MicroRNAs, RNA Modifications, RNA Editing. Bora E. Baysal MD, PhD Oncology for Scientists Lecture Tue, Oct 17, 2017, 3:30 PM - 5:00 PM

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1 MicroRNAs, RNA Modifications, RNA Editing Bora E. Baysal MD, PhD Oncology for Scientists Lecture Tue, Oct 17, 2017, 3:30 PM - 5:00 PM

2 Expanding world of RNAs mrna, messenger RNA (~20,000) trna, transfer RNA rrna, ribosomal RNA snrna, small nuclear RNA hnrna, heterogeneous nuclear RNA mirna, microrna (~2,000) sirna, short interfering RNA pirna, piwi-interacting RNA (~20,000, role in epigenetic silencing of selfish elements) lncrna, long noncoding RNA

3 Complexity of RNAs Transcription involves overlapping, intergenic and intronic sense and antisense small and large RNAs. lncrnas may have evolved from coding RNAs and vive-versa Many new classes of regulatory RNAs remain to be discovered. In contrast to DNA, RNA is single-stranded and fold into complex biologically significant 3-dimensional structures.

4 The rise of regulatory RNA

5 micrornas: MicroRNAs (mirnas) are small ( 22 nucleotide) highly conserved noncoding RNA molecules encoded in the genomes of plants and animals that regulate the expression of genes by binding to the 3'-untranslated regions (3'-UTR) of specific mrnas. Depending on the genomic loci, mirnas can be categorized into three types: intragenic (intra-mir), intergenic (intermir), and polycistronic (poly-mir). Association of mirna-bound RNA-induced silencing complex (mirisc) with target mrnas also cause deadenylationdependent target mrna decapping and degradation.

6 mirnas and their Targets mirnas are extensively processed and are bound and presented by the RNA-induced silencing complex (RISC). The mirisc then associates with a target mrna, usually within its 3 UTR. There is complementarity between the mrna and bases 2 8 of the mirna (the seed sequence). mirnas may function only under specific conditions (e.g. particular developmental stages, stress,response to certain environmental queues and extracellular signaling). Unclear whether mirnas have one primary mrna target or whether they exert their effect by targeting multiple mrnas at once.

7 mirna binding to mrna

8 BIOGENESIS OF mirnas RISC:RNA-induced silencing complex Drosha: an RNase-III processing enzyme Dicer: an RNase-III processing enzyme The association of mirnas with their target mrnas is mediated by Argonaute (AGO) proteins within the RISC in the cytoplasm.

9 mirna versus sirna

10 mirna based gene repression Translational repression and mrna degradation contribute to mirna based gene repression. Recent evidence suggest that translational repression of mirna targets is the primary repressive mechanism (mirnas may primarily interfere with initiation of translation). The effect of mirna gene knockout is often not obvious in the phenotype under normal conditions, only to become apparent in certain cellular contexts or under stress conditions.

11 Model of mirna-mediated control of gene expression.

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13 Anti-miRs versus mirna mimics in therapeutics LNA-modifed antimir directed against the liver-specifc mirna mir-122, which is required for replication of the hepatitis C virus (HCV)

14 RNA modifications RNA contains more than 100 distinct modifications that can modify RNA translation and splicing efficiency. Recent technical advances have revealed widespread and sparse modification of messenger RNAs with N6 -methyladenosine (m 6 A), 5- methylcytosine (m 5 C), and pseudouridine (Ψ). m 6 A is catalyzed by methyltransferase enzymes depending on flanking sequences, proximity to 3-end and secondary structure. m 6 A can be enzymatically removed by demethylase eraser enzymes (e.g. FTO). Pseudouridine sites in mrna are fewer than m 6 A, but there are more pseudouridine (pus) synthase enzymes. Pseudouridylation is thought to be irreversible. m 5 C is common in noncoding RNAs. There are marked species differences in prevalence of m 5 C in mrna: ~8000 m 5 C sites in human mrnas compared to a single mrna m 5 C site in budding yeast.

15 YTHDF2 increases turnover of m6a-modified mrna by promoting colocalization with decay factors.

16 Nucleotide modifications and detection strategies. Wendy V. Gilbert et al. Science 2016;352:

17 Diverse molecular functions of m6a, Ψ, and m5c in coding RNAs. Wendy V. Gilbert et al. Science 2016;352:

18 Evolving insights into RNA modifications and their functional diversity in the brain

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20 RNA Editing versus Central Dogma A post-transcriptional regulatory mechanism that alters the RNA sequences copied from DNA. Adenosine to inosine (A-to-I) and cytidine to uridine (C-to-U) are the most common types in mammals. Inosine and uridine are read as guanosine and thymidine by the translation machinery. Thus RNA editing has the capability to alter the inherited code in DNA.

21 Cytidine deamination Adenosine deamination

22 A>I RNA editing is essential for life and normal development: Transgenic mice with reduced GluR-B editing have severe epileptic seizures and die within 2 weeks of age. Catalyzed by a family of adenosine deaminases acting on RNA (ADARs) C>U RNA editing produces a shorter isoform of ApoB in intestinal cells: The only known physiological protein recoding by C>U RNA editing which is catalyzed by APOBEC1 cytidine deaminase

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24 Histograms showing the fold change in median expression of ADAR and ADARB1 in a cancer type compared with normal samples.

25 Inducible C>U RNA editing by APOBEC3A occurs in monocytes SDHB NBN TMEM131 Mo1 Mo2 Mo3 Ly1 Ly2

26 Candidate cytidine deaminases for inducible RNA editing in monocytes/macrophages -AID (activation-induced cytidine deaminase) is responsible for antibody hypermutations. -APOBEC3 genes play an important role in anti-viral innate immunity. -Functions of APOBEC2 and APOBEC4 are unknown.

27 In vitro cytidine deamination of SDHB RNA versus ssdna by APOBEC3A

28 Summary of various mechanisms through which RNA editing plays a role in the pathogenesis of cancer.

29 References: Baysal, B.E., Sharma, S., Hashemikhabir, S. and Janga, S.C., RNA Editing in Pathogenesis of Cancer. Cancer Research, 77(14), pp Gilbert WV, Bell TA, Schaening C. Messenger RNA modifications: Form, distribution, and function. Science Jun 17;352(6292): Morris, K.V. and Mattick, J.S., The rise of regulatory RNA. Nature reviews. Genetics, 15(6), p.423. Sand, M. (2014). The pathway of mirna maturation. mirna Maturation: Methods and Protocols, Sharma, S., Patnaik, S.K., Taggart, R.T., Kannisto, E.D., Enriquez, S.M., Gollnick, P. and Baysal, B.E., APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages. Nature communications, 6. Wilczynska, A., & Bushell, M The complexity of mirna-mediated repression. Cell death and differentiation, 22(1), 22.