Post Transcriptional Processing

Question 1

What types of RNA undergo processing events?

Answer 1

rRNA, tRNA and mRNA

Question 2

What is rRNA transcribed by and what is its role?

Answer 2

Transcribed by RNA pol 1 as a single 45S precursor transcript.

Question 3

What has to happen before the mature rRNA can be reached?

Answer 3

Subjected to modifications e.g of certain nucleotides which affects confirmation of rRNA and thus function.

Question 4

rRNA is assembled with ______.

Answer 4

Ribosomal proteins.

Question 5

What is pre-RNA? What is it cleaved into?

Answer 5

Pre-RNA is immature RNA and it is cleaved into 18S, 28S and 5.8S rRNA. (Physical card 24)

Question 6

What is tRNA transcribed by and how is it modified to become fully mature tRNA?

Answer 6

RNA pol III. Nucleotides from the 5’ and 3’ ends are cleaved and nucleotides of CCA are added into the 3’ end of mature tRNA. Some nucleotides are modified and the intron sequence is removed and products are ligated. Physical card 25.

Question 7

What role does processing have in the complexity of eukaryotes vs prokaryotes?

Answer 7

pre-mRNA transcripts are extensively processed in the nucleus and transported to the cytoplasm before translation. In prokaryotes, transcription and translation occur in the same place.

Question 8

What are the 3 main processing steps for mRNA?

Answer 8

1. Capping at the 5’ end - DNA strand transcribed.
2. Addition of the poly-A tail at 3’ end.
3. Splicing (removal of introns and linking of exons)

Question 9

What is the process of capping at the 5’ end and what are the implications of this?

Answer 9

Physical card 26

Question 10

What is the process of 3’ polyadenylation and what are the implications of this?

Answer 10

Physical card 27

Question 11

Which eukaryotic RNAs are not polyadenylated?

Answer 11

Histone mRNAs, rRNA, other smaller RNA. Only mRNA for our purposes is polyadenylated.

Question 12

How do histone mRNAs perform cleavage?

Answer 12

Have a stem-loop structure followed by a purine-rich sequence (A/G) to direct cleavage.

Question 13

What is the function of the A/G purine-rich sequence in the maturation of histone mRNA?

Answer 13

A/G recruit proteins - with mRNA which can bind to particular cleavage sequences - use endonucleases in the complex to cleave the pre-mRNA

Question 14

How can the poly (A) tail be exploited to do reverse transcription?

Answer 14

mRNA can be purified using oglio-dTs which bind to the poly(A) tail. As other RNA types don’t have a poly(A) tail we can use this to separate mRNA. Can dissociate two strands from oglio-dTs to purify mRNA or it can be processed while still bound.

Question 15

What is splicing?

Answer 15

Removes introns (non-coding regions) and joins the exons on either side (coding regions) to form mature mRNA.

Question 16

How is a splice site determined?

Answer 16

Consensus sequence at the ends of introns to identify splicing sites - Several sequences which tell machinery what is an exon/intron.

Question 17

What is the sequence for an intron? (Also look at physical card 28 for diagram)

Answer 17

Almost always starts with a GU sequence at 5’. At 3’ end conserved sequence followed by a pyramidal tract (series of U or C followed by any nucleotide, followed by C and the AG). Upstream there is an A in the branch site.

Question 18

What makes up the splicing machinery?

Answer 18

Spliceosome - large splicing complex consisting of small nuclear RNA (snRNA), over 300 proteins and pre-mRNA.

Question 19

How is incorrect splicing linked to disease?

Answer 19

Mutations affecting splicing cause at least 15% of all genetic diseases.

Question 20

Where can mutations occur in the splicing process?

Answer 20

In the pre-mRNA (e.g thalassemia - defective beta-globin products) or in the splicing factors.

Question 21

What is an example of how mutation can occur and what can this cause?

Answer 21

Physical card 29

Question 22

What is alternative splicing?

Answer 22

Although there is one gene, there are many mRNAs - many proteins. Not every exon will be included in all transcripts.

Question 23

How many of the human protein-coding genes are alternatively spliced?

Answer 23

Over 70%

Question 24

How many mRNAs can come from one pre-mRNA with n alternative splice sites?

Answer 24

2^n different mRNAs.

Question 25

Give an example of alternative splicing.

Answer 25

Physical card 30.

Question 26

How is mRNA transported from _______ to ______?

Answer 26

Nucleus to Cytoplasm. Through the nuclear pore complex - found in the membrane.