Molecular techniques for the study of the interaction between…….. RNA-Protein: RNA pull down Protein-RNA: RIP (RNA immunoprecipitation) Protein-RNA: CLIP (Cross-Linked RNA immunoprecipitation)

AIM: Identification of the protein interactors of an RNA Endogenous RNA pull down AIM: Identification of the protein interactors of an RNA ? Nuclear and Cytoplasmic RNAs Exogenous (in vitro) vs Endogenous (in vivo) RNA pull down The amount of cellular extract depends by the abundance of the RNA

Endogenous RNA pull down ? RNA PROTEINS Magnetic streptavidin beads Biotinilated Oligos residuo di biotina legato ad un UTP in posizione 5’ qRT PCR (or RNA-seq) WB (or Masspec) Probe Design 2 Collect cell extract 3. Binding step 4. Introduction of Streptavidin-magnetic beads 5. Pull down 6. Protein and RNA analysis

Exogenous RNA pull down

Protein-RNA interaction (RNA immunoprecipitation) RIP & CLIP (RNA immunoprecipitation) RNA Immunoprecipitation (RIP) is a powerful technique allowing the in vivo characterization of the physiological RNA targets of a given RNA-binding protein. Like ChIP, RIP is based on in vivo formaldehyde fixation of tissues or cells. Formaldehyde creates intermolecular and reversible bridges between biological components such as RNA and proteins. Therefore it fixes the physiological in vivo interactions between RNA- binding proteins and their RNA targets. Moreover it prevents the re-arrangement of RNA-protein complexes that occurs during lysate preparation. Cross-Linking and Immuno-Precipitation (CLIP) is a powerful technique allowing the in vivo characterization of the physiological binding site(s) of a given RNA-binding protein. CLIP is based on in vivo cross-linking of tissues or cells by UV irradiation. Unlike RIP, CLIP specifically characterizes direct protein-RNA interactions because UV irradiation only creates molecular bridges between proteins and nucleic acids that are in direct contacts. In addition, CLIP allows the characterization of the binding site of a given RNA-binding protein because it contains an additional step in which the RNAs are partially degraded with Rnases. This step leads to the formation of RNA-protein complexes containing only small RNA tags of 70 nucleotides comprising the binding site of the protein. Like RIP, CLIP requires specific and high-affinity antibodies to recover RNA-protein complexes containing a given RNA-binding protein by denaturing immunoprecipitation. Finally, the specific RNA targets of a given RNA protein are recovered and will be characterized by large scale cloning.

(RNA Immunoprecipitation) RIP (RNA Immunoprecipitation) AIM: Identification of the RNAs bound to a known Protein RBP ? Cytoplasmic or Nuclear extract Isolation of Ribonucleoprotein complexes The resulting data have a low resolution, also not directly associated RNAs could be immunoprecipitated, and the binding site in the co-purified RNA molecule remained unresolved. Variants: CLIP (UV-RIP) PAR-CLIP HITS-CLIP (CLIP-seq)

(RNA Immunoprecipitation) RIP (RNA Immunoprecipitation) Lysis Cells and collect cell extract 2 Prebinding between AntiBody and Beads 3. Introduction of cellular extract (Binding step) 4. Wash and Purification of RNA-protein complexes

CLIP (UV-RIP) 1. UV crosslink Cells or Tissue 2. Partial RNA digestion 3. Immunoprecipitate RBP and phosphorylate RNA 5’ end 4. Ligate the 5’ RNA adapter 5. Dephosphorylate RNA 3’ end 6. Purify RBP-RNA on SDS-PAGE 7. Digest the RBP 8. Ligate the 3’ RNA adapter 9. Purify RNA on urea-TBE gel 10. Reverse transcription 11. PCR 12. Illumina paired-end sequencing