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Approaches Used to Study Protein Involvement in Splicing

Approaches Used to Study Protein Involvement in Splicing. Biochemical Fractionation and Complementation - fractionate extract and assay each fraction for specific property Antibody Studies - immunoinhibition/immunodepletion (restoration)

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Approaches Used to Study Protein Involvement in Splicing

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  1. Approaches Used to Study Protein Involvement in Splicing • Biochemical Fractionation and Complementation • - fractionate extract and assay each fraction for specific property • Antibody Studies • - immunoinhibition/immunodepletion (restoration) • - co-immunoprecipitation (analyze both RNA and proteins) • RNA Binding Assays • - gel mobility shift • - UV crosslinking • - CLIP (crosslinking immunoprecipitation: identify RNA partners) • Analysis of Proteins in Isolated Splicing Complexes • - affinity selection of complexes formed on biotinylated premRNA • - affinity selection of ‘tagged’ splicing factors/ Mass Spectrometry • Methods to Study Protein/Protein Interactions • - Far Western, Co-immunoprecipitation, Yeast Two-hybrid • Genetic Approaches(study splicing-defective yeast mutants)

  2. Biochemical Fractionation snRNPs Protein Splicing Factors

  3. Reconstitution Assay + C NXT = unfractionated nuclear extract Complete = SF1, SF2, SF3, SF4 Conclusion: SF1 and SF3 are required for A complex assembly

  4. RNA + Protein RNA RNA + Protein p90 (non-specific) Protein + RNA fragment RNase p50 (U6-specific)

  5. Crosslinking Immunoprecipitation(CLIP Assays) Method to identify the RNAs that a particular RNA binding protein interacts with in vivo • - UV irradiate cells • Partial RNase digestion • Purify RNP (Ip) • Proteinase K/DNAse • Ligate RNA linkers 5’ + 3’ • Convert RNA to DNA (RT-PCR) • Subclone/Sequence DNAs • Database searching/Identification

  6. RNA Binding Motifs • RNA Recognition Motif (RRM) • -present in many RNA binding proteins (1-4 copies/protein) • - X-ray and NMR structure solved • - motif is ~80 amino acids • - hallmark features: octamer (RNP1) and hexamer (RNP2) • separated by ~ 30 amino acids • - RNP1 and 2 have characteristic array of aromatic amino • acids (Tyr, Phe) and several hydrophobic residues • -fold into a babbabstructure, forming an antiparallel • four-stranded b-sheet packed against two perpendicularly • oriented a-helices which are positioned on one side of the • b-sheet • -RNP1 and RNP2 are on the two central b-strands (b3and b1) • RGG (or GAR) Domain (Arginine and Glycine rich) • Zinc Fingers (CCHH) and Zinc Knuckle (CCHC)

  7. RRM Domain Structure Interaction of RRM From U1A and it’s Stem-loop Binding site (b-sheets form surface for RNA binding RNP1 and 2 contact the looped RNA) N C

  8. RNA BindingProteins GenerallyRecognizeSimple RNASecondaryStructuralElements(rather thandouble-helicalregions)

  9. Affinity Purification of Spliceosomal Proteins RNA Protein (3’ end 32P) (35S Met) Biotinylated premRNA (Ad or Tm) added to splicing extract under splicing conditions Streptavidin beads are then used to purify the complexes formed on biotinylated premRNA Results in co-purifying snRNAs and numerous “SAPs” (Spliceosome Associated Proteins) Method has been used to determine protein factors in different splicing complexes (e.g. E, A, B, C) snRNAs

  10. Mass Spectrometry of Purified Splicing Complexes Purify Splicing Complexes via selection of ‘tagged’ splicing factor Common protein ‘tags’/affinity method -epitope tags (myc, HA, FLAG) / antibodies -poly histidine (e.g. 6x) / nickel chromatography 2. Separate complexes (tagged protein and co-purifying proteins) on SDS gel 3. Excise individual protein bands 4. Digest with trypsin (cuts after Arg/Lys) Identify proteins by mass spectrometry (produces charged particles (ions) and uses electric/magnetic fields to measure mass of peptides) 6. Data base analysis Identify proteins from masses (or sequence) of peptides

  11. Stain 32P Identification of Protein/Protein Interactions Far Western Analysis 32P Autoradiography A) SC35 (an SR splicing factor) is 32P labeled by heart muscle kinase B) Radiolabeled SC35 interacts with a subset of the proteins immobilized on the membrane C) Control to ensure that there is proteins present Coomassie Blue Stain

  12. SC35 U1 70K Identification of Protein/Protein Interactions: Co-Immunoprecipitation Analysis 1 2 3 Antibody: U1 70K 35S-labeled Proteins: Lane 1: U1 70K Lane 2: U1 70K + SC35 Lane 3: SC35 Note: only protein(s) which Interact with antibody are shown Ab Conclusion: U1 70K protein physically interacts with SC35

  13. Identification of Protein/Protein Interactions In vivo: Yeast Two Hybrid Protein Interaction Trap

  14. Two Hybrid Interactions Between Different Splicing Factors

  15. Summary of Role of Protein in Spliceosome Assembly and Catalysis • ATP-dependent Helicases -disrupt RNA/RNA interactions • U2AF (65 and 35 kDa subunits) -interacts with polypyrimidine tract near 3’ splice sites -essential for U2 snRNP interaction at branchpoint -65 kDa subunit (RNA/protein), 35 kDa (protein/protein) • SR Proteins -Domain Structure: N-terminal RRM(s) and C-terminal SR domain often connected by a glycine-rich hinge -Key regulators of splicing (regulated by phosphorylation) -RNA/protein interactions and protein/protein interactions

  16. Domain Structure of Splicing ProteinsRRM andSR Domainsare Prevalent

  17. SR Proteins: ‘Glue’ of the Spliceosome • Cross Exon Recognition • SR proteins bind to exonic splicing enhancers (ESE) and recruit splicing factors • to splice sites (i.e. U1 snRNP to 5’ ss and U2AF to 3’ ss and YYYY tract • Cross Intron Recognition • SR proteins facilitate interactions between U1 snRNP and U2 snRNP and • bound U2AF recruits U2 snRNP to the branch site

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