eSymposia | RNA Editing and Modifications: From Biology to Therapy



Sessions

RNA eding of AZIN1 increases cellular aggressiveness in prostate cancer


RNA eding of AZIN1 increases cellular aggressiveness in prostate cancer RNA eding of AZIN1 increases cellular aggressiveness in prostate cancer Aram Ghalali1, Konrad H. Stopsack2, James M. Rice1a, Liangzhe Wang1, Shulin Wu3, Chin Lee Wu3, Bruce R. Zetter1 and Michael S. Rogers1* 1 Vascular Biology Program, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA, 2Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, USA 3Departement of Pathology, Massachusetts General Hospital, Boston, MA, USA, aCurrent Address: Silicon Therapeutics, Boston, MA, USA. We report here nuclear localization of AZIN1, a protein commonly modified by RNA editing in cancer cells, is associated with significantly increased risk of death in prostate cancer. We further find that this nuclear localization is caused by RNA editing of a single base in the AZIN1 mRNA, which in turn leads to a single Ser to Gly substitution in the AZIN1 protein. This change alone is also sufficient to increase the aggressiveness of prostate cancer cells. Our results reveal that, unexpectedly, this editing event changes the binding repertoire of AZIN1, rather than changing its affinity for known targets. Using droplet display PCR, we evaluated the presence of edited AZIN1 (edAZIN1) in aggressive prostate cancer using tissues with Gleason > 7, and found that 94% of these samples expressed edAZIN1. We further measured the expression and localization of AZIN1 in 202 prostate cancer specimens, along with 26 adjacent benign samples and found a negative association between nuclear localization and progression-free survival. Analysis of data from primary prostate cancer patients available via the Cancer Genome Atlas (n = 291 with edAZIN1 calls), the median proportion of edAZIN1 was 6.1% (interquartile range, 4.4 to 8.9). In multivariable models, edAZIN1 was higher with increasing expression of ADAR, a cancer-assoicated RNA editing enzyme, (by 2.8% points per interquartile range increase in ADAR expression; 95% CI, 2.3 to 3.4) and in tumors with higher genomic instability (by 0.6% points per interquartile range increase in copy number alteration burden; 95% CI, 0.3 to 0.9). Downstream, edAZIN1 was associated with higher Gleason grade, with a 2.8%-point difference in edAZIN1 between Gleason 3+3 and Gleason 9-10 tumors (95% CI, 1.2 to 4.5). Together, these results suggest that edAZIN1 is commonly expressed in prostate cancer cells and is associated with increased cellular ADAR expression, nuclear localization, and with increased cancer aggressiveness. We next sought to determine if editing causes increased cellular aggressiveness. We transfected prostate cancer cell lines (PC3, DU145) with constructs coding for wild-type, pseudoedited, and uneditable mRNAs for AZIN1. We found that only constructs capable of coding for edited AZIN1 increased cancer cell aggressiveness as measured by proliferation, invasion, and anchorage-independent growth. Constructs that were unable to undergo editing, showed no such increase. The mechanism underlying the increased aggressiveness of cells expressing AZIN1 has been proposed to be caused by higher affinity binding of edAZIN1 to its known substrates, relative to wild-type AZIN1, but this has not been shown directly. We therefore measured the affinity of edited and wild-type protein for the known target, antizyme, using a novel FRET sensor. Contrary to expectation, we found that editing decreased edAZIN affinity for antizyme, notwithstanding increased complex formation in vivo. We then used tandem affinity purification proteomics to identify selective AZIN1 and edAZIN1 interacting proteins. We identified an edAZIN1-specific complex containing several proteins that may be the driving force behind the nuclear shuttling of edAZIN. Tools developed in this study are now being used to explore the feasibility of developing small molecule drugs that interfere with AZIN1 binding to its preferred substrates and subsequently inhibit with cell growth pathways.

Speaker(s):

Dynamic RNA acetylation as a mechanism for RNA thermostabilization


Dynamic RNA acetylation as a mechanism for RNA thermostabilization N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. We recently developed ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. Our results indicate that in human and yeast mRNAs, ac4C sites are not detected but can be induced—at a conserved sequence motif—via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided 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 elucidating the role of this modification in biology and disease.

Speaker(s):

Adenosine deaminase ADAR2 modulates immune T cell functions


Adenosine deaminase ADAR2 modulates immune T cell functions Deamination of adenosine to inosine (A-to-I) is the most abundant form of RNA editing in mammalian cells and are catalyzed by adenosine deaminases (ADARs). RNA editing contributes to homeostatic innate immune and neurological functions, as well as pathologies in cancers. However, the upstream molecular mechanisms regulating this pathway remained to be elucidated. Here, we report that the Adarb1 locus encoding ADAR2 is significantly upregulated during adaptive immune T helper cell activation and polarization toward the T helper 17 (Th17) lineage. Mechanistically, transcription factor IRF4 together with the establishment of an intragenic super enhancer turns on Adarb1 transcription. Post-transcriptionally, Adarb1 transcripts were bound and regulated by RNA binding protein DDX5. Knocking out DDX5 dampens ADAR2 expression and reduced global RNA editing, resulting in dysregulated expression of HIF1α and loss of effector function in Th17 cells.

Speaker(s):

Cap-proximal N6-methylation protects viral mRNA against interferon beta pretreatment


Cap-proximal N6-methylation protects viral mRNA against interferon beta pretreatment Abstract: Vesicular stomatitis virus (VSV) mRNAs contain a 5’ cap structure m7GpppAm synthesized by the viral large polymerase protein (L). A host enzyme further N6-methylates viral mRNA cap structures to produce m7Gpppm6Am during infection. We report that the cellular methyltransferase PCIF1 carries out this modification on viral mRNA cap structures, modifying most of the viral mRNA in cells. We define cis acting requirements for modification of viral mRNA and demonstrate that neither mRNA stability nor translation are affected by this methylation. However, pretreatment of cells with interferon uncovered an advantage for viral mRNA in the presence of PCIF1. The suppressive effect of interferon on viral replication was blunted for PCIF1 modified viral mRNAs, facilitating ongoing viral gene expression. This study identifies a role for cap-proximal m6Am in countering innate immune effectors induced upon interferon beta treatment of cells in culture. Continuing work will uncover whether m6Am mediates the direct targeting of viral mRNA, or indirectly modifies the host ISG response. Authors and Affiliations: Michael A. Tartell 1,2, Konstantinos Boulias 3, Eric Lieberman Greer 3 and Sean P. J. Whelan 2 1: Program in Virology, Harvard Medical School, Boston MA, USA 2: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA 3: Department of Pediatrics, Boston Children’s Hospital, Boston MA, USA

Speaker(s):

Stereopure oligonucleotides support efficient ADAR-mediated RNA editing in non-human primates


Stereopure oligonucleotides support efficient ADAR-mediated RNA editing in non-human primates Prashant Monian1, Chikdu Shivalila1, Luciano Apponi1, Bijay Bhattarai1, Adam Bezigian1, David Boulay1, Michael Byrne1, Arindom Chatterjee1, Jigar Desai1, Frank Favaloro1, Jack Godfrey1, Andrew Hoss1, Naoki Iwamoto1, Jayakanthan Kumarasamy1, Pachamuthu Kandasamy1, Tomomi Kawamoto1, Anthony Lamattina1, Richard Looby1, Genliang Lu1, Jake Metterville1, Ronelle Murphy1, Snehlata Tripathi1, Stephany Standley1, Hailin Yang1, Yuan Yin1, Hui Yu1, Chandra Vargeese1,* 1Wave Life Sciences, *Corresponding author Recruiting endogenous RNA-editing enzymes using chemically modified oligonucleotides holds great promise for treating human disease. The ADAR (adenosine deaminases acting on RNA) family of enzymes catalyze adenine (A) to inosine (I) changes in coding and non-coding sequences throughout the transcriptome. Because inosine (I) is read as guanine (G) by the translational machinery, oligonucleotide-directed ADAR-mediated RNA editing has the potential to revert myriad disease-causing mutations through a variety of molecular mechanisms, including protein restoration, gene silencing, altered splicing, and protein alteration. PRISMTM, Wave Life Sciences’ proprietary discovery and drug development platform, enables us to develop stereopure oligonucleotides—those in which the chiral configuration of backbone linkages (e.g., Rp or Sp) are precisely controlled at each position—to target genetically defined diseases. We report the application of PRISM to develop stereopure oligonucleotides that support efficient ADAR-mediated RNA editing. We first explore the impact of oligonucleotide chemistries (modifications to the sugar and backbone), backbone stereochemistry and other aspects of design (e.g., length, mismatches) on the activity of RNA-editing oligonucleotides in an extensive structure-activity relationship (SAR) analysis using a dual-luciferase reporter system in vitro. We apply learnings from this SAR analysis to develop optimized, stereopure editing oligonucleotides: we demonstrate that stereopure oligonucleotides can direct endogenous ADAR enzymes to edit endogenous transcripts in vitro, and that oligonucleotides optimized based on our SAR analysis were more effective than stereorandom oligonucleotides. Stereopure oligonucleotides can elicit efficient site-directed RNA editing in primary human hepatocytes when delivered under gymnotic (i.e., free uptake) conditions or with an N-acetylgalactosamine (GalNAc) conjugate. We demonstrate that our design strategy is applicable to multiple sequences by showing ADAR-mediated editing of five endogenous transcripts, and we demonstrate that it is effective in multiple cell types in vitro, including primary human hepatocytes, primary human fibroblasts, and bronchial epithelial cells. Finally, we show that the most effective oligonucleotides identified in our in vitro studies support up to 50% editing of the b-actin transcript in vivo in the liver of non-human primates. Taken together, these preclinical investigations lay the foundations for development of PRISM-generated stereopure RNA-editing therapeutics with potential to treat human genetic disease. This work was funded by Wave Life Sciences.

Speaker(s):

Organizer Introduction and Keynote Address


Co-Chair(s): Speaker(s):

Organizer IntroductionOrganizer Introduction

Preview Available

Organizer Introduction


Co-Chair(s):

Keynote Address | Reversible RNA Methylation in Gene Expression Regulation


Chair(s): (s):

Targeting RNA Methylation in Cancer


Speaker(s):

Dynamic Regulation of the Epitranscriptome


Co-Chair(s):
Print Certificate
Completed on: token-completed_on
Print Transcript
Please select the appropriate credit type:
/
test_id: 
credits: 
completed on: 
rendered in: 
* - Indicates answer is required.
token-content

token-speaker-name
token-index
token-content
token-index
token-content
token-index
token-content
token-index
token-content
token-index
token-content
token-index
token-content
/
/
token-index
token-content
token-index
token-content