MicroRNAs, RNA Modifications, RNA Editing

Bora E. Baysal MD, PhD

Oncology for Scientists Lecture

Tue, Oct 17, 2017, 3:30 PM - 5:00 PM

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

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.

The rise of regulatory RNA

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 (inter-miR), and polycistronic (poly-miR).

• Association of miRNA-bound RNA-induced silencing complex (miRISC) with target mRNAs also cause deadenylation-dependent target mRNA decapping and degradation.

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.

miRNA binding to mRNA

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.

miRNA versus siRNA

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.

Model of miRNA-mediated control of gene expression.

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)

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 (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ).

• m6A is catalyzed by methyltransferase enzymes depending on flanking sequences, proximity to 3-end and secondary structure. m6A can be enzymatically removed by demethylase “eraser” enzymes (e.g. FTO).

• Pseudouridine sites in mRNA are fewer than m6A, but there are more pseudouridine (pus) synthase enzymes. Pseudouridylation is thought to be irreversible.

• m5C is common in noncoding RNAs. There are marked species differences in prevalence of m5C in mRNA: ~8000 m5C sites in human mRNAs compared to a single mRNA m5C site in budding yeast.

YTHDF2 increases turnover of m6A-modified mRNA by promoting colocalization with decay factors.

Nucleotide modifications and detection strategies.

Wendy V. Gilbert et al. Science 2016;352:1408-1412

Diverse molecular functions of m6A, Ψ, and m5C in coding RNAs.

Wendy V. Gilbert et al. Science 2016;352:1408-1412

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

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.

Cytidine deamination

Adenosine deamination

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

Histograms showing the fold change in median expression of ADAR and ADARB1 in a cancer

type compared with normal samples.

TMEM131 SDHB NBN

Mo1 Mo2 Mo3 Ly1 Ly2

Inducible C>U RNA editing by APOBEC3A occurs in monocytes

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.

In vitro cytidine deamination of SDHB RNA versus ssDNA by APOBEC3A

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

cancer.

References: Baysal, B.E., Sharma, S., Hashemikhabir, S. and Janga, S.C., 2017. RNA Editing in Pathogenesis of Cancer. Cancer Research, 77(14), pp.3733-3739. Gilbert WV, Bell TA, Schaening C. Messenger RNA modifications: Form, distribution, and function. Science. 2016 Jun 17;352(6292):1408-12. Morris, K.V. and Mattick, J.S., 2014. 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, 3-10. Sharma, S., Patnaik, S.K., Taggart, R.T., Kannisto, E.D., Enriquez, S.M., Gollnick, P. and Baysal, B.E., 2015. APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages. Nature communications, 6. Wilczynska, A., & Bushell, M. 2015. The complexity of miRNA-mediated repression. Cell death and differentiation, 22(1), 22.