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)

Biochemical Fractionation

snRNPs

Protein Splicing Factors

Reconstitution Assay

Conclusion:SF1 and SF3 are requiredfor A complex assembly

NXT = unfractionated nuclear extractComplete = SF1, SF2, SF3, SF4

+ C

p90 (non-specific)

p50 (U6-specific)

RNA +Protein

RNA

Protein +RNA fragment

RNA +Protein RNase

Crosslinking Immunoprecipitation(CLIP Assays)

- 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

Method to identify the RNAs that a particular RNA binding protein interacts with in vivo

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 structure, forming an antiparallel four-stranded -sheet packed against two perpendicularly oriented -helices which are positioned on one side of the -sheet -RNP1 and RNP2 are on the two central -strands (3and1 • RGG (or GAR) Domain (Arginine and Glycine rich)• Zinc Fingers (CCHH) and Zinc Knuckle (CCHC)

RRM DomainStructure

Interaction of RRM From U1A and it’s

Stem-loop Binding site(-sheets form surface for RNA bindingRNP1 and 2 contact the looped RNA)

N C

RNA BindingProteins

GenerallyRecognize

Simple RNASecondaryStructuralElements

(rather thandouble-helical

regions)

Affinity Purification of Spliceosomal Proteins RNA Protein

Biotinylated premRNA (Ad or Tm)added to splicing extract under splicing conditions

Streptavidin beads are then usedto purify the complexes formedon biotinylated premRNA

Results in co-purifying snRNAsand numerous “SAPs”(Spliceosome Associated Proteins)

Method has been used to determineprotein factors in different splicingcomplexes (e.g. E, A, B, C)

snRNAs

(3’ end 32P) (35S Met)

Mass Spectrometry of Purified Splicing Complexes 1. Purify Splicing Complexes via selection of

‘tagged’ splicing factorCommon 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)

5. 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

Identification of Protein/Protein Interactions

Far Western Analysis

A) SC35 (an SR splicing factor) is 32P labeled by heart muscle kinase

B) Radiolabeled SC35 interacts witha subset of the proteins immobilizedon the membrane

C) Control to ensure that there isproteins present

32PStain

32P Autoradiography

Coomassie Blue Stain

Identification of Protein/Protein Interactions:

Co-Immunoprecipitation Analysis

1 2 3 Antibody: U1 70K

35S-labeled Proteins: Lane 1: U1 70KLane 2: U1 70K + SC35Lane 3: SC35

Note: only protein(s) whichInteract with antibody areshown

Conclusion: U1 70K protein physically interacts with SC35

U170K

SC35

Ab

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

Two Hybrid Interactions Between Different Splicing Factors

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

Domain Structure of

Splicing Proteins

RRM andSR Domainsare Prevalent

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