AU Studieprojekt: Exploring molecular mechanisms in human disease

Exploring molecular mechanisms in human disease - Using CRISPR/Cas9 knock-out and knock-in technologies to screen and study gene function - - Functions of circular RNAs and their protein binding partners (RBPs) - Damgaard lab technician, 2 post doc, 3 PhD students, on average 2 MSc and 2 BSc Christian K Damgaard ckd@mbg.au.dk Room 404, Building 1130, 29888670 1 RBP1 RBP1 DMPK mRNA Myotonic dystrophy Processing bodies (PBs) Introduction Pre-mRNP 5’TOP-mRNA RBP1 Capping Splicing (snRNPs) hnRNPs AUG Polyadenylation UGA Nuclear membrane An RBP1 mRNP 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. Stress eIFs, eEFs, eRFs etc. Translation mRNA Processing body (PB) ( Localization?) RBP AUG UGA cell cycle arrest nutrient starvation An Regulatory factors RNases PB PB Deadenylation RBP1 PB Accumulation in RNA granules Decay TIA-1 TIA-1 TIAR TIAR TIAR TIAR TIA-1 A TIAR TIA-1 TIAR PB Regulated n RBP2 RBP1 TIAR RISC TIAR mRNA decay A n TIA-1 TIA-1 TIAR TIA-1 TIAR TIA-1 AUG miRNA UGA TIA-1 TIAR Stress granules (SGs) TIA-1 TIAR AAAn TIA-1 TIA-1 TIA-1 TIA-1 RBP1 An Dcp2 Ccr4//Not RBP1 Stress granule (SG) circRNA circular RNA sponge 5’->3’ decay 3’->5’ decay A N N N AUG N N N RBP1 N SG N N Xrn1 Circular RNAs in neurons and myotonic dystrophy Exosome Figure 1: Postranscriptional regulation of gene expression. After a processed mRNA has been exported to the cell cytoplasm it can become regulated by RNA binding proteins (RBPs) 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 regulated 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. circRNAs and RBPs are generally misregulated in disease RBP1 Hypotheses/questions How are circRNAs degraded? Endoclevage Scaffolding? RBP1 Phase separation? circRNA RBP1Protection by RBPs?? RBP Which RBPs regulate mRNA decay or translation? Effect on disease severity/progression?) Stress ? ? RBP ( RBP RBP AUG UGA Which RBPs interact with which circRNAs and what is the consequence at the cellular level, disease level? An Regulation of mRNA decay or translation by circRNAs? circRNA 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 CRISPR/Cas9-knockout CRISPR/Cas9-knock-in Stable inducible cell lines qRT-PCR Northern blotting RNA-seq RIP-seq RNA In Situ Hybridization Flow Cytometry Unstable 0.5 0.0 RPM 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: Examples: 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 Ribotagging Mag bead Figure 4: RNA-sequencing Differential Gene-expression FLAG RBP mRNA Decay Assays Subcellulær fractionation Protein-RNA binding Recombinant protein expression Figure 6: Immunofluorescence revealing co-localization between two proteins residing in processing bodies (PBs). Figure 5: CRISPR/Cas9 (or CRIPSR/Cas12a) used to generate knockouts or knockin cell lines. Figure 7: “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 0% Sucrose 50% Sucrose Zfp●609 ● Zfp●609 ● ● Cdy●l Rmst ●Zfp609 ● ● Cd●yl Tulp4 ● ● ● ● Nfi●x● Tulp4 s3 Nlrp5−ps Kat6● b Rasa2 Zfp827 1 ● ● ● ● ● Y Z AY51● 2917 ● ● Gli ● Ezh2 ● ● ● ● ● Ez●h2 ● ●●●● ● ● ● ● ● ● Nfix ● ● Med13l ● Rmst ● ●● ● ● ● ● ● ● ● ● ● 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 11: Flow cytometry - Click-It EdU proliferation assay Mono Figure 9: Polysome profiling RPM Figure 8: co-immunoprecipitation Figure 10: circRNA expression by RNA-sequencing Bachelor projects Using CRISPR/Cas9 to create knockout cell lines and subsequent functional characterization in cancer or myotonic dystrophy. Knockout or knockin human cell lines Examples: Knockout in cancer cells of various important RNA binding protein genes, followed by translation assays, proliferation assay (longer project: also transcriptome by RNAseq). circRNA abundance? Using CRISPR/Cas9 to create knock-in cell lines and subsequent functional characterization. Examples: Generation of GFP-tagged, immuno-tagged (e.g. FLAG or HA) or degron-tagged endogenous RNA binding proteins. We can then localize and follow proteins in live- cells. Immunoprecipitation to look for interaction partners or bound RNAs. Degron can be induced to remove degron-tagged endogenous protein within 2-3 hours and the immediate impact on cells can be tested (e.g. cancer cell proliferation, migration or invasion) potential. Localization Knockdown of specific RBPs or circRNAs and assay for importance in different established cellular processes. Do circRNAs affect fundamental processes in the cell and are given RBPs important for these? Proliferation