Lecture 23 - RNA Processing I

Question 1

Which RNA pol has a CTD?

Answer 1

RNA pol II

Question 2

How many repeats are in the human CTD?

Answer 2

52

Question 3

What is the sequence of the heptapeptide CTD repeat?

Answer 3

YSPTSPS

Question 4

What is responsible for the phosphorylation of the CTD?

Answer 4

TFIIH

Question 5

Which residues in the CTD are phosphorylated?

Answer 5

Serine 5 and Serine 2

Question 6

What does RNA pol II leave behind when elongation begins?

Answer 6

It leaves behind the promoter and all its transcriptional factors.

Question 7

What happens when RNA pol II reaches its first nucleosome?

Answer 7

It stops.

Question 8

What does RNA pol II do after it transcribes approximately 25 nucleotides? Why?

Answer 8

It pauses in order to make sure that the 5’ end of the pre-mRNA is intact (protected) before continuing (because RNAs are sensitive to digestion by nucleases).

Question 9

What is added to protect the 5’ end of pre-mRNA? How is it connected?

Answer 9

A 7 methylguanylate cap is added to the 5’ terminal nucleotide of the RNA molecule through a 5’-5’ phosphodiester linkage.

Question 10

What is added to the 7-methylguanylate cap? Where?

Answer 10

Methyl groups are added to the cap’s 2’ hydroxyl of the ribose group of the 1st base in animal cells and in higher plants. In vertebrates, the 2nd base is also methylated.

Question 11

What is the 1st RNA processing enzyme?

Answer 11

The Capping Enzyme (CAP)

Question 12

Which RNA processing enzyme protects the pre-mRNA, facilitates nuclear export, and enables recognition by the translation factors?

Answer 12

CAP

Question 13

What activates CAP?

Answer 13

CAP is activated by the phosphorylated ser5 of the CTD.

Question 14

What does the mRNA/CAP/5’-5’ linkage/methyl group complex interact with? What is this caused by?

Answer 14

The complex interacts with proteins (present in the nucleus) called hnRNPs that specifically protect mRNAs. They are attracted by ser5 phosphorylation.

Question 15

When is ser2 of the CTD phosphorylated?

Answer 15

Ser2 is phosphorylated once hnRNPs interact with the mRNA.

Question 16

What does the hyperphosphorylated CTD act as a landing pad for?

Answer 16

It acts as a landing pad for all proteins that will take place on a pre-mRNA, such as splicing, polyadenylation, and export factors.

Question 17

What proteins are responsible for fully phosphorylating the CTD?

Answer 17

P-TEFb, CDK9, and Cyclin T

Question 18

When will RNA pol II continue elongation after initially pausing?

Answer 18

RNA pol II will continue elongation once the CTD has been fully phosphorylated and it has ensured that all the necessary proteins are fully there.

Question 19

What is formed by an RNA associated with a protein?

Answer 19

Ribonucleoprotein Complex (RNP)

Question 20

What are RNA-protein interactions mediated by?

Answer 20

They are mediated by specific domains that fall into a number of classes. The major ones are the RRM domains (beta-sheets and helices), but there are other domains such as the KH domain and RGG repreats.

Question 21

What is the difference between introns in higher eukaryotes (mammals) and lower eukaryotes?

Answer 21

Higher eukaryotes have big and complex introns while lower eukaryotes have small introns (therefore more compact genomes).

Question 22

What do introns code for?

Answer 22

Introns encode for regulatory information. They have evolved regulatory elements that can be downstream or upstream.

Question 23

What regions of the transcript are present in mature mRNA?

Answer 23

Exons

Question 24

What experimental observation led to the belief that the TATA box is important?

Answer 24

The TATA box was thought to be important because of the number of cDNAs found for it. 40 years ago, the amount of genetic information known to us was very limited and the sequencing of cDNA was the most prominent was the genome was studied.

Question 25

What proved that introns exist?

Answer 25

When cDNA was base paired to the genomic sequence, some regions of the genomic DNA looped out, corresponding to the regions of introns that were not present in the cDNA.

Question 26

What must be present for an intron to be excised?

Answer 26

There must be 2 nucleotides at the 5’ boundary of an intro (G and U) and 2 nucleotides at the 3’ boundary (A and G) of the intron. If this GU AG rule is not respected, the intron will not be excised. As well, there must be a pyrimidine-rich region approximately 30 nucleotides upstream of the AG nucleotides with a branch point adenosine located around the region.

Question 27

What are snRNPs made of?

Answer 27

Proteins and Small Nuclear RNAs (snRNAs)

Question 28

What is the spliceosome made of?

Answer 28

5 snRNPs

Question 29

Why do the snRNPs in the spliceosome contain?

Answer 29

Each snRNP harbours snRNAs which are U-right (U1, U2, U4, U5, and U6) and six to ten proteins.

Question 30

How do snRNAs facilitate splicing?

Answer 30

First, U1 snRNA base pairs with the region around the exon-intron junction (the intron’s 5’ junction). Then, the U2 snRNA base pairs (limitedly) around the pyrimidine-rich region and particularly around the branch point A. It must pair with the point such that the point is not included in the hybrid and bulges out, putting a structural strain on the nucleotide.

Question 31

What happens when oligonucleotides complementrary to the sequence around the exon-intron boundary are introduced in vitro?

Answer 31

When this happens, splicing is disrupted.

Question 32

What happens in vitro when you mutate pre-mRNA around the 5’ splice site?

Answer 32

U1 snRNA can no longer base pair with the area and splicing is blocked.

Question 33

What happens if you make a compensatory mutation to U1 snRNA after mutating the exon-intron junction?

Answer 33

Making this compensatory mutation allows splicing to occur again.

Question 34

What happens in the first trans-esterification that occurs during splicing?

Answer 34

The hydroxyl group of the residue at the branch point will make a nucleophilic attack on the 5’ phosphate group of the 5’ intron nucleotide G (right at the exon-intron boundary) via the involvement of U2 snRNA. It gives rise to a 2’-5’ phosphorous ester linkage that leaves a free 3’ hydroxyl on the 5’ intron and leads to the formation of a lariat structure.

Question 35

What happens in the second trans-esterification reaction that occurs during splicing?

Answer 35

The free 3’ end of the 5’ exon nucleophilically attacks the 5’ phosphate group of the first residue in the following 3’ exon. The reaction has taken place at the intron-exon junction downstream that has been put into proximity through the reactions of snRNPs. As a result, a phosphodiester bond has been formed between the two exons and the lariat structure containing the intron has been removed.

Question 36

What are the end products of the two trans-esterification reactions?

Answer 36

The products are the excised lariat intron and a contiguous mRNA.

Question 37

Which complex facilitates splicing?

Answer 37

Spliceosome

Question 38

What do U1 snRNA and its associated ribonucleotide proteins (in the form of U1 snRNP) do?

Answer 38

They interact with the 5’ end of the intron to be spliced, mainly due to the base pairing between snRNA and the RNA around that intron junction.

Question 39

What does U2 snRNP do?

Answer 39

It interacts around branch point A (due to base pairing) and acts as a scaffold to bring in U4 snRNP, U5 snRNP, and U6 snRNP.

Question 40

Which snRNPs leave the active spliceosome before the first trans-esterification occurs?

Answer 40

U1 snRNP and U4 snRNP

Question 41

Which snRNPs can be recycled after the second trans-esterification?

Answer 41

U2 snRNP, U5 snRNP, and U6 snRNP

Question 42

Which specific enzyme cuts the lariat structure after splicing? Why does this require a special enzyme? What happens to the product after it is cut?

Answer 42

The lariat must be cut by a specific enzyme because of its “unusual” 2’-5’ linkage. We call the enzyme that breaks that linkage a debranching enzyme. It will give rise to a linear intron RNA that will be degraded by some exonucleases in the cell.

Question 43

How many ATP molecules are needed for splicing to occur?

Answer 43

0