Profiling of non-coding RNA classes Gunter Meister

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1 Profiling of non-coding RNA classes Gunter Meister RNA Biology Regensburg University Universitätsstrasse Regensburg

2 Overview Classes of non-coding RNAs Profiling strategies Validation Protein-RNA interactions

3 Non-coding RNAs small non-coding RNAs (18-35 nt in length) long non-coding RNAs (> 50 nt in length)

4 Non-coding RNAs small non-coding RNAs (18-35 nt in length) short interfering RNAs (sirnas) micrornas pirnas

5 Small RNA-guided post-transcriptional gene silencing

6 microrna-guided post-transcriptional gene silencing

7 Argonaute proteins Ago proteins in different organisms - highly conserved between species - many organisms encode multiple Ago proteins -Ago proteins have a molecular weight of about 100 kda - characterized by PAZ and PIWI domains (PPD proteins) S. pombe 1 D. melanogaster 5 C. Elegans 27 Mammals 8 PAZ-domain structure Ma, J. et al. Nature, 429, 2004

8 Structure of the A.fulgidus Argonaute protein Patel et al., 2007, Cold Spring Harb. Symp. Quant. Biol. 71 Wang et al. Nature, 2009

9 Argonaute proteins Ago proteins in different organisms - characterized by PAZ and PIWI domains (PPD proteins) - PAZ domains anchor the 3 ends of small RNAs S. pombe 1 D. melanogaster 5 C. Elegans 27 Mammals 8 - PIWI domain is structurally similar to RNase H and can cleave complementary RNAs - The MID domain anchors the 5 end of the small RNA - Ago proteins specifically bind small RNAs and are key-players in small RNA guided gene silencing

10 The human Argonaute protein family AGO-family, ubiquitously expressed PIWI-family, expressed in germ-line, associate with pirnas - ~ 80% sequence homology between human Ago1-4 - hago2: RNase-H-like Slicer subunit of RISC, the only human Argonaute protein mediating target cleavage

11 microrna-guided post-transcriptional gene silencing Huntzinger and Izaurralde, 2011, Nature Reviews Genetics

12 microrna-guided post-transcriptional gene silencing Huntzinger and Izaurralde, 2011, Nature Reviews Genetics

13 Non-coding RNAs small non-coding RNAs (18-35 nt in length) short interfering RNAs (sirnas) micrornas pirnas

14 pirna function interaction with Piwi-proteins (pirna = Piwi-interacting RNAs) control mobile genetic elements in the germline most likely by DNA-methylation important for synaptic plasticity in Aplysia

15 pirna function

16 Small RNA profiling strategies microarray analysis PCR-based strategies cloning and sequencing in situ hybridization

17 Microarray technology Planell-Saguer et al. 2011

18 Microarray technology Pros: fast and easy to handle different variations on the market (LNA-based etc.) uncomplicated data analysis Cons: not quantitative low selectivity and specificity (short length of small RNAs) no de novo identification of small RNAs

19 Small RNA profiling strategies microarray analysis PCR-based strategies cloning and sequencing in situ hybridization

20 mirna-qpcr technologies miscript mirna PCR System (Qiagen)

21 Stem-loop-primer system mirna-qpcr technologies

22 mirna-qpcr technologies Pros: fast and easy to handle small RNA amounts can be used uncomplicated data analysis quantitative Cons: highly sensitive method. Pipetting errors for low copy numbers stem-loop-primer PCR is rather expensive no de novo identification of small RNAs

23 Small RNA profiling strategies microarray analysis PCR-based strategies cloning and sequencing in situ hybridization

24 Small RNA cloning srnas from IP or total RNA Size fractionation 5 P OH 3

25 Small RNA cloning Marker radioactive RNA-Marker Total RNA Ctrl. 30min (10µg) Total RNA +OT 30min (10µg) Total RNA Ctrl. 30min (10µg) Total RNA +OT 30min (10µg) after cut 12% PAA gel Anne Dueck

26 Small RNA cloning srnas from IP or total RNA Size fractionation 5 P OH 3 5 P 3 T4 RNA Ligase truncated App 3 5 Adenylation 5 P 3 5 OH OH 3 T4 RNA Ligase 1 +ATP 5 3 RT-PCR

27 Small RNA cloning DNA library Ctrl. 30min DNA library +OT 30min srnas after cut 6% PAA gel Anne Dueck

28 Small RNA cloning Anne Dueck

29 Small RNA cloning Anne Dueck

30 mirna expression in glioblastoma stem-like cells Schraivogel et al. EMBO J, 2011

31 mirna expression in glioblastoma stem-like cells C Schraivogel et al. EMBO J, 2011

32 Small RNA cloning Pros: de novo small RNA identification small amounts of RNA can be used very cost efficient in conjunction with state-of-the-art sequencing platforms (5 Mio reads are sufficient for one library approx. 100 ) quantitative Cons: rather difficult cloning procedure (accessibility of the small RNA ends) data analysis requires bioinformatic support cloning biases (ligation, PCR etc.)

33 Small RNA profiling strategies microarray analysis PCR-based strategies cloning and sequencing in situ hybridization

34 mirna in situ hybridization Pena et al. Nature Methods, 2009

35 mirna in situ hybridization LNA-probes EDC cross linking in fixed tissue Pena et al. Nature Methods, 2009

36 mirna in situ hybridization Pros: detection of small RNAs in tissues can be used for paraffin-embedded tissues Cons: still not very reliable (especially for low abundant mirnas) not yet quantitative not useful for high-throughput profiling no de novo identification of non-coding RNAs

37 Overview Classes of non-coding RNAs Profiling strategies Validation Protein-RNA interactions

38 Northern blotting with EDC cross-linking

39 mirna identification by Northern blotting pre-mirna mirna Anne Dueck

40 mirna identification by Northern blotting Anne Dueck

41 Non-coding RNAs long non-coding RNAs (18-35 nt in length) classical non-coding RNAs (trnas, rrnas, snrnas, snornas, 7SL, 7SK RNAs lincrnas pseudogenes, Alu RNAs etc.

42 Non-coding RNAs Rinn and Chang, Annual Reviews Biochemistry, 2012

43 Long non-coding RNAs X chromosome inactivation

44 Long non-coding RNAs X chromosome inactivation Gagnon and Corey, Nucleic Acid Therapeutics, 2012

45 Long non-coding RNAs Rinn and Chang, Annual Reviews Biochemistry, 2012

46 lncrna profiling strategies microarray analysis PCR-based strategies cloning and sequencing in situ hybridization Northern blot for validation

47 lncrna MALAT-1 Daniele Hasler

48 Alu RNAs Daniele Hasler

49 Overview Classes of non-coding RNAs Profiling strategies Validation Protein-RNA interactions

50 Biochemically purified Ago protein complexes proteincomponents small RNAs associated mrnas

51 Experimental target mrna identification Nature Methods, 2007 mirna target mrnas have been predicted bio-informatically mirnas associate with partially complementary target sites prediction of targets is therefore difficult algorithms predict targets genome-wide. Only limited information on specific cell types or tissues

52 Target identification

53 Ago1-bound mrna analysis Ago1 Flag Ago1 Flag BL41 Jjoye

54 microrna-inhibition

55 Identification of potential mir-9* target mrnas (Beitzinger et al., 2007, Easow et al. 2007, Karginov et al. 2007)

56 Identification of potential mir-9* target mrnas Schraivogel et al. EMBO J, 2011

57 CAMTA1 is a target of mir-9 and mir-9*

58 Extended RNA interaction networks involving many classes of non-coding RNAs as well as proteins

59 Small RNAs

60 Small RNAs

61 RNA-protein interaction profiling HITS-CLIP PAR-CLIP

62 CLIP CLIP = Crosslinking and Immunoprecipitation HITS-CLIP = High-throughput sequencing of RNAs-CLIP PAR-CLIP = Photoactivatable Ribonucleoside enhanced -CLIP Chi et al., 2009 Hafner et al., 2010

63 PAR-CLIP - Treatment of HEK293T cells with 4SU - UV-crosslink at 365nm - IP of endogenous Ago2 - Isolation of associated mirnas/mrnas Chi et al., 2009 Hafner et al., 2010

64 summary non-coding RNAs can be profiled with various methods it is very important to validate the results with at least one different method Northern blots are the most direct way of validation and should be used RNA-protein interactions are analyzed by CLIP

65 Acknowledgements Laboratory of RNA Biology University of Regensburg

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