22nd Feb - RNABP Flashcards
How was the stepwise assembly of RNA splicing proteins elucidated?
Sedimentation velocity experiments
Native gel electrophoresis
Affinity purification
Outline the experiment that used sedimentation velocity to show stepwise assembly of splicing proteins
Sedimentation velocity is roughly proportional to the molecular mass of the complex, using a density gradient of sucrose to prevent pancaking. The RNA analysed showed a wave of sedimentation indicating step wise assembly, i.e. with increasing time of incubation the weight of the RNA increased
Outline the experiment that used native gel electrophoresis to show stepwise assembly of splicing proteins
Heparin was added before loading to displace weakly associated proteins
Longer incubation showed a gradual shift towards heavier bands
What is complex H?
A splicing independent complex, closely associated with splicing as they affect the positioning of the splice site corresponding to 22S peak in the gradient
How was affinity purification used to identify step wise assembly of the splicing complex?
Combined sedimentation velocity experiment with affinity purification
- Pre-mRNA tagged with MS2 binding sites
- Added nuclear extract containing MS2-MBP
- RNA bound to streptavidin beads; unbound protein washes out
- Intact complex eluted with maltose
Allowed the identification of the proteins in each fraction
What is useful about Gly-rich regions of the protein?
Will adopt whichever secondary structure is favourable therefore are ideal protein interaction domains
What are SR proteins?
Argine-serine rich proteins which bind directly to the RNA independent of splice sites
What is the structure of SR proteins?
RRM1, RRM2 , RS
What is the mechanism of SR proteins?
Phosphorylation of RS1 sends ASF/SF2 to the nucleus via a transporter where it translocates to nuclear speckles.
RS2 domain is phosphorylated for splicing and released from the nuclear speckle. When splicing is finished it is dephosphorylated and leaves the nucleus taking the spliced mRNA with it
What is the function of SR proteins in splicing?
Assists assembly and nuclear export of the mRNA
What are nuclear speckles?
Self-assembling aggregations of some splicing factors can be about 50-200 of them in the nucleus
What is an RRM?
An RNA binding domain
Describe an RRM
They have a beta sheet - alpha helix -beta sheet - beta sheet- alpha helix - beta sheet arrangement
Aromatic amino acids stack up against the bases providing strength to the interaction but does not affect specificity
What are UHMs?
U2AF homology motifs - protein interaction domains lacking electropositive potential on sheet and binding proteins via alpha helices
What is a KH type domain?
An RNA binding domain
Describe a KH RNABD
Based on beta-alpha-alpha-beta-beta-alpha
Slightly smaller than RRMs
Nucleic acids bind to the flexible linker region
What is SELEX?
Systematic evolution of ligands by exponential enrichment - a technique to produce ssRNA that specifically binds to target ligands
Outline the process of SELEX
- RNA library made of randomly generated sequences flanked by 3’ and 5’ ends
- Expand to target ligand - those that do not bind are removed by affinity chromatography
- RT PCR to make new pool of RNA from the winners
REPEAT for about 7 cycles
Why do aptamers selected by SELEX only show a preference not a specificity to the target?
The nature of the RNABD - phenylalanine will stack against anything
What is RNA compete?
A single competitive binding reaction in which an RBP is incubated with a vast excess of a complex pool of RNA. Then protein is recovered by affinity selection. Associated RNAs are then interrogated by microarray and computational analyses
What is CLIP?
Cross linking immunoprecipitation - uses UV to cross link and is an RNA version of ChIP
What are the SM core proteins?
Beta sheets which forma ring in which the RNA goes in the middle
Which snRNP matures in the nucleolus?
U6
Which snRNAs are expressed by PolII?
U1, U2, U4 and U5
Where are U1, U2, U4 and U5 matured?
In the cytoplasm
Sm ring forms a cap
