1. 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)

2. 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

3. 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

4. Exogenous RNA pull down

5. 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.

6. (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)

7. (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

8. 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