22nd Feb - RNABP Flashcards

1
Q

How was the stepwise assembly of RNA splicing proteins elucidated?

A

Sedimentation velocity experiments
Native gel electrophoresis
Affinity purification

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is complex H?

A

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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are SR proteins?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the structure of SR proteins?

A

RRM1, RRM2 , RS

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What is the mechanism of SR proteins?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What is the function of SR proteins in splicing?

A

Assists assembly and nuclear export of the mRNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What are nuclear speckles?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is an RRM?

A

An RNA binding domain

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Describe an RRM

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What are UHMs?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is a KH type domain?

A

An RNA binding domain

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Describe a KH RNABD

A

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

17
Q

What is SELEX?

A

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

18
Q

Outline the process of SELEX

A
  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
19
Q

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

A

The nature of the RNABD - phenylalanine will stack against anything

20
Q

What is RNA compete?

A

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

21
Q

What is CLIP?

A

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

22
Q

What are the SM core proteins?

A

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

23
Q

Which snRNP matures in the nucleolus?

A

U6

24
Q

Which snRNAs are expressed by PolII?

A

U1, U2, U4 and U5

25
Q

Where are U1, U2, U4 and U5 matured?

A

In the cytoplasm

Sm ring forms a cap