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Exploring your RNA World with an Emphasis on Non-coding Transcripts Benjamin Rodriguez, PhD Wei Li Lab, Baylor College of Medicine Molecular Biology Refresher.

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Presentation on theme: "Exploring your RNA World with an Emphasis on Non-coding Transcripts Benjamin Rodriguez, PhD Wei Li Lab, Baylor College of Medicine Molecular Biology Refresher."— Presentation transcript:

1 Exploring your RNA World with an Emphasis on Non-coding Transcripts Benjamin Rodriguez, PhD Wei Li Lab, Baylor College of Medicine Molecular Biology Refresher Course with Bioinformatics September 13th 2013

2 Course Materials: http://dldcc-web.brc.bcm.edu/lilab/benji/MBRB_2013/index.html Most up to date slides I will upload for all three of my lectures Browsers: http://genome.ucsc.edu/ http://epigenomegateway.wustl.edu/ Web-based analysis: http://mirtar.mbc.nctu.edu.tw/human/index.php http://lilab.research.bcm.edu/cpat/ Software, Sites, Materials

3 Outline Types of RNA transcripts and their functions Post-transcriptional regulation by microRNAs – Mouse models and clinical efficacy Noncoding RNAs and genomic imprinting – CDKN1C and Kcnqot1 Lab exercises: microRNA target prediction and assessing protein coding potential of novel transcripts

4 It’s an RNA World, Baby! Protein Translation mRNA tRNA rRNA RNA function and maturation snRNA snoRNA RNaseP Y RNA Rnase MRP RNA interference miRNA siRNA piRNA Regulatory RNAs lncRNA lincRNA Telomere synthesis Telomerase RNA

5 Functions of RNAs Protein Translation mRNA tRNA rRNA

6 Messenger RNA (mRNA) Protein translation Destiny dictated by post-transcriptional modifications “Cap and tail exits cell” mRNA methylation widespread and likely functional N6-methyladenosine (m6a) meRIP-Seq

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8 Ribosomal RNA (rRNA) 80% of cellular RNA Protein translation Transfer RNA (tRNA) 15% of cellular RNA

9 Functions of RNAs RNA function + maturation snRNA snoRNA RNaseP Y RNA Rnase MRP RNA-protein complexes Support cellular and molecular functions

10 Small nuclear RNA (snRNA) RNA function and maturation RNA component of the Spliceosome snRNP complex made up of 5 snRNAs and over 20 proteins Removes regions of non-coding mRNA (introns)

11 Small nucleolar RNA (snoRNA) RNA function and maturation Guides chemical modifications of other RNAs mainly rRNA, tRNA, and snRNA 2 main classes of snoRNA: H/ACA box, direct conversion of uridine to pseudouridine C/D box snoRNAs, help add methyl groups to RNAs

12 Ribonuclease P (RNaseP) RNA function and maturation RNA component of an RNA enzyme (Ribozyme) Cleaves a precursor sequence from tRNA molecules, generating mature tRNA Also required for RNA Pol III transcription of various small noncoding RNA genes (e.g., tRNA, 5S rRNA, SRP RNA, and U6 snRNA genes) Because they catalyze site-specific cleavage of RNA molecules, Ribozymes may have pharmaceutical applications

13 Y RNA RNA function and maturation Component of Ro Ribonucleoparticle (RoRNP) complex Chaperone regulating maturation of small ncRNAs Transcribed by RNA Pol III UV resistance in mammalian cells Essential for DNA replication Upregulated in human cancer tissue Required for increased proliferation of cancer cell lines

14 Ribonuclease MRP (RNase MRP) RNA function and maturation RNA component of RNase MRP Enzymatically active ribonucleoprotein Initiation of mitochondrial DNA replication In the nucleus, involved in precursor rRNA processing

15 Functions of RNAs RNA interference miRNA siRNA piRNA RNA inhibiting RNA

16 Small (short) interfering RNA (siRNA) RNA interference (RNAi) Biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Medicine for their work on RNAi in the nematode worm C. elegans RNAi helps defend cells against parasitic nucleotide sequences viruses and transposons Plays integral role in development as well as regulation of gene expression in general Technological applications Gene knockdown: study physiological role of individual genes Functional genomics: genome-wide RNAi screens Medicine: attractive, but RNAi delivery to tissues is difficult

17 MicroRNA (miRNA) miRNAs must first undergo extensive post-transcriptional modification before they are mature and functional Primary transcript (Pri-miRNA) mature miRNA pre-miRNA 1. Pri-miRNA are processed into 70-nucleotide precursors (pre-miRNA) 2. Precursor is cleaved to generate 21–25-nucleotide mature miRNAs

18 miRNA processing Pri-miRNA transcript is processed by RNAse III enzyme Drosha and dsRNA-binding protein DGCR8 Drosha cleaves these into 70-bp pre-miRNAs that consist of an imperfect stem-loop structure Step 1

19 Pre-miRNA exported from the nucleus into the cytoplasm by Exportin 5 (Exp5) Cleavage by Dicer yields small, imperfect dsRNA duplex (miRNA: miRNA * ) containing both mature miRNA strand and its complementary strand Step 2 miRNA processing

20 miRNA meet RISC RNA Induced Silencing Complex (RISC) Thermodynamics properties may dictate which strand is incorporated into RISC miRNA-RISC complex bind to 3’ UTRs of their targets May repress transcription through RNA cleavage or repress further translation of nascent protein peptides Method of repression may depend on extent of sequence complementarity between miRNA and target

21 miRNAs and cancer Mouse models implicate the gain or loss of individual microRNAs (miRNAs), miRNA clusters and the miRNA processing machinery in cancer Oncogenic miR-155, miR-21 and miR-17~92 Tumor-suppressive miR-15~16, LIN28, DICER Context-dependent miR-146 and miR-29 miRNAs and miRNA processing machinery involved in all stages of metastatic disease Studies uncovering miRNA function have led to their therapeutic application

22 Nature Reviews Cancer 11, 849-864 (December 2011) | doi:10.1038/nrc3166 Mouse models to evaluate in vivo miRNA functions

23 Opposing roles of miRNAs in cancer Oncogenic versus tumor-suppressive functions of miR-146 can be explained based on upstream nuclear factor κB (NF-κB) signals

24 Opposing roles of miRNAs in cancer Oncogenic miR-146 also targets BRCA1, thus preventing the pro- apoptotic effects of BRCA1 and resulting in a pro-survival response

25 Opposing roles of miRNAs in cancer Tumor-suppressive miR-29a targets multiple oncogenes, inhibiting growth and proliferation as well as aggressive disease (B-CLL)

26 Opposing roles of miRNAs in cancer Oncogenic miR-29a prevents cell adhesion through repressing peroxidasin homologue (PXDN)

27 Nature Reviews Cancer 11, 849-864 (December 2011) | doi:10.1038/nrc3166 Therapeutics: replacing tumor-suppressor miRNAs let-7 downregulated in multiple cancers Prime example of therapeutic efficacy

28 Nature Reviews Cancer 11, 849-864 (December 2011) | doi:10.1038/nrc3166 Therapeutics: some miRs best left alone miR-122 plays pleiotropic role in multiple pathophysiologies Administration may reduce HCC metastasis Antagomir decreases HCV viraemia (primates) and cholesterol levels (mouse)

29 Nature Reviews Cancer 11, 849-864 (December 2011) | doi:10.1038/nrc3166 miR-26a administration inhibited HCC proliferation, induced apoptosis and protected animals from disease progression Therapeutics: replacing tumor-suppressor miRNAs

30 Nature Reviews Cancer 11, 849-864 (December 2011) | doi:10.1038/nrc3166 Antagomir-miR-10b treatment had no effect on the primary breast tumor xenograft, but reduced pulmonary metastases by more than 80 % Therapeutics: targeting oncogenic miRNAs

31 Rational, combinatorial targeting multiple of multiple proteins in a leukemogenic pathway with a cocktail of pharmacologic inhibitors miR-29b targets DNMTs, thereby resulting in global DNA hypomethylation and reexpression of hypermethylated, silenced genes in AML Higher baseline expression of miR-29b associated with clinical response (P = 0.02) in a phase II decitabine trial of previously untreated adult AML patients Proteasome inhibitor bortezomib increases expression of miR-29b and decreases leukemia cell growth Phase I study of DAC and bortezomib in adults with relapsed or refractory AML (Aug 2013) Five of 23 patients achieved remission (CR and/or Cri) miR-29b: targeting a predictor of treatment response in AML

32 Telomere synthesis Telomerase extends ends of DNA molecules preventing DNA loss during replication Protein component possesses reverse transcriptase activity RNA component serves as a template for the telomere repeat Telomerase dysregulation is a hallmark of cancer Telomerase RNA

33 Regulatory RNAs Long non-coding RNA (lncRNA, lincRNA) Non-protein coding transcripts 200 nt to 100 kb in length. lncRNAs characterized thus far appear to function in diverse areas including epigenetics, alternative splicing, and nuclear import XIST plays a critical role in X-chromosome inactivation Kcnq1ot1 ncRNA is required for the maintenance of the silencing of ubiquitously imprinted genes (UIGs) at all developmental stages

34 Remember Genomic Imprinting? Imprinted genes occur in clusters and are often involved in growth control Paternal alleleMaternal allele Paternal allele Imprinting regulated by cis-acting elements (Imprinting Control Regions) and noncoding RNAs They act over long distances and control the imprinting of multiple genes

35 CDKN1C: Regulator of Embryonic Growth Nature 1997; 387(6629): 151-158 CDKN1C is required for postnatal survival Altered cell differentiation and proliferation in mice lacking CDKN1C Developmental defects consistent with a causative role for in Beckwith-Wiedemann syndrome Skeletal abnormalities Abdominal muscle defects Adrenomegaly +/+ +/- -/-

36 CDKN1C/KCNQ1OT1 Imprinted Domain Methylated maternal allele cannot function as a silencer or a promoter for KCNQ1OT1 and thus expresses CDKN1C Unmethylated paternal allele functions as a silencer and a promoter for KCNQ1OT1 transcription

37 Sense Gene Antisense transcript Antisense Transcripts are involved in Genomic Imprinting Linked to mechanisms of monoallelic gene expression, including genomic imprinting and X chromosome inactivation RNAs with sequence complementarity to other transcripts May regulate expression of target genes at level of transcription, mRNA processing, or translation

38 Discovery of a paired CDKN1C antisense transcript The CDKN1C-AS transcript aligns in cis to the CDKN1C promoter and gene body, spanning at least 2.5 kb in length.

39 E2 induces antisense CDKN1C expression in epigenetically reprogrammed breast cancer cells CDKN1C-AS significantly induced by 12 hr E2 treatment in MCF7 cells subjected to pharmacologic inhibition of DNA methylation and histone deacetylation (P < 0.01)

40 Assessing CDKN1C-AS function p57-ASΔ: contains a stop sequence downstream of CMV promoter that prevents CDKN1C-AS transcription p57-S: CDKN1C promoter drives transcription of GFP p57-AS: CMV promoter drives CDKN1C-AS transcription

41 Assessing CDKN1C-AS function Exogenous antisense transcript failed to repress GFP expression in cis

42 Assessing CDKN1C-AS function Exogenous antisense expression induced > 35-fold

43 Assessing CDKN1C-AS function CDKN1C-AS represses endogenous CDKN1C in trans Repression ameliorated by p57-ASΔ NS **

44 Proposed regulatory mechanism of CDKN1C-AS Formation of double-stranded RNA may negatively regulate stability, transport, and/or translation of sense CDKN1C transcript

45 E2 induces expression of KCNQ1OT1 ncRNA Expression of KCNQ1OT1 increased 1.6-fold following 12 hr E2 treatment (P < 0.005)

46 CTCF HDAC1 RNA Pol II Fraction of Input Vehicle E2 E2 induces recruitment of transcription regulator proteins to the 11p15.5 ICR IgG **

47 Epigenetic repression of CDKN1C through coordinated loop formation with the 11p15.5 ICR Binding of CTCF mediates long range epigenetic silencing through formation of chromatin loop structures 11p15.5 ICR recently shown to physically interact with the CDKN1C promoter in MCF7 cells Estrogen signaling can trigger complex loop formation between cis-regulatory regions and promoters

48 Epigenetic repression of CDKN1C through coordinated loop formation with the 11p15.5 ICR CTCF binding to ICR and CDKN1C locus forms a long-range intrachromosomal loop via dimerization of CTCF Ligand-bound ERα complex may mediate silencing through a secondary loop

49 Epigenetic repression of CDKN1C through coordinated loop formation with the 11p15.5 ICR ERα-mediated loop sequesters upstream tissue- specific enhancers Recruits PRC2 and HDAC1 CTCF serves as scaffold to secure PRC2 complex which methylates H3K27, leading to formation of a repressive chromatin state

50 Summary RNA transcripts perform a diverse set of functions in the cell independent of coding proteins MicroRNAs are promising therapeutic targets Noncoding RNAs play an important role in epigenetic mechanisms of transcriptional silencing Lab exercises: microRNA target prediction and assessing protein coding potential of novel transcripts

51 Laboratory Exercises microRNA target prediction http://mirtar.mbc.nctu.edu.tw/human/index.php Assessing protein coding potential of novel transcripts http://lilab.research.bcm.edu/cpat/

52 microRNA target prediction http://mirtar.mbc.nctu.edu.tw/human/index.php miRTar is an actively updated web-based program Flexible, easy to use interface Experimentally

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54 In this example, we search for miR-148a targets sites in the 3’ UTR of the DNMT3B gene

55 After submitting job, GUI presents input parameters Target linkage between microRNA and gene is represented in separate hyperlinks which retrieve different information microRNA target prediction

56 miRNA icon takes the user to mirBase, a database of multiple-species microRNA sequence, target, and annotation information

57 Gene icon provides detailed information on the gene, miRNA targets on transcripts (mRNA records corresponding to the searched gene), and an overlay of potential alternative splicing events microRNA target prediction

58 Target metrics, graphical and sequence representation

59 This graphical representation connects transcript to protein information on a single screen window microRNA target prediction

60 Searching for the various targets of multiple microRNAs

61 We searched 10 miRNAs versus 69 genes

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63 Choose KEGG to analyze pathway information for let-7a predicted target genes Kyoto Encyclopedia of Genes and Genomes Bioinformatics resource for linking genomes to life and the environment

64 Pathway enrichment analysis for let-7a-predicted targets Tired: no multiple-testing correction

65 Searching for targets of miR-29b which belong to the TGF-beta signaling pathway

66 Produces a summary table with hyperlinks Let us analyze KEGG and see if TGF-beta is the top result?

67 As you can see, your starting list is the most highly enriched result program should probably warning you about this Lists are not necessarily independent of one another Click on the TGF-beta hyperlink to bring up a KEGG pathway map

68 Diagram well summarizes lots of complex information Hyperlinks allow you to access individual gene results, similar to other modes

69 Can upload files of sequences or URLs Select example sequence in FASTA Set species to Human Choose submit

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71 Coding probability predicted to be > 99 % Let’s test mouse Kcnq1ot1! CPAT Output

72 Sequence pasted as BED or FASTA data Coding probability predicted to be < 12 % Our analysis further suggests mouse Kcnq1ot1 is a non- coding transcript


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