Profiling of non-coding RNA classes Gunter Meister RNA Biology Regensburg University Universitätsstrasse 31 93053 Regensburg
Overview Classes of non-coding RNAs Profiling strategies Validation Protein-RNA interactions
Non-coding RNAs small non-coding RNAs (18-35 nt in length) long non-coding RNAs (> 50 nt in length)
Non-coding RNAs small non-coding RNAs (18-35 nt in length) short interfering RNAs (sirnas) micrornas pirnas
Small RNA-guided post-transcriptional gene silencing
microrna-guided post-transcriptional gene silencing
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
Structure of the A.fulgidus Argonaute protein Patel et al., 2007, Cold Spring Harb. Symp. Quant. Biol. 71 Wang et al. Nature, 2009
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
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
microrna-guided post-transcriptional gene silencing Huntzinger and Izaurralde, 2011, Nature Reviews Genetics
microrna-guided post-transcriptional gene silencing Huntzinger and Izaurralde, 2011, Nature Reviews Genetics
Non-coding RNAs small non-coding RNAs (18-35 nt in length) short interfering RNAs (sirnas) micrornas pirnas
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
pirna function
Small RNA profiling strategies microarray analysis PCR-based strategies cloning and sequencing in situ hybridization
Microarray technology Planell-Saguer et al. 2011
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
Small RNA profiling strategies microarray analysis PCR-based strategies cloning and sequencing in situ hybridization
mirna-qpcr technologies miscript mirna PCR System (Qiagen)
Stem-loop-primer system mirna-qpcr technologies
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
Small RNA profiling strategies microarray analysis PCR-based strategies cloning and sequencing in situ hybridization
Small RNA cloning srnas from IP or total RNA Size fractionation 5 P OH 3
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) 200 150 100 75 50 35 25 20 after cut 12% PAA gel Anne Dueck
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
Small RNA cloning DNA library Ctrl. 30min DNA library +OT 30min 200 150 srnas 100 75 50 after cut 6% PAA gel Anne Dueck
Small RNA cloning Anne Dueck
Small RNA cloning Anne Dueck
mirna expression in glioblastoma stem-like cells Schraivogel et al. EMBO J, 2011
mirna expression in glioblastoma stem-like cells C Schraivogel et al. EMBO J, 2011
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.)
Small RNA profiling strategies microarray analysis PCR-based strategies cloning and sequencing in situ hybridization
mirna in situ hybridization Pena et al. Nature Methods, 2009
mirna in situ hybridization LNA-probes EDC cross linking in fixed tissue Pena et al. Nature Methods, 2009
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
Overview Classes of non-coding RNAs Profiling strategies Validation Protein-RNA interactions
Northern blotting with EDC cross-linking
mirna identification by Northern blotting pre-mirna mirna Anne Dueck
mirna identification by Northern blotting Anne Dueck
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.
Non-coding RNAs Rinn and Chang, Annual Reviews Biochemistry, 2012
Long non-coding RNAs X chromosome inactivation
Long non-coding RNAs X chromosome inactivation Gagnon and Corey, Nucleic Acid Therapeutics, 2012
Long non-coding RNAs Rinn and Chang, Annual Reviews Biochemistry, 2012
lncrna profiling strategies microarray analysis PCR-based strategies cloning and sequencing in situ hybridization Northern blot for validation
lncrna MALAT-1 Daniele Hasler
Alu RNAs Daniele Hasler
Overview Classes of non-coding RNAs Profiling strategies Validation Protein-RNA interactions
Biochemically purified Ago protein complexes proteincomponents small RNAs associated mrnas
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
Target identification
Ago1-bound mrna analysis Ago1 Flag Ago1 Flag BL41 Jjoye
microrna-inhibition
Identification of potential mir-9* target mrnas (Beitzinger et al., 2007, Easow et al. 2007, Karginov et al. 2007)
Identification of potential mir-9* target mrnas Schraivogel et al. EMBO J, 2011
CAMTA1 is a target of mir-9 and mir-9*
Extended RNA interaction networks involving many classes of non-coding RNAs as well as proteins
Small RNAs
Small RNAs
RNA-protein interaction profiling HITS-CLIP PAR-CLIP
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
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
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
Acknowledgements Laboratory of RNA Biology University of Regensburg