Eukaryotic RNA Splicing

Question 1

Where does transcription and translation take place?

Answer 1

Transcription = nucleus
Translation = cytoplasm

Question 2

What does RNA modification consist of?

Answer 2

5’ capping and 3’ polyadenylation

And splicing of introns and exon joining

Question 3

What is the process of splicing?

Answer 3

Splicing is cutting out introns (intruders) and joining of exo s

Question 4

Where are Eukaryotic mRNAs processed and spliced ?

Answer 4

In the nucleus

Question 5

What are the consensus sequences in the intron?

Answer 5

5’ (GU) and 3’ (AG)

Question 6

In what way does splicing work?

Answer 6

Pairwise fashion

Question 7

What would pairing wrong junctions result in?

Answer 7

It will result in the removal of exons by mistake

Question 8

What is the result of the spliceosome splices out the pair by “skipping”?

Answer 8

Alternative splicing - masked then we have transcripts missing

Question 9

Describe the 2 steps of splicing

Answer 9

1. • 5’ exon cleaved off
   • 2’ OH of intron A at branch
     site ATTACKS phosphodiester
     Bond at the 3’ exon/intron junction
     and joins 5’P of intron “G” forming another
     phosphodiester Bond to form a LARIAT
2. • 3’ OH of 5’ exon ATTACKS bond at 3’
     intron/exon junction.

This allows another phosphodiester Bond to form between the 2 exon and releasing of intron.

Question 10

What term is used to describe phosphodiester bonds transferred from one location to another?

Answer 10

Transesterification reactions and there are 2 of them.

Question 11

What does the spliceosome consist of?

Answer 11

A large complex composed of protein and snRNAs.

Question 12

How many snRNPs are in cloves in splicing and what are their names?

Answer 12

5 snRNPs - U1, U2, U4, U5, U6

Question 13

What does each snRNP consist of?

Answer 13

Each snRNP contains one snRNA + some associated proteins that make up the spliceosome.

+ about 70 splicing Factor proteins (not part of snRNPs)

Question 14

What is the main job of the snRNAs?

Answer 14

Help position spliceosome to make the right cleavages and bind in the right spot

Question 15

What is important in splicing to orient the spliceosome?

Answer 15

snRNA base pairing

Question 16

What do U5 loops interact with?

Answer 16

U5 is close to both Exxon’s and U5 loops interact with both Exons

Question 17

What do RNAs look like structurally?

Answer 17

RNAs have a secondary structure (fold up on themselves)

Question 18

Which snRNPs pair with 5’splice site?

Answer 18

U1 and U6

Question 19

What does U2 pair with?

Answer 19

Branch site

Question 20

During spliceosome assembly, what happens?

Answer 20

U1,U2, U4,U5, U6 all Bind and you get REARRANGEMENT and catalyze splicing

Question 21

What is the function of BBP (branch point binding protein)?

Answer 21

Binds branch point.

Question 22

U1 is recruited by ? To bind to 5’ splice site GU?

Answer 22

Recruited by RNA pol II

Question 23

What is U2AF

Answer 23

U2AF - a complex of 2 subunits (comes and goes) - one of the 70 accessory proteins

Question 24

Where does U2AF bind and what is its job?

Answer 24

U2AF binds to 3’ splice site,
Helps recruit U2 to come in,
Bumps out BBP and U2 binds to branch point.

Question 25

Where does U2 bind?

Answer 25

U2 snRNP displaces BBP and binds branch point.

Question 26

Where do U4, U5, and U6 bind?

Answer 26

Displace U2AF complex at 3’ splice site

Question 27

When does rearrangement take place?

Answer 27

After binding of U4, U5, U6

Question 28

Which snRNPs do the cleavage?

Answer 28

U2, U5, U6

Question 29

After rearrangement, which snRNPs leave?

Answer 29

snRNP U1 and U4 released.

Question 30

After rearrangement and release of snRNPs U1 and U4, what happens?

Answer 30

Transesterification reaction cleaves 5’ splice site and forms lariat.

Question 31

After transesterification cleavage of 5’ splice site and lariat formation what happens?

Answer 31

Further rearrangements bring splice sites together, two exons splice together and lariat released.

Question 32

What further attracts splicing complex?

Answer 32

Further phosphorylation of CTD RNA pol II attracts splicing complex

Question 33

After splicing occurs, what happens?

Answer 33

EJC (exon junction complex) is deposited at the splice junctions and “marks” transcript as spliced.

Question 34

How does mRNA get exported from nucleus to cytoplasm?

Answer 34

EJCs interact with export machinery AND ribosome. Proteins bind to EJC, escort mRNA through nuclear pores.

Question 35

What else are EJCs (exon junction complexes) also involved in, in addition to escorting mRNA through nuclear pores?

Answer 35

Also involved in nonsense mediated decay

Question 36

Where does nonsense mediated decay occur?

Answer 36

In the cytoplasm, ready to be translated.

Question 37

What is the “ pioneer round “ and how does it work?

Answer 37

1st Time RNA is translated. Ribosomes displace one EJC at a time all along as it moves along to translate message.

Question 38

What happens if an mRNA has a nonsense mutation in Pioneer round?

Answer 38

Not all the EJCs will get displaced by ribosomes. The EJCs that remain will recruit nonsense mediated decay enzymes ——- and mRNA will get degrade / message will not get translated. Transcript is degraded by NMD (nonsense mediated decay) pathway.