22nd Feb - RNABP

Question 1

How was the stepwise assembly of RNA splicing proteins elucidated?

Answer 1

Sedimentation velocity experiments
Native gel electrophoresis
Affinity purification

Question 2

Outline the experiment that used sedimentation velocity to show stepwise assembly of splicing proteins

Answer 2

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

Question 3

Outline the experiment that used native gel electrophoresis to show stepwise assembly of splicing proteins

Answer 3

Heparin was added before loading to displace weakly associated proteins
Longer incubation showed a gradual shift towards heavier bands

Question 4

What is complex H?

Answer 4

A splicing independent complex, closely associated with splicing as they affect the positioning of the splice site corresponding to 22S peak in the gradient

Question 5

How was affinity purification used to identify step wise assembly of the splicing complex?

Answer 5

Combined sedimentation velocity experiment with affinity purification

1. Pre-mRNA tagged with MS2 binding sites
2. Added nuclear extract containing MS2-MBP
3. RNA bound to streptavidin beads; unbound protein washes out
4. Intact complex eluted with maltose

Allowed the identification of the proteins in each fraction

Question 6

What is useful about Gly-rich regions of the protein?

Answer 6

Will adopt whichever secondary structure is favourable therefore are ideal protein interaction domains

Question 7

What are SR proteins?

Answer 7

Argine-serine rich proteins which bind directly to the RNA independent of splice sites

Question 8

What is the structure of SR proteins?

Answer 8

RRM1, RRM2 , RS

Question 9

What is the mechanism of SR proteins?

Answer 9

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

Question 10

What is the function of SR proteins in splicing?

Answer 10

Assists assembly and nuclear export of the mRNA

Question 11

What are nuclear speckles?

Answer 11

Self-assembling aggregations of some splicing factors can be about 50-200 of them in the nucleus

Question 12

What is an RRM?

Answer 12

An RNA binding domain

Question 13

Describe an RRM

Answer 13

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

Question 14

What are UHMs?

Answer 14

U2AF homology motifs - protein interaction domains lacking electropositive potential on sheet and binding proteins via alpha helices

Question 15

What is a KH type domain?

Answer 15

An RNA binding domain

Question 16

Describe a KH RNABD

Answer 16

Based on beta-alpha-alpha-beta-beta-alpha
Slightly smaller than RRMs
Nucleic acids bind to the flexible linker region

Question 17

What is SELEX?

Answer 17

Systematic evolution of ligands by exponential enrichment - a technique to produce ssRNA that specifically binds to target ligands

Question 18

Outline the process of SELEX

Answer 18

1. RNA library made of randomly generated sequences flanked by 3’ and 5’ ends
2. Expand to target ligand - those that do not bind are removed by affinity chromatography
3. RT PCR to make new pool of RNA from the winners
   REPEAT for about 7 cycles

Question 19

Why do aptamers selected by SELEX only show a preference not a specificity to the target?

Answer 19

The nature of the RNABD - phenylalanine will stack against anything

Question 20

What is RNA compete?

Answer 20

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

Question 21

What is CLIP?

Answer 21

Cross linking immunoprecipitation - uses UV to cross link and is an RNA version of ChIP

Question 22

What are the SM core proteins?

Answer 22

Beta sheets which forma ring in which the RNA goes in the middle

Question 23

Which snRNP matures in the nucleolus?

Answer 23

U6

Question 24

Which snRNAs are expressed by PolII?

Answer 24

U1, U2, U4 and U5

Question 25

Where are U1, U2, U4 and U5 matured?

Answer 25

In the cytoplasm

Sm ring forms a cap