3´ UTR. We stably integrated the reporter into HCT116 cells and performed an unbiased CRISPR knockout (KO) screen. We expect that KO of factors that promote MAT2A destabilization will increase GFP signal. In a pilot screen, we identified several translation-related proteins as candidate cytoplasmic MAT2A regulators. Translation and mRNA stability are coupled, so we hypothesize that MAT2A mRNA stability is regulated in concert with translation efficiency. After validation of these candidates, we will probe their mechanisms in MAT2A mRNA translation and cytoplasmic stability. More broadly, our work will further reveal the molecular mechanisms that regulate SAM homeostasis.

Presenting author email [email protected]

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RNA Modification & Editing 473 Uncovering a molecular mechanism to regulate tissue and development specific A-to-I RNA editing

Suba Rajendren, Alfa Dhakal, Pranathi Vadlamani, Jack Townsend, Heather Hundley Indiana University, Bloomington, IN, USA

Abstract

A-to-I RNA editing is one of the molecular mechanisms that create transcriptome diversity and can modulate gene expression in a dynamic cell-type specific manner. During mammalian brain development, editing levels at specific sites increase while the expression of editing enzymes, ADARs (Adenosine deaminases acting on RNAs) remain unchanged. The molecular mechanisms that mediate the spatio-temporal regulation of ADAR substrate binding and editing efficiency are largely unknown. In this study, we identified stage specific edited neural transcripts in C. elegans. Our gene expression analysis suggests that the stage-specific edited transcripts are differentially expressed during neural development, and nearly one-third of these differentially expressed genes are dependent on adr-2, the only known editing enzyme in C. elegans, for proper gene expression. We also identified transcripts that are edited throughout neural development. We found neural specific downregulation of adr-2 in C. elegans development. Strikingly, the majority of the sites in these transcripts showed increased editing in young adult neural cells. Our data suggests that ADR-1, a deaminase deficient ADAR protein is competing with ADR-2 for binding to the transcripts early in development and the neural specific downregulation of adr-1 in development leads to increased editing at these transcripts. Our data suggest a model where during neural development, ADR-2 levels overcome ADR-1 repression, resulting in increased editing of specific transcripts. Together, our findings reveal a tissue and development specific regulation of RNA editing and identify a molecular mechanism that regulates substrate recognition and editing efficiency by ADARs.

Presenting author email [email protected]

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RNA Modification & Editing 487 Widespread modification of bacterial mRNAs reveals potential functions of pseudouridine

Cassandra Schaening Burgos1, Gene-Wei Li1, Wendy Gilbert2 1MIT, Cambridge, MA, USA. 2Yale University, New Haven, CT, USA

Abstract

Pseudouridine (Ψ) is an ubiquitous RNA modification, present in the tRNAs and rRNAs of species across all domains of life, and is now known to be present in the mRNAs of diverse eukaryotes. However, the functional roles of Ψ in mRNA remain largely unknown. Here, we report the discovery of widespread modification of bacterial mRNAs with Ψ at positions poised to affect translation and regulation by small RNAs (sRNAs). Transcriptome-wide detection of Ψ in wild-type E coli revealed 201 high-confidence sites in mRNAs, located in coding sequences as well as untranslated regions. mRNAs are modified by at least four pseudouridine synthases (RluA, RluF, TruA, or TruB), with the remaining 7 synthases still being tested. The enzyme RluA targets several genes that are involved in translation, modifying their mRNAs at sites with sequence and structural similarity to its canonical tRNA and rRNA targets. Our preliminary analysis of several available datasets, which include sRNA-interactomes, RNA structureomes, and proteomics, implicate Ψ in the regulation of mRNAs via diverse mechanisms. From sRNA-interactome studies, we observed Ψ sites located in the binding sites of known small regulatory RNAs where Ψ is predicted to potentiate regulation by stabilizing the sRNA- mRNA duplex. Structure probing datasets suggest that pseudouridylated sites in mRNA are more frequently folded into secondary structures. Finally, proteomic datasets revealed potential translational recoding events at several RluA-dependent mRNA Ψ sites. Work is ongoing to assess the dependence of these events on the modification. Finally, structure probing datasets suggest that pseudouridylated sites in mRNA are more frequently folded into secondary structures. Overall, our work shows that pseudouridine is common in bacterial mRNAs and suggests ample opportunities for Ψ to regulate the life cycle of the transcripts that contain it.

Presenting author email [email protected]

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RNA Modification & Editing 566 Structure and function of yeast pseudouridine synthase 7

Meredith Purchal, Ryan McNassor, Hari Sharma, Taslima Khan, Leena Malik, Kristin Koutmou, Markos Koutmos University of Michigan, Ann Arbor, MI, USA

Abstract

Pseudouridine (Y) is among the most abundant post-transcriptional RNA modifications in cells and has long been known to make crucial contributions to non-coding RNA structure and function. Recently, several Ymodifications were identified in hundreds of mRNAs and we seek to describe how pseudouridine synthase (Pus) enzymes select and modify this new class of substrates. This work focuses on Pus7, a pseudouridine synthase that modifies a variety of mRNAs under cellular stress in addition to a diverse of noncoding RNAs in functionally relevant positions. We report the first x-ray structure of S. cerevisiae Pus7 (yPus7), where we observe that the core structure of the enzyme is highly conserved with the bacterial homologs, yet contains three additional domains with yet unknown functionality. Furthermore, we use kinetic and binding studies to uncover the molecular level determinants of yPus7 substrate selection. These studies revealed several residues important to Pus7 function (K61, F67, F307, N305, E71, and D256), and probe structural requirements of yPus7 substrates necessary for binding and pseudouridylation. Our studies suggest that yPus7 is a relatively promiscuous enzyme capable of interacting with a broad range of RNA sequences and structures.

Presenting author email [email protected]

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RNA Modification & Editing 585 Identifying the molecular fingerprints of rRNA modifications using nanopore sequencing

Oguzhan Begik1,2, Morghan Lucas1,3, Eva Maria Novoa1,3 1CRG, Barcelona, Spain. 2Garvan Institute of Medical Research, Sydney, NSW, Australia. 3UPF, Barcelona, Spain

Abstract

In the last few years, our ability to map RNA modifications transcriptome-wide has revolutionized our understanding of how these chemical entities shape cellular processes, modulate cancer risk, and govern cellular fate. Systematic efforts to characterize this regulatory layer have revealed that RNA modifications are far more widespread than previously thought, can be subjected to dynamic regulation, and can profoundly impact RNA processing stability and translation. A fundamental challenge in the field, however, is the lack of a generic approach for mapping and quantifying RNA modifications, as well as the lack of single molecule resolution. Third-generation sequencing technology such as Nanopore sequencing has emerged as a good alternative to the next-generation sequencing since it can provide full-length and native RNA information. Here we show that using third-generation sequencing, ribosomal RNA modifications can be detected with high accuracy, in the form of systematic errors and decreased base-calling qualities. We then further validate our detection method using KO systems. Moreover, we further validate our method by showing that these ‘errors’ are typically not observed in yeast snRNA-deficient strains (RNAs responsible for adding pseudouridylation on ribosomal RNAs), which lack the specific modifications at certain positions. Our results open new avenues to investigate the biological roles of RNA modifications in their native RNA context, as well as in the form of Reverse Transcription errors.

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RNA Modification & Editing 588 Fidgeting the uridines on long non-coding RNA

Gabriela Toomer, Huachen Gan, Joanna Sztuba-Solinska Auburn University, Auburn, Alabama, USA

Abstract

LncRNAs form a complex network of interactions with effectors to modulate a plethora of cellular pathways. However, the mechanisms that regulate the regulators that are lncRNAs are not well established. Polyadenylated nuclear (PAN) RNA is an abundant and stable transcript expressed by Kaposi’s sarcoma- associated herpesvirus (KSHV). PAN modulates cellular processes, i.e., cellular proliferation, immune response, to create an environment that benefits the pathogen. Even though PAN is recognized for its critical biological functions, only a few of the PAN interacting partners have been identified and studied from the protein-centric standpoint. Also, the influence of interacting molecules on PAN structure, function, and the mechanisms to regulate these interactions, remain undefined. We were the first to discover that the conformation of PAN, as well as the proteins associating with PAN, are distinct in different biological contexts (Sztuba-Solinska et al., 2017) The deposition of chemical moieties onto RNA, so-called epitranscriptomic modifications, can have profound effects on RNA. Pseudouridine (Ψ) is the most abundant epitranscriptomic signature, shown to affect RNA stability, structure, and protein binding. Numerous lncRNAs with critical cellular functions have been shown to carry Ψ, yet, the mechanism of Ψ formation and its influence over lncRNAs structure and cellular functions are elusive. We used the target-specific Ψ-seq analysis in combination with CMC-RT and ligation assisted PCR analysis of Ψ modification (CLAP) and showed that PAN lncRNA exists in multiple “pseudouridylation states” during latent and lytic stages of KSHV replication. Using RNA antisense purification for mapping the direct RNA:RNA interactions (RAP RNA) and RAP with mass spectrometry (RAP MS), we demonstrated that PAN is a part of the cellular interactome involving specific non-coding RNAs and enzymes that have been shown to regulate the installation of pseudouridine. We have established recombinant KSHV using a bacterial artificial system to explore the influence of Ψ on PAN lncRNA structure, stability, interactome network, and KSHV infectivity cycle. The information gained in this system will increase the understanding of lncRNA plasticity and will contribute to an underrepresented body of knowledge regarding the influence of epitranscriptomics on RNA structure and function.

Presenting author email [email protected]

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RNA Modification & Editing 593 RNA editing of immunogenic dsRNAs plays a primary role in autoimmune and immune-related diseases

Qin Li1, Michael Gloudemans1, François Aguet2, Tao Sun1, Gokul Ramaswami1, Yang Li1, Jonathan Pritchard1, Stephen Montgomery1, Jin Li1 1Stanford University, Stanford, CA, USA. 2Broad Institute of MIT and Harvard, Cambridge, MA, USA

Abstract

Genetic variants are inherently involved in disease etiology, but we often lack a thorough understanding of the molecular pathways through which they act. Here we focused on adenosine-to-inosine (A-to-I) RNA editing catalyzed by ADAR enzymes, a post-transcriptional mechanism vital for suppressing autoimmunity by labeling the endogenous double-stranded RNAs (dsRNAs) as self. Using the GTEx data across 49 human tissues, we identified and characterized cis-acting genetic variants associated with RNA editing levels. These RNA editing quantitative trait loci (QTLs), more than expression and splicing QTLs, are highly enriched in genome-wide association study (GWAS) signals of autoimmune and immune-related diseases. Colocalization of editing QTLs with GWAS risk loci enabled us to attribute causal variants to RNA editing and further pinpoint key dsRNA substrates in relevant tissues and diseases. We uncovered dsRNAs formed by inverted repeat Alus as expected, as well as by unexpected cis-natural antisense transcripts (cis-NATs) that are highly immunogenic without sufficient editing. Our findings indicate that RNA editing is a primary link between genetic variation and immune-related diseases.

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RNA Modification & Editing 595 PCIF1 catalyzes m6Am mRNA methylation to regulate gene expression

David Valle-Garcia1,2, Erdem Sendinc1, Abhinav Dhall1, Hao Chen1, Telmo Henriques3, Jose Navarrete-Perea4, Wanqiang Sheng1, Steven Gygi4, Karen Adelman3, Yang Shi1 1Division of Newborn Medicine and Epigenetics Program, Department of Medicine, Boston Children's Hospital, Boston, Massachusetts, USA. 2Current adress: Institute of Biotechnology, National Autonomous University of Mexico, Cuernavaca, Morelos, Mexico. 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. 4Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA

Abstract mRNA modifications play an important role in regulating gene expression. One of the most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Although m6Am was discovered in 1975, the enzyme responsible for its establishment was previously unknown. Using an evolutionary approach, we demonstrate that m6Am is a conserved mRNA modification mediated by the uncharacterized methyltransferase PCIF1 (Phosphorylated CTD Interacting Factor 1). Furthermore, we show that PCIF1 catalyzes only 5’ m6Am methylation in a Cap-dependent manner, but not internal m6A methylation in vitro and in vivo. To study the biological significance of m6Am, we developed a novel and robust methodology called m6Am-Exo-Seq to map its transcriptome-wide distribution. Our genome-wide mRNA methylation analyses revealed no global crosstalk between m6Am and m6A mRNA methylation events, suggesting that m6Am is functionally distinct from m6A. Finally, using in vitro and in vivo reporter assays as well in vivo proteomic quantification, we found that m6Am acts as a translational repression signal. Together, we identify the only human mRNA m6Am methyltransferase and demonstrate a novel mechanism of gene expression regulation through PCIF1-mediated m6Am mRNA methylation in eukaryotes.

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RNA Modification & Editing 599 Acetylation of cytidine residues boosts HIV-1 gene expression by increasing viral RNA stability

Kevin Tsai1, Ananda Ayyappan Jaguva Vasudevan1, Cecilia Martinez Campos1, Ann Emery2, Ronald Swanstrom2, Bryan Cullen1 1Duke University Medical Center, Durham, NC, USA. 2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Abstract

Epitranscriptomic RNA modifications, including methylation of A and C residues, are now recognized as key regulators of both cellular and viral mRNA function. Moreover, acetylation of the N4 position of cytidine (ac4C) was recently reported to increase the translation and stability of cellular mRNAs. Here, we show that ac4C and N-acetyltransferase 10 (NAT10), the enzyme that adds ac4C to RNAs, have been subverted by human immunodeficiency virus 1 (HIV-1) to increase viral gene expression. HIV-1 transcripts are modified with ac4C at multiple discreet sites, and silent mutagenesis of these ac4C sites led to decreased HIV-1 gene expression. Similarly, loss of ac4C from viral transcripts due to depletion of NAT10 inhibited HIV-1 replication by reducing viral RNA stability. Interestingly, the NAT10 inhibitor Remodelin could inhibit HIV-1 replication at levels that have no effect on cell viability, thus identifying ac4C addition as a potential novel target for antiviral drug development.

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[email protected]

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RNA Modification & Editing 630 VIRMA-dependent N6-methyladenosine modifications regulate the expression of long non-coding RNAs CCAT1 and CCAT2 in prostate cancer

Daniela Barros-Silva1, João Lobo1, Catarina Guimarães-Teixeira1, Isa Carneiro2, Jorge Oliveira3, Elena S. Martens-Uzunova4, Rui Henrique1, Carmen Jerónimo1 1Research Center of Portuguese Oncology Institute of Porto, Porto, Portugal. 2Department of Pathology, Portuguese Oncology Institute of Porto, Porto, Portugal. 3Department of Urology, Portuguese Oncology Institute of Porto, Porto, Portugal. 4Department of Urology, Cancer Institute, Erasmus MC University Medical Center, Rotterdam, Netherlands

Abstract

RNA methylation at position N6 in adenosine (m6A) and its associated methyltransferase complex (MTC) are involved in tumorigenesis. We aimed to explore m6A biological function for long non-coding RNAs (lncRNAs) in prostate cancer (PCa) and its clinical significance. m6A and MTC levels in PCa cells were characterized by ELISA and western blot. Putative m6A-regulated lncRNAs were identified and validated by lncRNA profiler qPCR array and bioinformatics analysis, followed by m6A/RNA co-immunoprecipitation. Impact of m6A depletion on RNA stability was assessed by Actinomycin D assay. The association of m6A-levels with PCa prognosis was examined in clinical samples. Higher m6A-levels and VIRMA overexpression were detected in metastatic castration-resistant PCa (mCRPC) cells (p < 0.05). VIRMA knockdown in PC-3 cells significantly decreased m6A- levels (p = 0.0317), attenuated malignant phenotype and suppressed the expression of oncogenic lncRNAs CCAT1 and CCAT2 (p < 0.00001). VIRMA depletion and m6A reduction decreased the stability and abundance of CCAT1/2 transcripts. Higher expression of VIRMA, CCAT1, and CCAT2 as a group variable was an independent predictor of poor prognosis (HR = 9.083, CI95% 1.911–43.183, p = 0.006). VIRMA is a critical factor sustaining m6A-levels in PCa cells. VIRMA downregulation attenuates the aggressive phenotype of PCa by overall reduction of m6A-levels decreasing stability and abundance of oncogenic lncRNAs.

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RNA Modification & Editing 638 N6-methyladenosine and YTHDC1 regulate the effects of lncRNA HOTAIR in breast cancer cells

Allison M Porman Swain, Justin T Roberts, Aaron M Johnson Department of Biochemistry & Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

Abstract

N6-methyladenosine (m6A) is one of the most abundant RNA modifications that has important roles in normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. To investigate a potential role for m6A on the lncRNA HOTAIR, we mapped m6A at single nucleotide sites in HOTAIR-expressing breast cancer cells using m6A enhanced cross-linking immunoprecipitation (meCLIP). We identify at least 14 m6A modified adenosine residues within HOTAIR and show that modification of HOTAIR is mediated by the m6A transferase METTL3/14 complex. A single m6A site in domain 2 of HOTAIR was consistently identified in m6A mapping experiments. When compared to cells overexpressing wild-type HOTAIR, overexpression of a mutated form of HOTAIR with a single A-to-U mutation is unable to enhance breast cancer cell proliferation or soft agar colony formation. We observe interaction between the nuclear m6A reader YTHDC1 and HOTAIR in breast cancer cells, and a single A-to-U mutation resulted in a decreased interaction of in vitro m6A-modified domain 2 of HOTAIR with YTHDC1. Proliferation of cells overexpressing mutant HOTAIR is enhanced by overexpression of YTHDC1 and decreased by knock-down of YTHDC1. We also show that overexpression of YTHDC1 results in increased soft agar colony formation which is enhanced when cells express wild-type HOTAIR. Knockdown of YTHDC1 or mutating multiple m6A sites within HOTAIR results in decreased overall expression levels of HOTAIR. At the molecular level, a single A-to-U mutation decreases chromatin retention of HOTAIR, which is recovered by overexpression of YTHDC1. Finally, in a reporter cell line where HOTAIR is tethered upstream of luciferase to mimic repression by HOTAIR, knockdown of YTHDC1 leads to an increase in luciferase expression. Altogether, these data suggest a mechanism whereby m6A is important in regulating the function of HOTAIR via mediating interaction of YTHDC1 with specific m6A sites. This interaction in turn regulates the stability and chromatin retention of HOTAIR as well as the repression of its target genes. We propose a mechanism whereby YTHDC1 mediates transcriptional interference by HOTAIR to initiate repression and formation at its targets.

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RNA Modification & Editing 641 Structure of yeast Pseudouridine Synthase 1 bound to a target RNA and characterization of its role in mRNA modification

Sebastian Grünberg1, Lindsey A. Doyle2, Jingmin Jin1, Nan Dai1, Ivan R. Correa Jr.1, Barry L. Stoddard2, Erbay Yigit1 1New England Biolabs, Inc., Ipswich, MA, USA. 2Fred Hutch, Seattle, WA, USA

Abstract

Pseudouridine (), the most abundant RNA modification, can be found in all forms of RNA and is the result of the isomerization of uridine by pseudouridine synthases (PUS). The function of in rRNA and tRNA has been extensively studied. However, findings that alterations in levels are associated with multiple human diseases and cancer, a proposed role for in splicing, and the use of and other RNA modifications in the emerging field of RNA therapeutics recently put the spotlight on the role of modifications in mRNA. Research in our lab is aimed at characterizing PUS enzymes and understanding the function of in mRNA. Here we present the crystal structure of Saccharomyces cerevisiae PUS1 bound to RNA at 2.4 Å resolution. This is the first structure of a eukaryotic PUS enzyme in complex with its target RNA and revealed multiple insights into how RNA interacts with the conserved residues guiding the RNA into the active site. The new structure also highlights the differences on how bacterial PUS1 homolog TruA and eukaryotic PUS1 interact with its target RNA. In addition, we identified and characterized multiple standalone PUS enzymes and their role in pseudouridylation of mRNA. While most PUS variants show a high substrate specificity for their natural targets, several of the PUS enzymes we purified are able to convert uridine to in various mRNA templates to high levels in vitro. Our data show that closely related PUS variants display distinct sequence preference, suggesting that various PUS enzymes could be used to specifically install modifications in target sequences, e.g. in mRNA used in therapeutic applications. We believe that understanding the enzymatic mechanism, sequence targeting, and regulation of pseudouridylation of mRNA by PUS enzymes will provide new insights into the role of and RNA modifications in general.

Presenting author email [email protected]

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RNA Modification & Editing 642 Germline NPM1 mutations reveal the central role of 2’-O-methylation and translation regulation in the pathogenesis of human stem cell disorders

Daphna Nachmani1,2, Tom Vulliamy3, Jean Soulier4, Brunangelo Falini5, Inderjeet Dokal3, Keisuke Ito6, John Clohessy1, Pier Paolo Pandolfi1 1Harvard Medical School, Boston, MA, USA. 2Hebrew Univeristy, Jerusalem, Israel. 3Queen Mary University of London, London, United Kingdom. 4Hôpital Saint-Louis, Paris, France. 5University of Perugia, Perugia, Italy. 6Albert Einstein College of Medicine, Bronx, NY, USA

Abstract

RNA modifications are emerging as key determinants of development and disease. However, compelling genetic demonstrations of their relevance to human disease, are lacking. Here, we combine human genetics with in-depth molecular studies and in vivo mouse models to reveal the role of rRNA 2’-O-methylation (2’-O- Me) in the pathogenesis of human stem cell disorders. We identify nucleophosmin (NPM1) as an essential regulator of 2’-O-Me on rRNA by directly bind C/D box small nucleolar RNAs (snoRNAs), and thereby modulating translation. We demonstrate both in vitro and in vivo the importance of 2’-O-Me regulated translation for cellular growth, hematopoietic stem cells (HSC) maintenance and differentiation. Inactivation of Npm1 in adult HSCs led to altered 2’-O-Me and impaired ribosomal function, resulting in bone marrow failure (BMF) and leukemia susceptibility. On this basis, we identify NPM1 germ-line mutations in dyskeratosis congenita (DC) patients presenting with BMF, and characterize them as selectively deficient in snoRNA binding. We find that CRISPR knock-in mice harboring the DC germ-line NPM1 mutation phenocopy both hematological and non-hematological DC features. Our findings provide a direct demonstration of the role of 2’-O-Me in stem cell function and disease pathogenesis.

Presenting author email [email protected]

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RNA Modification & Editing 679 In-Vivo Mutational Analysis of Archaeal Box C/D Ribonucleoprotein Complex

Michael Bosmeny, Parinati Kharel, Ramesh Gupta Southern Illinois University, Carbondale, Illinois, USA

Abstract

The methylation of RNA results in a number of effects, including changes in RNA stability, the interaction of the tRNA codon and anticodon, and may even be related to tRNA localization in the cell. RNA can be methylated by single proteins in all organisms. Furthermore, box C/D ribonucleoprotein (RNP) complexes can also produce these methylations in Archaea and eukaryotes. In Archaea, the box C/D RNP is composed of three proteins, Fibrillarin, Nop5, and L7ae, as well as a guide RNA. Mutations of these proteins has enabled us to better understand the roles and interactions of the components of the complex. The ability of altered box C/D RNPs to methylate certain tRNA or rRNA modification sites are here tested in-vivo. Haloferax volcanii was altered to lack the native Fibrillarin or Nop5 proteins. Alanine-substitution mutant genes were expressed in these strains to rescue the missing protein. The ability of these mutant strains to methylate at known box C/D modification sites was tested by a limited-dNTP-mediated reverse transcription reaction. Tested sites included Cm34 of elongator tRNA-Met, Cm34 and Um39 of tRNA-Trp, and Gm1934 of the 23S ribosomal RNA. Mutants were selected primarily based on homology modeling of the archaeal box C/D complex, choosing sites of predicted strong protein-protein or protein-RNA interactions. Mutants that did not allow modification include those that targeted the Fibrillarin/S-adenosyl methionine interaction, and those that inhibit Nop5’s ability to bind and align the guide RNA.

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RNA Modification & Editing 692 Biogenesis and functions of aminocarboxypropyluridine in tRNA

Mayuko Takakura1, Kensuke Ishiguro2, Shinichiro Akichika1, Kenjyo Miyauchi1, Tsutomu Suzuki1 1the University of Tokyo, Tokyo, Japan. 2the University of Tokyo, Toyko, Japan

Abstract

Transfer (t)RNAs contain a wide variety of post-transcriptional modifications, which play critical roles in tRNA stability and functions. 3-(3-amino-3-carboxypropyl)uridine (acp3U) is a highly conserved modification found in variable- and D-loops of tRNAs. Biogenesis and functions of acp3U have not been extensively investigated. Using a reverse-genetic approach supported by comparative genomics, we find here that the Escherichia coli yfiP gene, which we rename tapT (tRNA aminocarboxypropyltransferase), is responsible for acp3U formation in tRNA. Recombinant TapT synthesizes acp3U at position 47 of tRNAs in the presence of S-adenosylmethionine. Biochemical experiments reveal that acp3U47 confers thermal stability on tRNA. Curiously, the ΔtapT strain exhibits genome instability under continuous heat stress. We also find that the human homologs of tapT, DTWD1 and DTWD2, are responsible for acp3U formation at positions 20 and 20a of tRNAs, respectively. Double knockout cells of DTWD1 and DTWD2 exhibit growth retardation, indicating that acp3U is physiologically important in mammals.

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RNA Modification & Editing 697 Transcriptome-wide reprogramming of N6-methyladenosine modification by the mouse microbiome

Xiaoyun Wang1, Tao Pan2 1South China Normal University, Guangzhou, Guangdong, China. 2The University of Chicago, Chicago, IL, USA

Abstract

Microbiome affects many aspects of human health and disease and elicits a wide range of host responses including remarkable epigenetic changes such as DNA methylation, histone modification and non-coding RNA expression. A still poorly explored area of microbiome-host interaction is the response of host RNA modification. N6-Methyladenosine (m6A) is the most abundant mRNA modification in mammalian cells, occurring at approximately 3 modified adenosine residues per transcript. We investigated the host response marked by the m6A in the transcriptome to the presence of microbiome in mice. We employed one group of germ-free (GF) mice to identify the host response to the absence, and another group of specific pathogen-free (SPF) mice to identify the host response to the presence of microbiome. We harvested three tissues of GF and SPF mice of the same genetic background and 4 weeks of age, brain, intestine, and liver, and performed m6A analysis in polyA-selected RNA by liquid chromatography/mass spectrometry to determine the total m6A/A ratios and by the m6A-MeRIP sequencing to determine the transcriptomic m6A pattern and distribution. Brain showed the highest m6A content for both GF and SPF mice, and brain and intestine showed higher m6A content in the GF mice, also, our m6A-MeRIP results of all three tissues showed the well-known m6A pattern across the mRNA transcripts such as the strong enrichment of m6A peaks at the junction of coding region and 3’ UTR. More m6A peaks were present in all three mRNA regions in GF than SPF brain. To obtain mechanistic understanding of the m6A changes in GF and SPF tissues we measured the levels of the mRNA m6A writer proteins METTL3 and METTL14, and the m6A eraser proteins FTO and ALKBH5. We found that both m6A writer and both m6A eraser proteins were highly overexpressed in the GF compared to the SFP brain. All together, these results indicate that the presence of microbiome has a profound influence on the cellular mRNA m6A patterns in a tissue dependent manner. Future studies will reveal the specific microbial species and the molecular mechanisms that regulate the host m6A methylome.

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RNA Modification & Editing 713 Loss of Cnot6l impairs inosine RNA modifications in mouse oocytes

Nehemiah Alvarez1,2, Lane Christenson1, Pavla Brachova1 1University of Kansas Medical Center, Kansas City, KS, USA. 2De Novo Genomics, Kansas City, KS, USA

Abstract

Oocyte meiotic maturation is regulated by a balance of RNA storage and translational activity. Through a process that is incompletely understood, a stock of maternal mRNA undergoes translational activation, followed by deadenylation and mRNA decay, facilitating maternal mRNA clearance. After translational activation, transcripts become deadenylated by the CCR4-NOT complex through a translationally coupled mechanism. Knockout of Cnot6l, a component of the CCR4-NOT complex, results in mRNA decay defects during MI entry. Knockout of Btg4, another component of the CCR4-NOT complex, results in mRNA decay defects in the early embryo. Our previous work in oocytes, as well as published work from others, established that inosine RNA modifications can impact mRNA stability through a translation mechanism. Since the Cnot6l and Btg4 knockout mice result in over-translation and stabilization of mRNA, we hypothesized that in these mutant backgrounds, we would observe an increase in inosine modifications in mRNA. To test this, we used a computational approach to identify inosine RNA modifications in total and polysomal RNA-seq data during meiotic maturation (GV, MI, and MII stages, n=2/stage) in wild-type, Cnot6l-/-, and Btg4-/- mice. Surprisingly, we observed strong defects in inosine modifications in oocytes from Cnot6l-/-, but not in Btg4- /- mice. Among common transcripts, inosine modifications were significantly reduced in Cnot6l-/- GV oocytes (WT=1249±232, Cnot6l-/-=91±27, Btg4-/- =1161±110, p<0.05 one-way ANOVA). Additionally, sequencing of the polysome-associated RNA revealed clearance of inosine modified mRNA (GV=1666±30; MI=850.5±5.5; MII=709.5±6.5; p<0.05 one-way ANOVA). Efficiency of inosine RNA modifications also decreased at these stages (GV=83%, MI=38%, and MII=32%). Our results suggest a novel connection between the components of the deadenylation and inosine RNA modification machinery during oocyte maturation.

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RNA Modification & Editing 726 2’-OMe modification revealed that siRNA seed region is divided into two functionally different domains

Yoshiaki Kobayashi, Kumiko Ui-Tei Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan

Abstract

In RNA interference (RNAi), a small interfering RNA (siRNA) is loaded onto Argonaute (AGO) protein, and the siRNA guide strand base pairs with a target mRNA with perfect sequence complementarity to repress its expression through cleavage by AGO protein. On the other hand, siRNA often exhibits off-target effect which represses the expression of unintended mRNAs with partial sequence complementarities in the seed region (2- 8 nucleotides from the 5’ end) of the guide strand. Previously, we have reported that the degree of off-target effect is correlated positively with the thermodynamic stability in base-pairing between the seed region of the guide strand and unintended mRNA. Also, we reported that off-target effect is avoided by the introduction of 2´-O-methyl (2´-OMe) modifications in the seed region, since they induce steric hindrance in the duplex formation on the AGO protein without affecting the RNAi activity. In this study, the detailed mechanism to avoid off-target effect was examined by systematic introduction of 2´- OMe modifications in the seed region. As a result, it was revealed that seed region is composed of two functionally different regions: The modifications at positions 2-5 induced steric hindrance in duplex formation on the AGO protein, but the modifications at positions 6-8 contributed to form stable base-pairing with both target and off-target mRNAs, leading to their repression.

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RNA Modification & Editing 734 Chemistry to discover and decode RNA acetylation

Supuni Thalalla Gamage, Justin Thomas, Keri Bryson, Abigail Thorpe, Jordan Meier National Cancer Institute, Frederick, MD, USA

Abstract

NAT10 is an essential and highly conserved acetyltransferase enzyme that has been found to catalyze modification of cytidine residues in RNA. Recently, our lab has developed first in class affinity reagents for studying N4-acetylcytidine (ac4C) which have been applied to identify a number of novel NAT10 RNA targets. However, a major limitation of these studies is their inability to define sites of ac4C with single nucleotide resolution. Towards that goal, here we present the development of chemistry to detect ac4C in RNA by sequencing. Our initial studies have found that hydride donors such as sodium borohydride (NaBH4) efficiently reduce the ac4C . This ac4C derivative in turn causes polymerase stops and mutations during reverse transcription that can be detected by sequencing. Building on this method, we have developed variants of this chemistry that show faster kinetics and cause increased misincorporation at known ac4C sites in ribosomal RNA. Applying these for single-nucleotide resolution, quantitative analysis of ac4C in RNA provides new insights into the role of snoRNA in cytidine acetylation. Overall, these methods have the potential to expand our understanding of RNA acetylation in gene regulation and provide new insights into the emerging “epitranscriptome.”

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RNA Modification & Editing 754 Identification of novel RNA Polymerase II CTDinteraction sites on the mRNA

Marcus Bage, Rajaei Almohammed, Victoria Cowling, Andrei Pisliakov University of Dundee, Dundee, United Kingdom

Abstract

Recruitment of the mRNA Capping Enzyme (CE/RNGTT) to the site of transcription is essential for the formation of the 5’ mRNA cap, which in turn ensures efficient transcription, splicing, , nuclear export and translation of mRNA in eukaryotic cells. The CE is recruited and activated by the Serine-5 phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. Through the use of molecular dynamics simulations and enhanced sampling techniques, we provide a systematic and detailed characterisation of the human CE-CTD interface, describing the effect of the CTD phosphorylation state, length and orientation on this interaction. Our computational analyses identify novel CTD interaction sites on the human CE surface and quantify their relative contributions to CTD binding. We also identify differences in the CTD binding conformation when phosphorylated at either the Serine-2 or Serine-5 positions, thus providing insights into how the CE reads the CTD code. The computational findings are then validated by binding and activity assays. These novel CTD interaction sites are compared with cocrystal structures of the CE-CTD complex in different eukaryotic taxa, leading to the conclusion that this interface is considerably more conserved than previous structures have indicated.

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RNA Modification & Editing 768 M6A RNA Demethylase Regulates Histone Ubiquitination to Support Osteosarcoma Growth and Progression

Pooja Yadav, Panneerdoss Subbarayalu, Subapriya Rajamanickam, Vijay Eedunuri, Yidong Chen, Manjeet Rao UT Health San Antonio, San Antonio, Tx, USA

Abstract

Osteosarcoma (OS) is the most common primary bone tumor which predominantly affects children and adolescents. Introduction of chemotherapy in 1970s dramatically improved prognosis for OS patients. However, there has not been any significant advancement in last three decades and survival outcomes have stayed stagnated for years. Chemotherapy is highly toxic and non-specific which leads to long-term debilitating side effects. Thus, more efficacious and less toxic therapeutic approaches based on OS biology are urgently needed.

N6-methyladenosine (m6A) RNA methylation is the most common RNA modification found on mRNA and long non-coding RNA that can regulate almost all aspect of RNA metabolism. It is becoming apparent that m6A modifying proteins play critical role during tumorigenesis. However, the understanding of their function in multiple tumor types is still not complete. We found that RNA demethylase AlkB homolog 5 (ALKBH5) is uniquely amplified in sarcomas and its expression is elevated in osteosarcoma tumors. We demonstrate that depletion of ALKBH5 inhibits the OS growth and migration without affecting the viability of normal human fetal osteoblast cells. Supporting our in vitro data, silencing of ALKBH5 reduced tumor xenograft growth in mice. Interestingly, ALKBH5 depletion reduced the expression of genes involved in cell-cycle progression, DNA replication and repair. ALKBH5 knockdown induced cell-cycle arrest and replication stress in OS cells. Furthermore, ALKBH5 depletion led to reduced DNA double-strand break repair capacity of OS cells. Mechanistically, we identified epigenetic modifiers-USP22 and RNF40 to be direct targets of ALKBH5 m6A demethylase activity. ALKBH5 knockdown decreased USP22 and RNF40 expression, and increased histone H2A monoubiquitination which inhibited the transcription of key cell-cycle and DNA repair genes. Consistent with that, USP22 and RNF40 overexpression rescued the effect of ALKBH5 silencing on OS cell growth. Taken together, our study found association between m6A RNA methylation and epigenetic modification which work in concert to regulate pro-tumorigenic gene expression and posits ALKBH5 as a potential therapeutic candidate for OS.

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RNA Modification & Editing 777 Manipulation of Regnase-1 mRNA stability alleviates inflammatory responses in diseased models

Ka Man Carman Tse, Xiaotong Cui, Takuya Uehata, Takeuchi Osamu Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, Japan

Abstract

Post-transcriptional regulation of gene expression exerts controls over immune responses and have important roles in fine-tuning the inflammatory events to prevent undesired tissue damage and maintain homeostasis. Several studies have shown that RNA-binding protein Regnase-1 (also known as Zc3h12a or MCPIP1) can suppress inflammation by destabilizing pro-inflammatory mRNAs and T cell activation transcripts through binding to the conserved stem-loop structures in their 3’UTRs. In addition, it is found that upon TLR stimulation, Regnase-1 can negatively regulate its own mRNA expression through associating to the stem- loops present in its 3’UTR.

Here we found that disruption of the stem-loop structures in Regnase-1 3’UTR structural elements can abrogate the Regnase-1 mediated self-degradation. Introduction of antisense oligonucleotides targeting Regnase-1 stem loop structures increases the Regnase-1 stability in purified bone marrow-derived macrophages (BMDMs) and in luciferase reporter assay. In addition, we observed that antisense oligonucleotide treatment can suppress the expression of inflammatory cytokines which 3’UTRs are targeted by Regnase-1 (eg. Il-6, Il-1b) in control and LPS-stimulated BMDMs. Therefore, disrupting the Regnase-1 secondary structures can increase its stability and reduce the half-lives of pro-inflammatory gene transcripts. We also found that Regnase-1 manipulation in trans could relieve the inflammatory responses in LPS-induced lung injury model and experimental autoimmune encephalomyelitis mouse model. Together our findings present an attractive therapeutic window for using antisense oligonucleotides in targeting Regnase-1 in acute and choric inflammatory diseases.

Presenting author email [email protected]

Topic category

RNA Modification & Editing 824 Use of ribonucleases to investigate RNA modifications and structure

Tien-Hao Chen, Sebastian Gruenberg, Nan Dai, Kelly Becker, John W Gourdeau, John Buswell, Ivan R. Corrêa Jr., Brett Robb, Erbay Yigit New England Biolabs, Ipswich, MA, USA

Abstract

RNA modifications and structures are the two hallmarks that play key roles in cellular processes. Among more than 150 modifications identified in all domains of life, only a few have been characterized extensively; the functions of most of which remain poorly understood. In addition, it is likely that there are more unknown modifications awaiting discovery. The standard method for accurate identification of modified nucleotides includes RNA isolation, nuclease digestion of RNA to oligonucleotides, followed by mass spectrometry (MS) analysis. Appropriate selection of ribonucleases to generate desired RNA fragments is critical for MS analyses. However, currently only a limited number of ribonucleases are available, often leading to insufficiently digested oligonucleotides. In this work, we characterized three ribonucleases that a) recognize specific sequences or structures, b) can generate alternative RNA digests for MS analysis, and c) distinguish certain modifications from the unmodified nucleotides. We are currently evaluating these ribonucleases in their ability to facilitate MS analyses of modified and structured RNA. In addition, we re-evaluated the venerable ribonuclease E. coli RNase I, a reportedly single-strand specific endoribonuclease with no sequence preference. We show two novel calcium-dependent activities of RNase I that expand our understanding of its substrate specificity and suggest a regulatory role for calcium for RNase I function.

Presenting author email [email protected]

Topic category

RNA Modification & Editing 849 A highly compensatory network of 13 RNA methyltransferases maintain the 5-methyl cytosine epitranscriptome in the hyperthermophilic archaeon, Thermococcus kodakarensis

Kristin Scott, Thomas Santangelo Colorado State University, Fort Collins, Co, USA

Abstract

RNA 5-methyl cytosine (m5C) plays an important role in many biologically and pathologically relevant processes. However, the distribution, function, and genesis of m5C RNA residues remains largely unknown. Bisulfite conversion followed by an ultra-deep sequencing approach revealed 641 high confidence m5C sites in the epitranscriptome of the hyperthermophilic archaeon, Thermococcus kodakarensis. Distinct methylation patterns between different types of RNA and between stationary and exponentially growing cells was observed, indicating the epitranscriptome dynamically responses to environmental queues. We additionally bisulfite-sequenced 13 non-essential RNA methyltransferase (RMTase) deletion strains to correlate individual methylation events with the specific enzymatic activity. We correlated 473 m5C sites with one or more RMTase, whereas 180 m5C sites were not differentially methylated in any deletion strain. We found significant overlap in target sites between RMTases, indicating a high level of redundancy and cooperation to establish and maintain the epitranscriptome. The extensive cooperation between RMTases highlights the biological importance of the epitranscriptome, and provides a radically different view of epitranscriptome formation and maintenance in the environmental extremes.

Presenting author email [email protected]

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RNA Modification & Editing 873 RNA Phosphorothioate Modification in and Eukaryotes

Ying Wu SUNY-Albany, Albany, USA

Abstract

RNA modifications play important roles in RNA structures and regulation of gene expression and translation. We report the first RNA modification on the phosphate, the RNA phosphorothioate (PS) modification, discovered in both prokaryotes and eukaryotes. The PS modification is also first reported on nucleic acids of eukaryotes. The GpsG modification exists in the Rp configuration and was quantified with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By knocking out the DndA gene in E. coli, we show the Dnd clusters that regulate DNA PS modification may also play roles in RNA PS modification. We also show that the GpsG modification locates on rRNA in E. coli, L. lactis, and HeLa cells, and it is not detected in rRNA-depleted total RNAs from these cells.

Presenting author email [email protected]

Topic category

RNA Modification & Editing 881 Functional characterization of the human tRNA methyltransferases TRMT10A and TRMT10B

Elisa Vilardo1, Fabian Amman2, Ursula Toth1, Annika Kotter3, Mark Helm3, Walter Rossmanith1 1Medical University of Vienna, Vienna, Austria. 2University of Vienna, Vienna, Austria. 3Johannes Gutenberg- University, Mainz, Germany

Abstract

To date, more than 150 RNA modifications have been described, most of which are found in tRNAs. TRM10 is a family of methyltransferases responsible for the N1-methylation of purines at position 9 of tRNAs in Archaea and Eukaryotes. The encodes three TRM10-type enzymes, namely TRMT10A, TRMT10B, and TRMT10C. We previously characterized in details the mitochondrial form TRMT10C, whereas the functional significance of the two putative nuclear enzymes TRMT10A and TRMT10B remained unexplored. Here we used KO cell lines and a NGS-based approach to identify the targets of the two enzymes. We showed that TRMT10A is m1G9-specific and methylates a subset of nuclear-encoded tRNAs, whilst TRMT10B is the first m1A9-specific tRNA methyltransferase found in eukaryotes and is responsible for the modification of a single nuclear- encoded tRNA. Additionally, we show that the lack of G9 methylation causes a reduction in the steady-state levels of the initiator tRNAiMet‑CAT and an alteration in its further post-transcriptional modification. In conclusion, our work clarifies the role of TRMT10A and TRMT10B in vivo, and we provides evidence that the loss of TRMT10A affects the pool of cytosolic tRNAs that are required for protein translation.

Presenting author email [email protected]

Topic category

RNA Modification & Editing 888 Dynamic RNA acetylation as a mechanism for RNA thermostabilization

Aldema Sas-Chen1, Justin Thomas2, Donna Matzov1, Toshiaki Isobe3, Thomas Santangelo4, Moran Shalev- Benami1, Jordan Meier2, Schraga Schwartz1 1weizmann institute of science, rehovot, Israel. 2NIH, Frederick MD, USA. 3Tokyo Metropolitan University, Tokyo, Japan. 4Colorado State University, Colorado, USA

Abstract

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification, present on tRNA, rRNA and recently investigated in eukaryotic mRNA. We report ac4C-seq, a chemical genomic method for single- nucleotide resolution, transcriptome-wide quantitative mapping of ac4C. While we did not find detectable ac4C sites in human and yeast mRNAs, ac4C was induced via ectopic overexpression of eukaryotic acetyltransferase complexes, invariably at a conserved sequence motif. In contrast, cross-evolutionary profiling reveals unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, ncRNA and mRNA from hyperthermophilic archaea. Ac4C is dramatically induced in response to temperature, and acetyltransferase- deficient archaeal strains exhibit temperature-dependent growth defects. Cryo-EM visualization of WT and acetyltransferase-deficient archaeal ribosomes furnishes structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for unravelling this modification’s role in biology and disease.

Presenting author email [email protected]

Topic category

RNA Modification & Editing 893 Detection of m6A RNA modifications in complex viral sequences using long- read direct RNA sequencing reveals role in splicing efficiency

Alexander Price1, Katharina Hayer1, Alexa McIntyre2, Nandan Gokhale3, Ashley Della Fera1, Christopher Mason2, Stacy Horner3, Angus Wilson4, Daniel Depledge4, Matthew Weitzman5 1Children's Hospital of Philadelphia, Philadelphia, PA, USA. 2Weill Cornell Medicine, New York, NY, USA. 3Duke University, Durham, NC, USA. 4New York University, New York, NY, USA. 5University of Pennsylvania, Philadelphia, PA, USA

Abstract

The discovery of the reversible nature of N6-methyladenosine (m6A) modification of RNA has fundamentally altered our view of mRNA regulation. The m6A modification is added post-transcriptionally to RNA to allow the binding of m6A-specific “reader” proteins that have been implicated in diverse processes such as RNA splicing, nuclear export, stability, and translation. Adenovirus is a common respiratory pathogen that disproportionately affects children and the immune compromised. RNAs generated by this DNA virus are known to be marked by m6A, but the location and outcome of this modification on adenoviral infection has never been deciphered. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6A mapping using short-read sequencing, we profiled m6A within the adenovirus transcriptome using a combination of conventional techniques and Oxford Nanopore direct RNA long-read sequencing. This novel technique provides a new methodology for m6A discovery within individual transcript isoforms at single nucleotide resolution. Our technique revealed that both early and late viral transcripts contain m6A, however depletion of m6A writer METTL3 has a disproportionate impact on viral late transcripts by reducing their splicing efficiency. These data demonstrate that m6A modification of viral RNA is important during the late stage of adenoviral infection and provide a potential novel therapeutic option for viral infections that are heavily reliant on host RNA splicing machinery.

Presenting author email [email protected]

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RNA Modification & Editing 900 N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells

Abdulkadir Abakir, Alexey Ruzov Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham, Nottingham, United Kingdom

Abstract

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m6A-containing R-loops accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle, and that the m6A reader promoting mRNA degradation, YTHDF2, interacts with R-loopenriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double- strand breaks, in mammalian cells. Our results suggest that m6A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability.

Presenting author email [email protected]

Topic category

RNA Modification & Editing 920 Structural Modeling of t6A Biosynthesis System and Flexibility Analysis of TsaC2 in Borrelia burgdorferi sensu lato complex

Ashley Groshong1, Kelly Hawley1,2, Melissa Caimano1,3, Amit Luthra1 1Department of Medicine, UConn Health, Farmington, CT, USA. 2Division of Infectious Diseases and Immunology, Connecticut Children’s Medical Center, Hartford, CT, USA. 3Molecular Biology and Biophysics UConn Health, Farmington, CT 06030 USA, Farmington, Ct, USA

Abstract

The universal N(6)-threonylcarbamoyladenosine (t6A) modification at position 37 of ANN-decoding tRNAs plays a central role in translational fidelity through precise recognition of cognate codons and stabilization of codon- anticodon interactions. In bacteria, t6A biosynthesis starts with the formation of the unstable intermediate threonylcarbamoyl adenylate (TC-AMP), a reaction catalyzed by the universally conserved enzyme TsaC/TsaC2 (formerly YrdC/Sua5), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of the tRNA by a TC-transfer complex comprised of TsaB, TsaD and TsaE. TsaC is a one-domain protein, whereas TsaC2 has bipartite architecture made of an N-terminal domain homologous to TsaC, a linker region, and a C-terminal Sua5 domain. We started the present study by performing an extensive bioinformatics analysis of bacterial TsaC/C2 genes. A noteworthy observation from this analysis was that some strains of the Borrelia burgdorferi sensu lato complex (Bb), the causative agent of Lyme disease, encode two forms of TsaC2. The Lyme disease spirochetes harbor up to 21 linear and circular plasmids along with its linear chromosome. Surprisingly, there are three genes encoding TsaC2 with two major forms. TsaC2-A is encoded on the chromosome (bb0734), while two copies of TsaC2-B are plasmid-borne (bbt06 and bbu11). Both forms of TsaC2 in Bb have high amino acid identity, except TsaC2-B which lacks the conserved linker motif (Pro-Gly-Met). Of note, the conserved Pro–Gly– Met motif is essential for the in vitro ATPase activity of TsaC2 proteins. Therefore, we cloned, expressed and purified TsaC2-B and determined its solution structure in the absence of any substrate. Based on scattering experiments, we also demonstrated that TsaC2 exhibits a highly dynamic structure in solution in which its N- terminal and Sua5 domains dissociate, via the intrinsically disordered linker region. Next, we constructed homology models of all components (TsaB, TsaC2-A, TsaC2-B, TsaD and TsaE) of t6A modification machinery for the diverse species of Borrelia. Structural comparison of 3D models of Borrelia’s t6A machinery with Thermotoga maritima (TsaB2D2E2) and Escherichia coli (TsaB1D1E1) suggests that Bb appears to have a divergent repertoire of t6A components.

Presenting author email

[email protected]

Topic category

RNA Modification & Editing 939 Analytical and bioinformatics platform for RNA modification profiling by mass spectrometry.

Anna Popova, Luigi D’Ascenzo, James Williamson Scripps Research, La Jolla, CA, USA

Abstract

RNA modifications, by expanding the repertoire of RNA structure and interactions, play important role in biology and therapeutic design. At the cellular level, modifications were linked to antibiotic resistance, RNA biogenesis, translational fidelity, and gene expression. Importantly, stoichiometry of many RNA modifications is responsive to cellular and environmental cues, and can vary across cell types and disease states. Here we present the experimental and bioinformatics tools for mass spectrometry (MS) based identification and quantitative analysis of RNA modifications in abundant RNAs.

RNA modification analysis is based on endonucleolytic cleavage of the purified RNA of interest into small 3-15 nt fragments that are chromatographically separated and sequenced via LC-MS/MS. Compared to the field of protein identification, analysis of the tandem MS spectra of RNA has been significantly lagging behind, due to more complex fragmentation patterns and high redundancy of RNA (4 nt vs. 20 aa in proteomics). To address the need, we developed Pytheas, a software for annotation of RNA MS/MS spectra and score-based assignment to the oligonucleotide sequences present in the in-silico digest library. Pytheas is using an empirical scoring function that has been trained to successfully distinguish between the target and decoy sequences, and it is equipped with statistical and data visualization tools to quickly assess quality of the entire LC-MS/MS dataset. Pytheas performance has been tested using data collected on several instruments and RNAs of different origin, including synthetic oligonucleotides, in-vitro transcribed RNA, rRNA, and tRNA. As an example, 16S and 23S isolated from B. Subtilis will be used to show how Pytheas can be implemented for the discovery and sequence placement of previously unannotated methylated nucleosides and “mass-silent” pseudouridines using both stable-isotope labeling and chemical derivatization.

In summary, Pytheas is a highly customizable software, that improves identification of known modifications and assists in the discovery process. Pytheas is developed to support custom isotope-labeling schemes and is fully compatible with relative RNA quantitation. While at present the software is focused on “native” RNA modifications, we are highly interested in expanding Pytheas to accommodate analysis of diverse RNA derivatives for synthetic biology and RNA therapeutic applications.

Presenting author email [email protected]

Topic category

RNA Modification & Editing 958 METTL4 catalyzes m6Am methylation in U2 snRNA to regulate pre-mRNA splicing

Yeek Teck Goh1, Casslynn W.Q. Koh1, Donald Yuhui Sim2, Xavier Roca22, W.S. Sho Goh1 1Genome Institute of Singapore, Singapore, Singapore, Singapore. 2Nanyang Technological University, Singapore, Singapore, Singapore

Abstract

N6-methylation of 2’-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2’-O- dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome- wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4’s in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as overall 3’ splice-site weakness with certain motif positions more affected than others. This study establishes that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.

Presenting author email [email protected]

Topic category

RNA Modification & Editing 965 A unified model for the function of YTHDF proteins in the regulation of m⁶A-modified mRNA

SARA ZACCARA, SAMIE JAFFREY Weill Medical College of Cornell University, New York, New York, USA

Abstract

N6-methyladenosine (m⁶A) is the most abundant mRNA nucleotide modification and regulates critical aspects of cellular physiology and differentiation. m⁶A is thought to mediate its effects through a complex network of interactions between different m⁶A sites and three functionally distinct cytoplasmic YTHDF m⁶A binding proteins (DF1, DF2, and DF3). In contrast to the prevailing model, we show that DF proteins bind the same m⁶A-modified mRNAs, rather than different mRNAs. Furthermore, we find that DF proteins do not induce translation in HeLa cells. Instead, the DF paralogs act cooperatively to mediate mRNA degradation and cellular differentiation. The ability of DF proteins to regulate stability and differentiation becomes evident only when all three DF paralogs are simultaneously depleted. Our studies reveal a unified model of m⁶A function in which all m⁶A-modified mRNAs are subjected to the combined action of the YTHDF proteins in proportion to the number of m⁶A sites.

Presenting author email [email protected]

Topic category

RNA Modification & Editing 142 T-psi-C - new, community updated database of tRNA sequences and structures

Marcin Sajek1, Tomasz Wozniak1, Mathias Sprinzl2, Jadwiga Jaruzelska1, Jan Barciszewski3,4 1Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland. 2Bayreuth University, Bayreuth, Germany. 3Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland. 4NanoBioMedical Centre of the Adam Mickiewicz University, Poznan, Poland

Abstract tRNAs have been widely studied for their role as genetic code decoders in the ribosome during translation. Currently, the tRNA research field is experiencing a renaissance, and much of this interest has focused on the tissue-specific regulation of translation decoding efficiency, due to expression level changes and/or nucleoside modification, which are often connected with human disease. Yet, existing tRNA databases have not been updated for more than 10 years, so they do not contain this new functional information and have not kept pace with the rate of discovery in this field. Therefore, a regularly updated database that contains information about newly discovered characteristics of tRNA molecules and can be regularly updated is strongly needed. T-psi-C database (http://tpsic.igcz.poznan.pl) offers three major advantages over previous tRNA databases: 1/ an updated set of tRNA sequences, including data from high-throughput techniques; 2/ 3D structures of tRNA molecules retrieved from PDB or 3D structural models generated by homology modeling; 3/ the capacity for continuous updating with newly obtained tRNA sequences and structures by any member of the scientific community. It also contains its own application programming interface (API), which allows users to retrieve or upload data in JSON format. Altogether, T-psi-C is user-friendly, easy to develop and an up-to-date source of knowledge about tRNAs

Presenting author email [email protected]

Topic category tRNA: Processing and Function 425 Characterization of the polynucleotide kinase Clp1 family proteins in Prokaryotes

Motofumi Saito, Asako Sato, Akio Kanai Inst. Adv. Biosci., Keio Univ., Tsuruoka, Yamagata, Japan

Abstract

Clp1, a polyribonucleotide 5'-hydroxyl-kinase (PNK) in eukaryotes, is involved in pre-tRNA splicing and mRNA 3'-end formation. However, our knowledge of how these Clp1 family proteins evolved and diversified is limited. Recently we detected 3,557 Clp1 family proteins in the three domains of life, Eukarya, Archaea, and Bacteria (Ref). Many were from Archaea and Eukarya, but a few were found in restricted, phylogenetically diverse bacterial species. According to our analysis using representative complete genomes, the proportion of species with Clp1 family proteins was 100% in Eukarya, 50% in Archaea, and 8.3% in Bacteria. The domain structures of the Clp1 family proteins differ between the three domains of life. In terms of the Clp1 family protein structures, the PNK domain is highly conserved among all family members. Although eukaryotic Clp1 also has conserved N-terminal and C-terminal domains, and the PNK domain is located in the middle of the protein, there is no conserved N-terminal domain in Prokaryotes and the PNK domain is located close to the N- terminus. We found that the size distribution of the N-termini had two peaks corresponding to short (<60 aa) and long (>61 aa) forms in Prokaryotes. The short peak mainly consisted of bacterial Clp1 proteins and the longer peak mainly consisted of archaeal Clp1 proteins. Since no experimental study has reported that a bacterial Clp1 protein actually has PNK activity, we demonstrated the activity of bacterial Thermus scotoductus Clp1 (Ts-Clp1). Ts-Clp1 preferentially phosphorylates single-stranded RNA (ssRNA) oligonucleotides (Km value for ATP, 2.5 µM), or single-stranded DNA (ssDNA) at higher enzyme concentrations. In contrast, several studies have reported that eukaryotic Homo sapiens-Clp1 mainly phosphorylate ssRNA but not ssDNA under typical reaction conditions. Therefore, we speculate that at least the N-terminal domain in eukaryotes may contribute the substrate specificity for ssRNA.

Ref: Saito, M., Sato, A., Nagata, S., Tamaki, S., Tomita, M., Suzuki, H., and Kanai, A. (2019) Genome Biology and Evolution 11(10): 2713-2726.

Presenting author email [email protected]

Topic category tRNA: Processing and Function 514 Characterization of tRNAs and tRNA-derived Small RNAs in Human Cerebral Cortical Organoids

Alex Bagi, Todd Lowe University of California Santa Cruz, Santa Cruz, CA, USA

Abstract

The evolution of the primate lineage has led to a rapid expansion of neuronal regulatory gene regions (nRRs) with hyper-amplification of neural-specific protein domains (such as DUF1220) in humans. The human chromosome 1q21.1 region falls into this category, noted for containing NOTCH2NL (N2NL), a human-specific neural-critical gene family believed to be a major contributor to human brain evolution. High-resolution sequencing of the human 1q21.1 nRR has allowed us to identify a collection of transfer RNA (tRNA) genes that appear to be involved in the rapid expansion of this region across the primate lineages. Known for their canonical roles in protein translation, tRNAs have recently been ascribed additional functions in regulating gene expression. For example, a pivotal study in mice showed that severe neurodegeneration could be caused by a mutation in a tRNA gene that was characterized as producing the majority of Arg-UCU transcripts in the brain, demonstrating the first highly tissue-critical tRNA gene. Additionally, tRNA genes have been shown to affect chromatin accessibility of nearby protein-coding genes at the DNA level. With the advent of new tRNA sequencing methods such as ARM-seq and DM-tRNA-seq, there is an opportunity to better understand the activity and differential expression of tRNAs and tdRNAs across tissues and throughout development. Here, we present differentially regulated tdRNAs using ARM-seq and compare results from diverse samples including orthogonal human cerebral cortical organoids and stem-cell lines. Analysis of relative expression profiles of tRNAs and tdRNAs in the context of human neuronal development was performed using the tRNA Analysis of eXpression (tRAX) pipeline. Using a combination of approaches including analyses of the genomic context and epigenetic chromatin state as well as relative expression profiling, we have identified significant tRNA changes in these developing organoid models. In addition to the previously studied Arg-UCU transcript, we found unique expression profiles across human neuro-development for over twenty other specific tRNAs and tdRNAs. This deeper look into tRNAs encoded in human nRRs supports an expanded, integral role for tRNA genes in human brain development and regulation, beyond simple protein translation.

Presenting author email [email protected]

Topic category tRNA: Processing and Function 545 Analysis of ARM-seq and DM-tRNA-seq using tRAX allows for detection of tissue-specific tRNA/tdRNA expression and modifications

Jonathan Howard, Andrew Holmes, Todd Lowe University of California, Santa Cruz, Santa Cruz, California, USA

Abstract

Transfer RNAs (tRNAs) are the largest, most complex non-coding RNA family, universal to all living systems. Often regarded as passive players in protein expression, tRNAs have recently been shown to have new roles in regulatory pathways outside their fundamental role in mRNA translation. New evidence suggests tRNAs undergo tissue-specific expression, processing, and modifications, resulting in a constellation of tRNAs and tRNA-derived small RNAs (tdRNAs), with the potential to regulate diverse pathways in response to changes in the cellular or extracellular environment. Furthermore, dysregulation of both tRNA and tdRNA expression has been observed and implicated in a variety of diseases. Unfortunately, eukaryotic tRNA expression and modification data is scarce due to historic lack of high-throughput, low-cost molecular detection methods effective for these heavily modified RNAs. Moreover, tissue specificity of certain tRNAs makes in vivo study of their expression and regulatory effects difficult. Here, we present new findings using the ARM-seq (AlkB- facilitated RNA Methylation sequencing) and DM-tRNA-seq (Demethylase-thermostable group II intron RT tRNA sequencing) methodologies, along with the tRNA Analysis of eXpression (tRAX) computational pipeline, allowing a first in-depth look at both mature tRNA and tdRNA expression from a variety of mouse tissues. We present tissue-specific and isodecoder-specific expression patterns for both mature tRNAs and tdRNAs, including the previously described CNS-enriched tRNA-Arg-TCT-4, and the newly recognized heart-enriched tRNA-Gly-GCC-1. Additionally, we detect subsets of tdRNAs, representing 5’ and 3’ tRNA halves, that are sensitive to both 5’ phosphorylation and 3’ de-phosphorylation using ARM-seq library preparation. Furthermore, computational analyses of DM-tRNA-seq data further support the effectiveness of using nucleotide misincorporation data to infer tRNA methylation dynamics. With this approach, we observed extensive differences in predicted modification states between tRNAs isoacceptors and isodecoder types. Overall, our data provides one of the first global assessments of both mature tRNA and tdRNAs from normal tissues, and should provide a foundation for future research into disease-associated changes in tRNA/tdRNA expression.

Presenting author email [email protected]

Topic category tRNA: Processing and Function 591 Alteration of the premature tRNA landscape by gammaherpesvirus infection

Jessica Tucker1, Aaron Schaller1, Ian Willis2, Britt Glaunsinger1 1UC Berkeley, Berkeley, CA, USA. 2Albert Einstein College of Medicine, Bronx, NY, USA

Abstract

Transfer RNAs (tRNAs) are transcribed by RNA polymerase III (RNAPIII) and play a central role in decoding our genome, yet their expression and non-canonical function remain understudied. Many DNA tumor enhance the activity of RNAPIII, yet whether infection alters tRNA expression is largely unknown. Here, we present the first genome wide analysis of how viral infection alters the tRNAome. Using a tRNA-specific sequencing method (DM-tRNA-seq), we find that the murine gammaherpesvirus MHV68 induces global changes in pre-tRNA expression with 14% of tRNA genes upregulated more than 3-fold, indicating that differential tRNA gene induction is a characteristic of DNA virus infection. Elevated pre-tRNA expression corresponds to increased RNAPIII occupancy for the subset of tRNA genes tested; additionally, post- transcriptional mechanisms contribute to the accumulation of pre-tRNA species. Pre-tRNA accumulation, but not RNAPIII recruitment, requires gammaherpesvirus-induced degradation of host mRNAs by the virally encoded mRNA endonuclease muSOX. Our work suggests that depletion of pre-tRNA maturation or turnover machinery contributes to robust accumulation of pre-tRNAs in infected cells. Because RNAPIII activity is required for efficient viral gene expression and replication, we hypothesize that excess pre-tRNAs in infected cells play a functional role during infection and are currently exploring whether pre-tRNAs sequester host proteins or are cleaved into tRNA fragments. Collectively, these findings reveal pervasive changes to tRNA expression during DNA virus infection and highlight the potential of using viruses to explore tRNA biology.

Presenting author email [email protected]

Topic category tRNA: Processing and Function 703 How does yeast regulate translation? The study of tRNA-derived fragments, that can regulate ribosome-associated aminoacyl-tRNA synthetases.

Anna Maria Mleczko1, Piotr Celichowski2, Kamilla Bąkowska - Żywicka1 1Institute of Bioorganic Chemistry Polish Academy of Sciences, Poznań, Poland. 2Department of Histology and Embryology, Poznan University of Medical Sciences, Poznań, Poland

Abstract

Ribosome-associated noncoding (ranc) RNAs are a novel class of short regulatory RNAs with functions and origins that have not been well studied. In this present study, we functionally characterized the molecular activity of Saccharomyces cerevisiae transfer RNA (tRNA)-derived fragments (tRFs) during protein biosynthesis. Our results indicate ribosome-associated tRFs derived from both 5' (ranc-5'-tRFs) and 3'-part of tRNAs (ranc-3'-tRFs) have regulatory roles during translation. We demonstrated five 3'-tRFs and one 5'-tRF associate with a small ribosomal subunit and aminoacyl-tRNA synthetases (aa-RSs) in yeast. Furthermore, we discovered that four yeast aa-RSs interact directly with yeast ribosomes. tRFs interactions with ribosome- associated aa-RSs correlate with impaired efficiency of tRNA aminoacylation. This work was supported by the FNP (POMOST/2011-4/1 to K.B.-Ż.), NCN (2014/13/D/NZ1/00061 to K.B.-Ż.), IBCH PAN (19/GM/2017 to A.M.M) and by MNiSW under the KNOW program.

Presenting author email [email protected]

Topic category tRNA: Processing and Function 707 Human TRIT1 (tRNA isopentenyltransferase 1) exhibits substrate specific restriction against tRNATrpCCA

Abdul Khalique, Sandy Mattijssen, Alexander F. Haddad, Shereen Chaudhry, Richard J. Maraia Eunice Kennedy Shriver National Institute of Child Health and Human Development, of the National Institutes of Health (NIH), Bethesda, Maryland, USA

Abstract

Human TRIT1 is a highly conserved tRNA isopentenyltranferase (IPTase) that modifies subsets of cytoplasmic and mitochondrial tRNAs by adding an isopentenyl group onto the adenine at position 37 (i6A37), immediately 3' to the anticodon. The tRNA-i6A37 modification increases the translational fidelity and efficiency by optimizing codon-anticodon fit within the ribosome, which promotes maintenance of the correct reading frame during protein synthesis. The importance of this modification is illustrated by the pathogenic mutation, R323Q in human TRIT1 that decreases i6A37 modification efficiency and leads to mitochondrial dysfunction due to impaired tRNA-i6A37-dependent mitochondrial mRNA translation. Interestingly, the profile of IPTase tRNA substrates vary among different eukaryotic species with regard to their relative numbers and identities in the cytoplasm and mitochondria. The tRNA sequence, A36-A37-A38, is the conserved motif recognized by IPTases, and is the major determinant for i6A37 modification. However, an exception to this recognition rule exists. It was documented that S. cerevisiae tRNATrpCCA is unmodified despite the presence of A36-A37-A38, whereas S. pombe tRNATrpCCA is efficiently modified. This indicates variability in the eukaryotic IPTase recognition systems, and moreover that the S. cerevisiae IPTase, Mod5, is specifically inhibited/restricted by the anticodon of tRNATrp-CCA, which is unusual as an IPTase substrate in that it contains pyrimidines in both the 35 and 36 positions. The full substrate specificity of TRIT1 had remained unknown.

To better understand the spectrum of TRIT1 substrate activity we used i6A37-dependent tRNA-mediated suppression assay (TMS) and i6A37-sensitive quantitative northern blotting to examine its activities in S. pombe and S. cerevisiae lacking their endogenous IPTases, on a diversity of tRNA substrates. We found that TRIT1 exhibits substrate-specific restriction against tRNATrpCCA but not other tRNAs in S. pombe. Further analysis of tRNATrp CCA by point mutation at U32C followed by quantitative i6A37-sensitive northern blotting suggests that position 32 is a conditional determinant of substrate-specific i6A37 modification by the restrictive IPTases, Mod5, and TRIT1. Our results provide new insights into IPTases and the role of anticodon loop recognition in their substrate-specific activities, as well as the phylogenetic-variable tRNA-i6A37 profiles, that may help us better understand disease states due to IPTase deficiency.

Presenting author email [email protected]

Topic category tRNA: Processing and Function 748 Carboxy-terminal variations in human tRNA nucleotidyltransferase (TRNT1) affect substrate binding and catalysis

Matthew Leibovitch1, Michael Chung2, Pamela J Hanic-Joyce1, Paul BM Joyce1,3 1Concordia University, Department of Chemistry and Biochemistry, Montreal, Quebec, Canada. 2Concordia University, Department of Biology, Montreal, Quebec, Canada. 3Centre for Structural and Functional Genomics, Concordia University, Montreal, Quebec, Canada

Abstract

A 3’-terminal cytidine, cytidine, adenosine (CCA) sequence is required for aminoacylation of all transfer RNAs (tRNAs). This sequence is added, during tRNA biogenesis or repair, by tRNA nucleotidyltransferase (tRNA-NT) enzymes. Based on sequence conservation and catalytic mechanisms, tRNA-NTs belong to two groups, the Class I archaebacterial enzymes and the Class II eubacterial and eukaryotic enzymes. Solved crystal structures of Class II enzymes reveal a seahorse-shaped protein with head, neck, body and tail domains. The amino- terminal head and neck domains contain five conserved motifs (A to E) that play specific roles in nucleotide binding and catalysis. The body and tail domains show much less sequence conservation but crystallographic data suggest a role in tRNA binding and orientation. Here the human Class II tRNA-NT, TRNT1, serves as a model to explore the function of C-terminal tail residues in enzyme activity. We show that a C-terminal truncation of 10 amino acids, removing the C-terminal α-helix, results in an approximately 3-fold drop in kcat and increases in KM and KD values for tRNA as compared to the native enzyme, reflecting changes in tRNA binding. When expressed in yeast, the shortened enzyme supports growth. Removing the last two α-helices from TRNT1 causes an 8-fold decrease in kcat, dramatic increases in KM and KD and a loss of viability when expressed in yeast. Neither truncation measurably affects the higher order structure of the remainder of the protein, as measured by circular dichroism or fluorescence spectroscopy. We also explore two naturally- occurring C-terminal variants identified in SIFD patients. First, a frameshift mutation that shortens the protein by nine amino acids has limited effects on substrate binding, reduces activity by approximately 10-fold and does not support growth in yeast. Second, a single amino acid substitution, K416E, at the end of the penultimate α-helix, results in a 37% reduction in kcat, an approximately 6- and 40-fold increase in KM and KD, respectively, and supports yeast growth. Together these data highlight the importance of carboxy-terminal residues in tRNA-NT activity.

Presenting author email [email protected]

Topic category tRNA: Processing and Function 751 Analysis of two putative tRNA nucleotidyltransferases from Dictyostelium discoideum reveals both active and inactive enzymes

Nathalie E Reid1, Maria A Xavier-Soares2, Meriem Beniani1, Paul BM Joyce1,3 1Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada. 2Department of Biology, Concordia University, Montreal, Quebec, Canada. 3Centre for Structural and Functional Genomics, Concordia University, Montreal, Quebec, Canada

Abstract

All transfer RNAs (tRNAs) must end in a 3’-cytidine, cytidine, adenosine (CCA) sequence that is required for aminoacylation. Sequence analysis reveals that most organisms have a single gene encoding a tRNA nucleotidyltransferase responsible for CCA addition, although certain organisms have separate genes encoding CC- and A-adding activities. This two-enzyme system was seen first in bacteria (1), but more recently was reported in eukaryotes (2,3). The presence of CCA-, CC- and/or A-adding enzymes in a diverse range of organisms raises interesting evolutionary questions and has inspired models for the origins of the bacterial (4) and eukaryotic (3,5) enzymes. Here we analyzed proteins, expressed from genes encoding two putative tRNA nucleotidyltransferases from the Amoebozoan Dictyostelium discoideum (3,6). Exploring this third clade, in addition to the previously characterized Choanoflagellata (3) and Fungi (3,6), provides more information on the evolution of eukaryotic tRNA nucleotidyltransferases. Interestingly, one of these Dictyostelium proteins lacked the flexible loop, seen in CCA- and A-adding enzymes but missing from certain CC-adding enzymes, suggesting the potential for CC addition by this enzyme. The other protein contained the flexible loop and therefore seemed likely to be either a CCA- or A-adding enzyme. Here we show that the latter enzyme has CCA-adding activity in vitro and supports growth in vivo in a yeast complementation assay. In contrast, the former protein, missing the flexible loop, is inactive in vitro and does not allow yeast growth. We discuss how this enzyme has lost its activity and explore the evolutionary relatedness of these Amoebozoan proteins to other eukaryotic tRNA nucleotidyltransferases. 1) Tomita K, Weiner AM (2001) Science 294:1334-1336. 2) Reid NE et al. (2019) Biochem Biophys Res Commun. 508: 785-790. 3) Erber L et al. (2020) Int. J. Mol. Sci. 21: 462; https://doi.org/10.3390/ijms21020462 4) Jones G (2019) Journal of Molecular 87: 254–270. 5) Betat H et al. (2015) Nucl Acids Res. 43: 6739–6746. 6) Leibovitch et al. (2013) Biochem J 453: 401–412.

Presenting author email [email protected]

Topic category tRNA: Processing and Function 813 A novel assay reveals the pathways for tRNAPhe retrograde nuclear import and re-export in yeast

Regina Nostramo1,2, Anita K. Hopper1,2 1The Ohio State University, Columbus, OH, USA. 2Center for RNA Biology, Columbus, OH, USA

Abstract

In eukaryotes, tRNAs are transcribed in the nucleus and subsequently exported to the cytoplasm where they serve as essential adaptor molecules in protein synthesis. However, once in the cytoplasm, tRNA can be trafficked back into the nucleus in an evolutionarily conserved process termed tRNA retrograde nuclear import. Several functions of this process have been identified, including maturation of select tRNA species, tRNA quality control, translation regulation, and response to cellular stress. The tRNA import pathway is also hijacked by viruses such as HIV-1 for their own nuclear import. Currently, the pathway(s) for tRNA retrograde import, and the subsequent re-export step, is largely unknown. Therefore, we developed an assay in S. cerevisiae to identify the proteins involved in these processes by taking advantage of the unique modification, , found at position 37 of mature tRNAPhe. The presence of this modification on tRNAPhe serves as a road map of previous travel bidirectionally between the nucleus and cytoplasm and can be detected by abasic site formation followed by aniline-induced cleavage. In support of previous genetic data, our assay reveals that the karyopherin Mtr10 mediates retrograde import of tRNAPhe, both constitutively as well as under stress conditions, such as amino acid starvation. Conversely, the Hsp70 protein Ssa2 functions in the import of tRNAPhe specifically under conditions of amino acid deprivation. Furthermore, we found that tRNAPhe is re- exported by Crm1 and Mex67, but not by the well-known tRNA exporters Los1 or Msn5. These findings indicate that similar to primary tRNA nuclear export, the re-export process occurs in a tRNA species-specific manner. Together, our findings elucidate the pathways for tRNAPhe retrograde import and re-export and validate an assay that can be used on a genome-wide level in yeast to identify additional gene products involved in these tRNA trafficking events under stress and non-stress conditions.

Presenting author email [email protected]

Topic category tRNA: Processing and Function 830 Expanding our Understanding of tRNA Modification Genes in Bacillus subtilis

Marshall Jaroch University of Florida, Gainesville, FL, United States Minor Outlying Islands

Abstract

Extensive knowledge on both the nature and position of tRNA modifications has been limited to two bacteria, Escherichia coli and Mycoplasma capricolum. Bacillus subtilis sp subtilis strain 168 is the model gram-positive bacteria and the list of genes involved in tRNA modification in this organism is still incomplete. Mass- spectrometry analysis of bulk tRNA extracted from B. subtilis combined with next generation sequencing technologies and comparative genomic analyses led to the identification of 41 tRNA modification genes with associated confidence scores (V. de Crécy-Lagard, R. Ross and Y. Motorin, unpublished data). With this list of genes in hand, we set out to survey the function of tRNA modifications in B. subtilis by mining the available “omics” data such as essentiality data, phenotypic screens, TnSeq data and gene expression data. In this study, we specifically analyze a phenotype screen that tested several conditions (growth, C or N sources, temperature sensitivity)(Cell Syst 2017, 4(3):291-305.e7) and transcriptomic data available in the Colombos database (http://colombos.net/). Analysis of these data sets has led us to validate previous functions assigned to the tRNA modifications and to propose that the expression of specific tRNA modification genes can vary depending on growth conditions. These observations can help to direct further experimental studies and to continue to elucidate the functions for poorly understood genes in B. subtilis.

Presenting author email [email protected]

Topic category tRNA: Processing and Function 857 Dicistronic tRNA-mRNA Transcript Expression Analysis in Grapevine (Vitis vinifera) Suggests Environmental Regulation and Evolutionary Conservation in Plants

Pastor Jullian Fabres1, Na Sai1, Stephen Pederson1, Fei Zheng1, James Breen1, Matthew Gilliham1, Penny Tricker1, Rakesh David1, Carlos Rodríguez López2 1University of Adelaide, Adelaide, SA, Australia. 2University of Kentucky, Lexington, KY, USA

Abstract

Co-transcription of RNA molecules is not a common feature in the plant nuclear genome. However, polycistronic transcripts have been found in plants, mostly of non-coding RNA species and more recently the transcript of tRNA-mRNA molecules. Evidence suggests that dicistronic tRNA-mRNA transcripts are able to move through the vascular system as a possible mechanism to communicate between distant tissues. Little is known to what extent dicistronic transcripts of tRNA and mRNA species are expressed in field-grown plants, or the factors contributing to their expression. We analysed tRNA-mRNA dicistronic transcripts in the major horticultural crop grapevine (Vitis vinifera) using a novel pipeline developed to identify dicistronic transcripts from high-throughput RNA sequencing data. We analysed leaf and berry samples from 22 commercial vineyards covering six sub-regions of the Barossa Wine Growing Region, Australia. We identified 19 dicistronic tRNA-mRNA molecules that their abundance was tissue and geographic sub-region specific. In leaf tissue, the expression patterns of dicistronic tRNA-mRNAs significantly correlated with tRNA expression, suggesting that transcriptional regulation of their expression might be linked. We also found evidence of evolutionary conservation of dicistronic candidates in grapevine, and previously reported dicistronic transcripts in Arabidopsis, indicating a syntenic genomic arrangement of tRNAs and protein coding genes between species.

Presenting author email [email protected]

Topic category tRNA: Processing and Function 952 Identifying mitochondrial tRNAs in vertebrates using tRNAscan-SE

Patricia Chan, Allysia Mak, Todd Lowe University of California Santa Cruz, Santa Cruz, CA, USA

Abstract

Mitochondrial genomes in vertebrates, representing the majority of available sequenced mitochondrial genomes, typically include 22 tRNA (mt-tRNA) genes. Each mt-tRNA decodes one of the twenty amino acids, except for leucine and serine which are decoded by two mt-tRNAs with distinct anticodons. Previous studies have shown that sequence and structural variations, such as abbreviated or complete loss of the stem and loops of the D- and/or T-arms, causes numerous mt-tRNAs to deviate from the canonical cloverleaf structure. Although the prior version of tRNAscan-SE has an “organelle search mode”, it used of a general covariance model that was trained on a mix of eukaryotic cytosolic, bacterial, and archaeal tRNAs, and cannot detect mt- tRNAs with degenerate structures missing D- or T-arms. To effectively identify mt-tRNAs in vertebrates with improved accuracy and speed, tRNAscan-SE 2.0 includes a mt-tRNA search mode that utilizes 22 individual covariance models created from curated tRNA alignments and the Infernal software package, one for each isotype/anticodon. Query sequences are scanned with the full set of covariance models and the best, most significant non-overlapping hits are presented with gene coordinates, tRNA isotype/anticodon, and secondary structure annotations. To assess the performance of mt-tRNA gene prediction, we applied the detection method to 3,345 mitochondrial genomes in vertebrates which contain 73,674 annotated mt-tRNA genes in NCBI RefSeq. Prediction results were compared with existing algorithms including ARWEN, MiTFi, and tRNAscan-SE 1.3 “organelle search mode”. Both tRNAscan-SE 2.0 and MiTFi performed best with 99% of predictions consistent with RefSeq annotations. However, tRNAscan-SE 2.0 searches over 100 times faster than MiTFi. While NCBI RefSeq has a well-established collection of high-quality mitochondrial genomes, the use of slightly sub-optimal gene prediction tools, BLAST searches, and manual curation across thousands of mitochondrial genomes can lead to some mis-annotations. Upon manual inspection of inconsistencies between RefSeq and tRNAscan-SE or MiTFi predictions, we found that over 97% were due to incorrect strandedness or isotype classification errors in RefSeq. This new functionality in tRNAscan-SE 2.0 provides a fast, improved method to identify mt-tRNA genes in vertebrates, with the flexibility to expand its scope to detect mt-tRNAs in other eukaryotic clades.

Presenting author email [email protected]

Topic category tRNA: Processing and Function 23 Investigating FET protein phase separation by developing an E. coli model

Rachel Victor, Jacob Schwartz University of Arizona, Tucson, AZ, USA

Abstract

The RNA binding protein FUS (Fused in Sarcoma) has several roles in regulating RNA in cells, including RNA processing and transcription. Additionally, FUS has prominent roles in the neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as well as sarcoma development. FUS is a mostly disordered protein and readily phase separates as part of its cellular function. Thus, understanding the unique mechanisms employed by FUS has been challenging and has limited our understanding of disease mechanisms. We express FUS in E. coli and employ chemical crosslinking and chromatographic separation to observe FUS transitioning from a monomer to phase separated state. This E. coli model recapitulates the natural, highly crowded environment found in cells and can be a platform for large and parallel evaluation of protein phase separation, by offering rapid cell and protein production, sub-cellular separation of proteins and nucleic acids into small complexes and granules, and structure/function relationships for disordered RNA- binding proteins that regulate gene transcription. From this approach, we have found evidence that FUS forms higher order assemblies in E. coli.

Presenting author email [email protected]

Topic category

Granules & Condensates 24 Granules of disordered fusion proteins regulate transcription in Ewing sarcoma

Nasiha Ahmed, Lucas Harrell, Rachel Victor, Tarjani Thaker, Thomas Tomasiak, Jacob Schwartz The University of Arizona, Tucson, AZ, USA

Abstract

Chromosomal translocation events drive many types of childhood cancers. Ewing sarcoma is a pediatric bone cancer driven by a chromosomal translocation where the N-terminal low-complexity domain of the RNA-binding protein EWSR1 fuses to the DNA-binding domain of the ETS transcription factor FLI1. This fusion creates the aberrant transcription factor EWS-FLI1, which drives tumor formation by altering the transcriptional landscape. Interestingly, constitutive expression of EWSR1 is retained in Ewing sarcoma cells. The low-complexity domain found in EWSR1 and EWS-FLI1 is intrinsically-disordered and can mediate protein-protein interactions with itself and other protein partners. This domain also has the robust propensity to phase separate. Despite the common domain between the two proteins, little is known about how EWSR1 effects EWS-FLI1 activity. We performed RNA-sequencing to reveal that EWSR1 and EWS-FLI1 coregulate a large pool of genes in Ewing sarcoma cells. The loss of EWSR1 inhibits anchorage-independent growth and suggests that EWSR1 plays a role in the oncogenic effect of EWS-FLI1. Finally, we have found the existence of EWSR1, EWS-FLI1, and RNA Pol II in a novel granular body in Ewing sarcoma cell lines and have resolved these granules by transmission electron microscopy. This interaction of EWSR1 and EWS-FLI1 presents a straight-forward model that merges wild-type and fusion protein properties to alter transcriptional output and drive Ewing sarcoma biology.

Presenting author email [email protected]

Topic category

Granules & Condensates 267 Rapid and reversible phase-separation of multivalent proteins during osmotic cell volume change modulates RNA metabolism

Sethu Pitchiaya1, Ameya Jalihal1, Pushpinder Bawa1, Karan Bedi1, Lanbo Xiao1, Xia Jiang1, Marcin Cieslik1, Mats Ljungman1, Arul Chinnaiyan1,2, Nils Walter1 1University of Michigan, Ann Arbor, Michigan, USA. 2Howard Hughes Medical Institute, Ann Arbor, Michigan, USA

Abstract

Mammalian cells dynamically (within minutes) respond to a variety of environmental stresses by typically inducing membrane-less ribonucleoprotein assemblies called processing bodies (PBs) and stress granules (SGs). Here, we find that the trimeric PB protein and RNA decapping coactivator DCP1A rapidly (within ~10 s) phase-separates in mammalian cells during hyperosmotic stress and quickly dissolves upon isosmotic rescue (over ~100 s). Strikingly, this intracellular hyperosmotic phase separation (HOPS) is not exhibited by other PB components, is distinct from SG assembly and correlates with the degree of cell volume compression across several cell types. High-throughput immunofluorescence and GFP-imaging suggests that HOPS is exhibited broadly by homo-multimeric (valency ≥ 2) proteins. Notably, HOPS leads to the nuclear sequestration of multimeric pre-mRNA component CPSF6 away from actively transcribing RNA polymerases, rationalizing hyperosmolarity-induced global impairment of transcription termination. Together, our data suggest that the multimeric proteome is rapidly reorganized into membrane-less protein assemblies by physiological processes and environmental stresses that alter macromolecular crowding. Overall, our work reveals an unexpected mode of globally programmed phase separation that adapts the cell to volume change, modulates RNA metabolism and mediates spatial gene regulation.

Presenting author email [email protected]

Topic category

Granules & Condensates 279 Discrete functional RNA encoded domains in LLPS droplets

Christine Roden The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Abstract

Many phase-separated droplets form from RNA-binding proteins and RNA. There is evidence of spatial order within RNA droplets such as the nucleolus, P- and stress granules. It is not well understood how higher order organization is governed by protein/RNA-interactions, if internal order is a common droplet feature, and how internal order contributes to function. Spatially-distinct territories within a droplet emerge from two or more co-existing states with distinct material properties (cores and shells). We hypothesize that shells govern surface interactions between droplets, gate entry/exit into droplets and control surface biochemistry. In the case of mRNA, localization to droplet shells could control translation. Ashbya gossypii, a multinucleate fungus, uses RNA: protein droplets to pattern cytoplasm. Droplets containing G1 cyclin RNA CLN3 regulate Ashbya nuclear cycle asynchrony by spatially/temporally restricting CLN3 translation. We do not fully understand how CLN3 RNA sequence/structure encodes translational regulation and immiscibility with a second type of droplet within shared cytoplasm. We show that reconstitution of Whi3 protein and CLN3 RNA results in gel-like droplets with CLN3 RNAs patterned in spatially distinct domains, cores and shells. We developed an assay to test translation in droplets to study how shells control translation. In vitro, core CLN3 RNA is irreversibly trapped within Whi3 droplets and inaccessible to translation machinery whereas shell mRNA is weakly accessible. This suggests that in vivo an unknown trans factor is required to release core RNA at G1 or that only shell RNA is translatable. We also observed that CLN3 shell RNA sequence regulates immiscibility by specifying growth and coalescence rates. Collectively, these observations provide key insights into how cells use droplet shell RNA to regulate translation and sorting for function in vivo. Finally, intradroplet spatial organization may be generalizable to many droplet types.

Presenting author email [email protected]

Topic category

Granules & Condensates 291 Investigating the interplay between RNA and Protein Kinase R in stress granule assembly

Sean Ihn, Hyun Lee, Alex Palazzo University of Toronto, Toronto, Ontario, Canada

Abstract

Biomolecular condensates form via liquid-liquid phase separation, a process in which weak, intermolecular and multivalent interactions allow RNA and proteins to de-mix from the bulk solution. One well-studied condensate is the stress granule (SG), a cytoplasmic condensate that sequesters various proteins and RNAs in response to cellular stress. Although extensive research has been dedicated into understanding how certain proteins contribute to condensate assembly, few studies have examined the role of RNA in this process. It has been proposed that polysome disassociation and impaired translation resulting from cellular stress lead to the accumulation of polysome-free RNAs, which then make weak multivalent interactions with each other to drive SG formation. I set out to test this idea and demonstrated that microinjecting free reporter RNAs into the cytoplasm of mammalian cells rapidly induces SG formation, thereby supporting the central role of free RNA in SG assembly. Interestingly, cells injected with reporter RNAs had higher levels of eIF2α phosphorylation, a marker for cellular stress. Protein Kinase R (PKR) is an eIF2α-kinase that is activated by double-stranded RNA. To determine whether PKR phosphorylates eIF2α after RNA injection, I treated cells with C16, an inhibitor of PKR activity, prior to injection. My results suggest that eIF2α-phosphorylation is unaffected by PKR inhibition. Furthermore, when RNAs were injected at high concentrations, SG formation occurred despite PKR inhibition; however, PKR inhibition prevented SG formation when reporter RNAs were injected at low concentration. Therefore, my preliminary data suggests that PKR may enhance SG formation in the presence of low levels of free RNA, while at high levels of free RNA, which likely promotes numerous RNA-RNA interactions, SG formation occurs regardless of PKR activity. In addition, this effect may be independent of eIF2α- phosphorylation, indicating that PKR likely acts on a novel substrate to promote SG formation. In summary, my work provides new insights into how PKR may act to potentiate cellular phase-separation and enhance SG assembly.

Presenting author email [email protected]

Topic category

Granules & Condensates 629 Multivalent interactions with RNA drive recruitment and stabilization of an RBP in novel phase separated condensates in Xenopus oocytes

Sarah Cabral, Kimberly Mowry Brown University, Providence, RI, USA

Abstract

RNA localization is a key biological strategy for organizing the cytoplasm and generating both cellular and developmental polarity. In Xenopus oocytes, RNAs required for germ layer patterning localize in novel phase separated structures, termed localization bodies (L-bodies). L-bodies are heterotypic and multiphase, containing many copies of non-dynamic, localizing RNAs, surrounded by a comparatively dynamic protein layer. Within L-bodies, proteins exhibit a range of mobilities, with direct binding to non-dynamic RNAs correlating with decreased protein mobility. Notably, PTBP3, an RNA binding protein containing four RNA recognition motifs (RRMs), is the least dynamic of all proteins tested via FRAP and is highly enriched in L-body cores. Binding of PTB to RNA is required for both RNA and PTBP3 localization to L-bodies, as well as their low mobilities within them. Using point mutations in each of the four PTBP3-RNA binding interfaces, we have found that RNA binding to RRMs 3 or 4, but not 1 or 2, is sufficient to drive PTBP3 localization to L-bodies. Importantly, the multivalency of RNA-protein interactions with both RRM3 and RRM4 is required for the low mobility of PTBP3 within the L-body. Our results support a role for localizing RNAs as a scaffold component in L-bodies and for multivalent interactions with the RNA scaffold as a key driver of L-body protein dynamics.

Presenting author email [email protected]

Topic category

Granules & Condensates 704 Defining how Kapβ2 mitigates FUS aggregation and toxicity

Charlotte Fare, James Shorter University of Pennsylvania, Philadelphia, PA, USA

Abstract

FUS is a nuclear RNA-binding protein (RBP), and its cytoplasmic mislocalization and aggregation is associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). FUS bears a proline-tyrosine nuclear localization signal (PY-NLS), which is recognized by the nuclear import receptor Karyopherin-β2 (Kapβ2). Recent work has demonstrated that, in addition to regulating the subcellular localization of proteins with a PY-NLS, Kapβ2 is also an impressive protein chaperone that can disaggregate and remodel its cargo. To understand how the nuclear import and chaperone activities of Kapβ2 are related to one another, we designed truncated Kapβ2 proteins to probe the structure of both Kapβ2 and the PY-NLS of FUS. Using a yeast model system and biochemical assays, we find that a C-terminal portion of Kapβ2 is sufficient to rescue toxicity, restore nuclear import, and prevent aggregation of FUS. Furthermore, we discovered that truncations lacking residues known to contact the PY-NLS are able to partially chaperone FUS, but cannot restore nuclear localization to the same extent as the full-length protein.

Presenting author email [email protected]

Topic category

Granules & Condensates 800 Germ plasm localized RNAs are conserved across a multi-genera vertebrate clade

Christina Hansen, Jacob Kurek, Trevor Chamberlain, Francisco Pelegri University of Wisconsin - Madison, Madison, WI, USA

Abstract

In many animal species, germ cell specification requires the inheritance of germ plasm, a biomolecular condensate containing maternally-derived RNAs and proteins. In the model vertebrate system Danio rerio (zebrafish), germ plasm is organized at two primary levels: i) as homotypic ribonucleoparticle (RNP) microstructures and ii) as massive supramolecular aggregates comprised of RNPs and associated components. However, the precise composition of germ plasm components and how they influence germ cell fate remains poorly understood. To date, 13 RNAs have been reported to localize to the germ plasm masses that form in the furrows of the earliest embryonic cell divisions in zebrafish; however, studies in other model organisms have shown that while a few of these germ plasm components, such as products of the gene nanos, are highly conserved across animal lineages, others appear to be species-specific. Here, we aim to determine if, within a restricted phylogenetic space, RNA components of germ plasm are conserved as a core set. In order to address this question, we use the Danio and Devario genera as a model vertebrate phylogenetic system by systematically testing for the presence of known zebrafish RNAs in three other species within this clade: the orange-finned danio D. kyathit, the pearl danio D. albolineatus, and the giant danio Devario aequipinnatus. We find that all tested zebrafish germ plasm RNAs are also localized to the germ plasm masses in each of these Danionin species, indicating a high level of conservation of germ plasm components at this phylogenetic scale. Our data also indicate that the patterns of germ plasm RNP localization and organization across early embryonic development are conserved within this clade.

Presenting author email [email protected]

Topic category

Granules & Condensates 818 Single-molecule imaging reveals translation of mRNAs localized to stress granules

Daniel Mateju, Jeffrey Chao FMI, Basel, Switzerland

Abstract

Cellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively non-translating mRNA. Here, we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. While our data confirms that non-translating mRNAs are preferentially recruited to SGs, we find evidence for translation of mRNA localized to SGs. Our data indicate that SG-associated translation is not rare and that the entire translation cycle (initiation, elongation and termination) can occur on SG-localized transcripts. Furthermore, translating mRNAs can be observed transitioning between the and SGs without changing their translational status. Together, these results argue against a direct role for SGs in inhibition of mRNA translation.

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Topic category

Granules & Condensates 892 Treatment of cancer cells with Lapatinib negatively regulates general translation and induces stress granules formation

Pauline Adjibade1,2, Rachid Mazroui3 1Université Laval, Quebec, QC, Canada. 2McGill university, Montreal, QC, Canada. 3Université Laval, Quebec, Canada

Abstract

Stress granules (SG) are cytoplasmic RNA granules that form during various types of stress known to inhibit general translation, including oxidative stress, hypoxia, endoplasmic reticulum stress, ionizing radiations or viral infection. Induction of these SG promotes cell survival in part through sequestration of proapoptotic molecules, resulting in the inactivation of cell death pathways. SG also form in cancer cells, but studies investigating their formation upon treatment with chemotherapeutics are very limited.. Here we identified Lapatinib (Tykerb / Tyverb ®), a tyrosine kinase inhibitor used for the treatment of breast cancers as a new inducer of SG in breast cancer cells. Lapatinib-induced SG formation correlates with the inhibition of general translation initiation which involves the phosphorylation of the translation initiation factor eIF2α through the kinase PERK. Disrupting PERK-SG formation by PERK depletion experiments sensitizes breast cancer cells to Lapatinib. This study further supports the assumption that treatment with anticancer drugs activates the SG pathway, which may constitute an intrinsic stress response used by cancer cells to resist treatment

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Granules & Condensates 925 Regulation of eukaryotic mRNA decapping by condensate formation

Ryan Tibble, John Gross UCSF, San Francisco, CA, USA

Abstract

Removal of the 7-methylguanosine cap by the Dcp1/Dcp2 often commits mRNA to 5'-3' degradation. Decapping is tightly controlled through numerous protein-protein interactions and many of these decay cofactors localize to membraneless P-bodies. Despite many advances in our understanding of P-bodies and other RNA granules, their heterotypic composition and dynamic behavior has made it difficult to construct a molecular understanding of their function. To address this, we reconstitute a minimal decapping condensate and demonstrate activity can be modulated by droplet composition. These differences in activity can be explained by the underlying interactions driving droplet assembly. Furthermore, these interactions directly influence the ability of Dcp2 to perform catalysis. Together, our results suggest atomic level restructuring of the decapping complex is intimately linked to mesoscale organization with direct consequences for activity. This synergistic relationship provides insights into how the formation of macroscopic assemblies, including P- bodies, imparts cells with the ability to exquisitely regulate gene expression.

Presenting author email [email protected]

Topic category

Granules & Condensates 963 Role of Liat1 phase separation in ribosome biogenesis

Akshaya Arva, Yasar Kasu, Christopher Brower Texas Woman's University, Denton, Texas, USA

Abstract

LIAT1 (Ligand of ATE1) was discovered by its interaction with ATE1, involved in the N-degron pathway of protein degradation. The characterization and functional significance of LIAT1 awaits discovery. While the C- terminal half of LIAT1 is necessary for ATE1-binding, the N-terminal half is predicted to be disordered. Intrinsically disordered proteins have recently been shown to participate in liquid phase separation which facilitates the formation of membrane-less intracellular compartments such as the nucleolus. Here, we show that the N-terminal half of LIAT1 is intrinsically disordered and harbors conserved poly-lysine and poly- glutamate regions. Using bimolecular fluorescence complementation (BiFC) and immunocytochemistry, we found that LIAT1 self-associates both in the cytosol and in the nucleus. We also found that the N-terminal intrinsically disordered domain (IDD) of LIAT1 is sufficient to target it to the nucleolus. We also identified sequence determinants within the IDD of LIAT1 important for these activities. Nucleolar fractionation of Liat1 from cell lines and various mice tissues shows Liat1 can form high molecular species in nucleolus and ~37KDa band in cytosol. Consistent with a function in the nucleolus, LIAT1 also interacts with ribosomal proteins. In sum, this study supports a role for LIAT1 in ribosome biogenesis.

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Topic category

Granules & Condensates