AU Studieprojekt: Exploring post-transcriptional events in human disease

Aarhus Universitet (AU)

Studieprojekt/speciale
Østjylland (Aarhus)

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Exploring post-transcriptional events in human disease Damgaard lab technician, 2 post docs, 3 PhD students, on average 2 MSc and 2 BSc 1 Christian Kroun Damgaard ckd@mbg.au.dk Room 404, Building 1130 29888670 Molekylærbiologi: Ja/Yes Molekylær medicin: Ja/Yes Bioteknologi: Nej/No Introduction The eukaryotic cell possesses numerous gene-regulatory mechanisms to control cell function according to given conditions and environmental cues. These include rapid changes in gene-expression elicited at almost every thinkable level inside the cell - events often deregulated in disease. Historically, much attention has been given to the regulation of transcrip- tion and mRNA processing events, which in turn produce a tremendous diversity from metazoan genes. These important regulated events aside, there is now increasing evidence that cytoplasmic processes, including regulation of both global and local protein translation and mRNA stability are crucial modulatory instruments for the cell during development, cell growth and as primary responses to environmental changes. These pro- cesses are governed by, both RNA-binding proteins (RBPs) and large classes of ncRNAs, including circular RNAs (circRNAs), long non-coding RNAs (lncRNAs) and microRNAs (miRNAs). In my laboratory we study the function of all these types of RNA- and protein regulators in tightly con- trolled translation and mRNA decay and assess how their deregulated function impact diseases like myotonic dystrophy (DM1), Neurodegenera- tive disease and cancer. RBP1 cell cycle arrest nutrient starvation RBP1 RBP1 DMPK mRNA Myotonic dystrophy Pre-mRNP 5 ’TOP-mRNA Capping Splicing (snRNPs) hnRNPs Stress AUG Polyadenylation UGA Nuclear membrane An mRNP AUG UGA TIA An mRNP remodeling? eIFs, eEFs, eRFs etc. TranslationAA mRNA (Localization?) AUG Processing body (PB) UGA AAAn TIA-1 TIAR Regulatory factors TIAR TIA-1ATn IAR TIAR TIAR RNases PB RBP2 RBP1 TIAR TIA-1 TIA-1 TIA-1 TIA-1 TIA-1 TIA-1 TIAR PB TIA-1 TIA-1 lncRNA AUG lncRNA TIAR A n TIAR TIAR TIAR TIAR PB Decay TIA-1 TIA-1 TIA-1 ? TIA-1 DefauAltA An Regulated mRNA decay UGA Stress granule AAAn 3 ’->5’ decay Exosome Ccr4//Not DeadenylationA A AUG UGA Dcp2 Processing bodies (PBs) Stress granules (SGs) Pan2/3 A(PARN) A0 A RISC Decapping Dcp2 AUG miRNA AAAn UGA A Ccr4/Not AUG UGA 5’->3’ decay 3’->5’ decay A A0 ’->5’ decay RBP1 N N N N 3 N AUG N 5’->3’ decay N N Exosome N A N N N circular RNA sponge AUG Xrn1 N N N N N N Exosome Xrn1 RBP1 PB SG Circular RNAs in neuronal differentiation and myotonic dystrophy Figure 1: Postranscriptional regulation of gene expression. After a processed mRNA has been exported to the cell cytoplasm it can become regulated at the level of localization, translation and decay. This is mediated by several regulatory factors including translation- (e.g. green/orange TIA-proteins), decay factors (pacmen) and RNA binding proteins (e.g. “AU-rich binding proteins” (AUBPs)). Current work in the lab is concerned with the regulation of mRNA localization, translation and decay. Many regula- ted mRNAs accumulate in cytoplasmic foci termed processing bodies (PBs) and stress induce accumulation of certain mRNAs i stress granules (SGs). Both granules contain repressed mRNAs but PB tethered mRNAs are thought to undergo rapid decay. Stress granules: mRNA-dependent aggregation of stalled translational complexes (48S) occuring under stress conditions (oxidative stress, UV-irradiation a.o.). TIA-1 and TIAR are essential for SG-aggregation, which occurs via their prion-like domain. Processing Body: mRNA-dependent aggregation of decapping enzyme (hDcp2) and its co-factors: hDcp1a, b, Edc3, Hedls, Lsm4, Rck/p54 among others. ARE-mRNAs, siRNA targets, miRNA targets, NMD substrates accumulate in PBs when decay factors are limiting. CUG-expanded DMPK mRNA in DM1 1 2 3 4 5 PB 0 2 4 8 20 Methods SG/PB SG Chase/hrs Control 0 2 4 8 20 2.5 2.0 1.5 1.0 (n=13) (n=16) (n=33) (n=9) (n=29) Control Stable Mammalian cell culture RNAi Unstable 0.5 0.0 RPM CRISPR/Cas9 -knockout/knock-in Stable inducible cell lines qRT-PCR Northern blotting RNA-seq RIP-seq RNA In Situ Hybridization Flow Cytometry 0.6 Ezh2 (chr6:47576562−47577667) 0.4 Zfp827 (chr8:79118174−79136663) Ezh2 (chr6:47576535−47577667) Figure 3: mRNA pulse-chase decay assay. Reporter mRNA expression is pulsed/chased. 0.2 0 Figure 2: RNA Fluroescent In Situ Hybridization (RNA-FISH) for visualizing “stress granules” (SGs), “processing bodies” (PBs) or sequestered nuclear mRNPs from patients with myotonic dystrophy (DM1). Slc8a1 (chr17:81647808−81649638) Ankib1 (chr5:3747020−3772787) Nfix (chr8:84771783−84772315) Hdgfrp3 (chr7:81893798−81905636) Rmst (chr10:92075169−92136690) Magi1 (chr6:93792346−93815825) Tulp4 (chr17:6137210−6139156) Input IP NanoString analysis Immunofluorescence Western blotting (fluorescence) Protein/RNA co-localization Protein Immunoprecipitation RNA Immunoprecipitation Polysome fractionation mRNA Decay Assays Subcellulær fractionation Protein-RNA binding Recombinant protein expression Mag bead Figure 4: RNA-sequencing Differential Gene-expression FLAG RBP Figure 5: Immunofluorescence revealing co-localization between two proteins residing in processing bodies (PBs). Figure 6: “mRNA Immunoprecipitation” (RIP). Northern/qRT-PCR/RNA-seq. Input IP mESC mNPC mN8 Sucrose gradient ● ● ● 1 0 0 0 0 .00 .75 .50 .25 X Cytoplasmic lysate Zfp●609 ● Zfp●609 ● ● Cdy●l Rmst ●Zfp609 ● ● Cd●yl Tulp4 ● ● ● ● Nfi●x● Tulp4 s3 Nlrp5−ps Kat6● b Rasa2 Zfp827 1 0% Sucrose ● ● ● ● ● Y Z AY51● 2917 ● ● Gli ● Ezh2 ● ● ● ● ● Ez●h2 ● ●●●● ● ● ● ● ● ● Nfix ● ● Med13l ● Rmst ● ●● 50% Sucrose ● ● ● ● ● ● ● ● ● ● Med13l Srgap1 ● ● ● ● M●apk●4 Ma● gi1 ● ● E● zh2 ● ● ● ● ● ● ● ● ● ● Elf2 ● ● ● ● ●● ●● A●n●k●s●1b Aff3 ● ●● ● ● ● ● ● ● Glis3 ● ● ● ● NrxnS1lc8a1 ● ●● ● ● ●●●●Lr●c●●h● ●3● ● Zfp827 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● a●t6●b● ●Hipk3 ● ● ● ●● ● ● ● K ● Lrch3 ● ●● ● ●● ●● ●● ● ●● ●●●●● ●●●● E●●lf2 ● ● ●A● nk● ib1 ● Med13l ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● Mb●o●●at2 ● GigyMf2agi1 ● ● ● ● ● ● ● M● boat2 ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ●● ● ● ● ● Ankib1 ● ● ● P ● ● ● ● ● ●● ● ● ● ● ●● ● Monosomes Polysomes ●● ● ● ●● ● ● ● Kat6b ● ● ● ● ● ● ● Zfp● 148 ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ●●● ●● ●●●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●●● ●● ● ● ● ● ● ●● ●● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●●● ●●●●● ● ● ● ● ● ● ● ● ●●●● ● ●● ● ●● ● ●● ● ● ●● ● ● ●●●●●●●●●● ●● ● ● ●● ●● ● ● ● ● ●● ●●●● ● ● ● ● ●●● ● ● ● ● ●●● ● ●●● ● ● ● ● ●● ● ●● ● ● ●●●● ● ● ● ● ● ● ● ● ●● ●● ●● ●●●●●●●● ● ●●● ●●●● ● ● ●●●●●●●● ● ●● ●● ●● ●● ● ● ● ● ● ●● ●● ●● ● ● ●●● ● ● ● ●●● ●●● ● ●● ● ● ● ● ● ●●●● ● ●●●●● ●● ● ●● ● ● ● ● ● ● ● ● ● ●● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ●● ● ● ● ●●●●● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●● ● ● ●●●●● ● ●●●● ●● ● ● ● ● ● ●●●●●●●●●● ●●● ●●●● ●●●● ● ● ● ● ● ● ● ●●●●●● ● ● ● ●●● ● ● ● ● ● ●● ● ●● ●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ●●● ●●●● ●● ● ● ● ●●●● ●● ● ● ● ●●●●●●● ●●● ●●●● ● ● ●● ● ● ● ●●●● ● ● ● ● ●● ● ●●●●● ●●● ● ● ●●●● ●●● ● ●●● ● ● ●●● ●● ● ● ● ●● ● ●● ●● ●●● ● ● ● ● ●●●● ● ● ● ● ● ● ● ●●● ●●● ●●●● ● ●● ● ● ● ●● ●● ●●●●●●●●●●●●● ● ● ●● ●●●●●● ●●● ●●●●●●● ●● ●●●● ●●●●● ● ● ● ● ● ● ●● ● ● ● ● ● ●●●●●●●●●● ● ●●●●●●●●● ●● ● ●●●● ●●●● ●● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ●●● ●● ●●● ● ●●●●● ● ● ● ●●●●●● ●●● ● ● ● ● ● ● ● ●●●●●●●● ● ● ● ●● ●●● ● ● ●●●●● ● ● ●●●●● ● ● ● ● ● ● ●●● ● ● ●● ● ● ●●●● ●● ●●●●●●●● ●●●●●●●●●●●●● ●●● ● ● ● ● ● ●●● ● Poly ● ● ● ● ● ●●●● ● ● ●●●●●●● ● ●●●● ●●●●●●● ● ●● ●● ●●●●● ●● ● ●● ● ● ●● ● ●● ● ●●● ● ●●●● ● ●● ●●●●● ●● ●● ●●●● ● ●●● ●● ●●●●●●● ● ● ●●●● ●●●●●●●●●●●●●●●● ● ● ● ● ●●●●● ● ●● ● ● ●● ●● ●● ● ● ●● ● ● ● ● ● ●●●●●●● ● ●● ●●●● ● ● ● ● ●●● ● ● ● ● ● ● ●●●●● ●●●●●●●●● ● ● ● ● ● ● ● ●●●●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●●● ●●●●●●●●●●● ●● ● ● ● ● ● ● ● ● ●●●●●●●●●● ● ● ● ● ●● ●●●●●●●●●●●●● ● ● ● Q ● ● ●● ● ● ● ●● ● ●●●●●●●● ● ● ● ● ● ● ● ● ● ● ● ●●●● ● ● ● ● ● ●●● ●● ●● ● ● ● ●● ● ●●● ● ● ● ●● ●● ● ● ● ●●● ● ● ●● ● ● ● ● ● ● .00 0 .01 0.10 1.00 0.01 0.10 1.00 0.01 0.10 1.00 Figure 10: Flow cytometry - Click-It EdU proliferation assay Mono Figure 8: Polysome profiling RPM Figure 7: co-immunoprecipitation Figure 9: circRNA expression by RNA-sequencing Myotonic dystrophy type 1 - DDX6 and circRNAs Projects TF 1 ) Studying mechanisms regulating neuronal diꢀerentiation - do circular RNAs play a role? Transcription factors: Sequestration? Disease-related changes Patient fibroblasts CUG- BP1 ds-RNA: Activation hnRNPs/DDX5/17 (Transcription) CUG- BP1 of PKR binding/upregulation (Splicing) (Global translation) Project 1: Project 2: Project 3: Project 4: RIP using RBP or tag (FLAG or Streptag) speciꢁc antibody - detection of candidate RNAs or RNA-seq followed by functional study. Eꢀect of manipulating with RBP levels (knockdown/out or overexpression)? Functional studies of RBP/sponge interactions in neuronal cells using cellular imaging and biochemical approaches. RNA-FISH, SUNTAG-FISH orimmunoꢂuorescence Speciꢁc knockdown or overexpression of candidate circRNA or RBP and assess functional impact on cell biology and gene-expression. CUG-BP1: Binding/upregulation Splicing) hnRNPhnRNP F ( G C MBNL1: H U U Sequestration C G G CU (Splicing, mRNA decay, U MBNL1 C G Antisense transcription miRNA/RNAi response) polyadenylation) G C U C G U G C ( U U C G C G DDX5 DDX17 DDX5: Unwinding? Non-ATG translation polyglutamine peptides cytoplasm (Toxicity) U U C G G C MBNL1 U U C G C G Stimulation of MBNL1 binding (Splicing) MBNL1 U U C G !" #$% &' DDX17 G M C UBNL1 G C U MBNL1 C G Upf1 U U G Identiꢁcation of the circRNA interactome by RNA pull-down followed by Mass Spectrometry (MS) C DDX5 MBNL1 G C UDDX6U C G C Upf1: Steady-state and foci dynamics mRNA decay - NMD?) G MBNL1 MBNL1MBNL1 U U C G C G ( U U G C C G 2 ) Studying mechanisms regulating gene expression during cellular stress U U G C DDX6 MBNL1 C G Stau1 Stau1 U U C G G C Patient muscle + Differentiation-related changes U U Stau1:Binding/upregulation C G C U DDX6: Binding/unwinding? Stripping off MBNL1? (Splicing/nuclear export) G ( Splicing/nuclear export/translation U C G G C Project 1: Identiꢁcation and functional characterization of genes aꢀected by LARP1 knockout. Compare RNA-seq, RIP-seq and CLIP data to identify direct and indirect eꢀects. mRNA decay - SMD?) Figure 10: Molecular mechanisms in DM1. Project 2: Project 3: CRISPR/Cas9 mediated introduction of degron in LARP1 and TIA/R proteins to facilitate fast depletion (2-4 hrs). Functional analyses of mass-spec identiꢁed LARP1/TIA-1/TIAR interaction partners using global and biochemical assays. BGH p(A) NeoR SV40 p(A) ( CTG)262 eGFP-CTG262 CMV CMV eGFP eGFP SV40 SV40 SV40 p(A) eGFP-CTG5 BGH p(A) NeoR (CTG)5 5CUG 262CUG 3 ) Studying mechanisms deregulating gene-expression in Myotonic Dystrophy type 1/2 (DM1 and DM2) Project 1: Project 2: Using existing circRNA inventory of DM1 and DM1 to manipulate circRNA levels followed by cell biological assays ( proliferation, apoptosis, muscular diꢀerentiation) and biochemical assays (gene-expression, splicing or translation). Deꢁning degradation speciꢁcity of DM1 mutated RNA - involvement of the exosome and targeting complexes. Figure 12: DDX6 knockdown increases DMPK mRNA aggre- gation in patient cells. Figure 13:Differential expression of circRNAs in C2C12 cells with or with disease-related CUG expansions. Figure 11: Creation of model cell line containing CTG repeats and transgene expression levels


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Aarhus Universitet (AU)
Aarhus Universitet (AU)
Aarhus Universitet blev grundlagt i 1928 og er i dag i den absolutte verdenselite på flere forskningsfelter. Aarhus Universitet (AU) er blandt verdens 10 bedste universiteter grundlagt inden for de seneste 100 år og har en lang tradition for partnerskaber med nogle af verdens bedste forskningsinstitutioner og universitetsnetværk.

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