RNA Splicing

Question 1

What is the main type of intron found in eukaryotes?

Answer 1

GU-AG intron.

Question 2

How many classes of introns are there in eukaryotes?

Answer 2

7.

Question 3

What type of reactions are involved in the splicing mechanism?

Answer 3

Two transesterfication reactions.

Question 4

What is the spliceosome mechanism?

Answer 4

1. U1 binds 5’ splice site and U2 binds to the A branch site.2. U4, U5, U6 bind to the intron, completing spliceosome assembly. 3. 5’ splice site is cut, 5’ end of intron is connected to the branch site- forming a lariat structure. U1 and U4 are released, U5 and U6 change positions. 4. 3’ splice site is cut and exons are ligated together. 5. Lariat intron is released with the remaining parts of the spliceosome.

Question 5

What is the spliceosome made of?

Answer 5

5 snRNAs that associate with proteins to form snRNPs- U1, U2, U4, U5 and U6 which vary in length from 60-300nts.

Question 6

What are snRNPs?

Answer 6

snRNAs + proteins + mRNA.

Question 7

Give an example of how mutations in the splice site can cause disease.

Answer 7

Incorrect splicing of the β globin gene can cause thalessemia.

Question 8

Give an example of a gene that undergoes alternative splicing.

Answer 8

α-tropomyosin- can have at least 9 different mRNA transcripts, different tropomyosins are found in different cell types.

Question 9

What is alternative splicing?

Answer 9

Different combinations of exons producing different mRNA transcripts. This alters the structure and function of the proteins produced.

Question 10

Where are group I introns found?

Answer 10

In pre-mRNA.

Question 11

Where are group II introns found?

Answer 11

In organelle genomes.

Question 12

What feature of both group I and group II introns supports the RNA world theory?

Answer 12

They are autocatalytic so can catalyse their own removal from RNA.

Question 13

Which transcription factors are used by RNA polymerase III to produce U6 snRNP?

Answer 13

Oct1/PBP (bind upstream of the TATA box) and TFIIIB.

Question 14

What are the similarities between RNA synthesis in prokaryotes and eukaryotes?

Answer 14

3 eukaryotic RNAPs structurally similar to prokaryotic RNAP. Similar RNA polymerisation mechanisms.

Question 15

What is the GU often flanked by in GU-AG intron?

Answer 15

Purine rich regions.

Question 16

Give a feature of the 3’ splice site.

Answer 16

12-14nt polypyrimidine tract.

Question 17

Give common features of eukaryotic introns.

Answer 17

Polypyrimidine tract (12-14 nts) and branch site adenine (30 nt upstream of acceptor.

Question 18

Where are snRNPs assembled?

Answer 18

Regulatory steps in both nucleus and cytoplasm.

Question 19

Explain the assembly of U1, U2, U4 and U5 snRNPs.

Answer 19

Bind to 7 Sm ribonucleoproteins (B, D1, D2, D3, E, F, G) to form a toroidal ring. This protects the snRNA from nucleases and is necessary for nuclear import of the complex. Further regulatory steps occur in the cytosol and the mature snRNPs are imported back into the nuclease for splicing.

Question 20

Which part of the snRNP provides specificity?

Answer 20

The snRNA recognises the 5’ and 3’ ends and branch site of introns.

Question 21

Where are the snRNAs produced and how are they transported to the cytoplasm?

Answer 21

Produced in nucleus by RNAPII - contain m7-G cap which serves as a nuclear export signal.

Question 22

What is the minor introns consensus sequence?

Answer 22

AU-AC

Question 23

What is the conserved branch site sequence?

Answer 23

5’-UCCUUAAC-3’

Question 24

What snRNAs do minor introns use?

Answer 24

U11, U12, U4atac and U6atac snRNAs
(these are analogues of U1, U2, U4 and U6).
AND requires U5 major snRNA.

Question 25

What are self splicing introns?

Answer 25

They catalyse their own excision from mRNA, tRNA and rRNA pre mRNA.

Question 26

Which types of eukaryotes are these introns predominantly found in?

Answer 26

Single cell organisms and plants.

Question 27

Why would self splicing introns be termed ‘ribozymes’?

Answer 27

They are autocatalytic.

Question 28

What are the two types of self splicing introns?

Answer 28

Group I and Group II

Question 29

What is the most common self splicing intron?

Answer 29

Group I.

Question 30

How do group I introns perform splicing?

Answer 30

An exogenous guanosine (exoG) attacks the phosphodiester bond at the 5’ splice site.
The 3’ OH group of exon 1 is then aligned to attack the 3’ splice site.

Question 31

Which organisms are group II introns predominantly found in?

Answer 31

Mostly found in fungi, plants and even some prokaryotes.

Question 32

What types of RNA are group II introns found in?

Answer 32

pre-mRNA and pre-rRNA.

Question 33

How do group II introns perform splicing?

Answer 33

Occurs by similar mechanism as major intron splicing - 2 transesterification reactions with lariat loop.

Question 34

Group II introns can also retrohome - what does this mean?

Answer 34

Insert themselves into the genome as ssRNA - then are reverse transcribed by the associated intron encoded protein.

Question 35

What is the length of pre-tRNA introns and what structural feature do they contain?

Answer 35

14-60nt in length and contain an anticodon loop.

Question 36

How are pre-tRNA introns spliced?

Answer 36

No transesterification - RNA is cut at 2 sites by an endonuclease and the 3’ end of the upstream exon converted by phosphodiesterase to OH.
OH group at 5’ of the downstream exon converted by kinase to 5’P.
joined together by RNA ligase.