Lecture 12: Post-transcriptional modification in eukaryotes

Question 1

What is pre-mRNA?

Answer 1

mRNA immediately after transcription, which still contains introns

Question 2

In what kind of cells is pre-mRNA found?

Answer 2

Eukaryotic cells only

Question 3

What are hnRNPs?

Answer 3

Heterogeneous ribonucleoproteins; they’re a set of nuclear proteins that associate with pre-mRNA (pre-mRNA isn’t free to roam around the nucleus)

Question 4

What are hnRNAs?

Answer 4

Heterogeneous nuclear RNAs; refers to pre-mRNA and other nuclear RNAs of various sizes

Question 5

Components of RNA processing

Answer 5

5’ cap, splicing, 3’Poly(A) tail addition

Question 6

Purpose of phosphorylation of RNA Pol II before transcription

Answer 6

Allow RNA processing proteins (capping, splicing, etc) to bind, release GTFs except TFIID, move RNA Pol II forward and start transcribing

Question 7

Precisely where is RNA Pol II phosphorylated?

Answer 7

CTD domain of RPB1 subunit at Ser5

Question 8

Bond involved in 5’ cap

Answer 8

5’-5’ triphosphate linkage of 7-methylguanylate (GTP) residue

Question 9

Functions of 5’ cap

Answer 9

Transport out of the nucleus, initiate translation (machinery must recognize the cap), and protection from 5’ to 3’ exonucleases

Question 10

Reactions that cap the 5’ end (3)

Answer 10

Removal of phosphate, addition of GMP, addition of methyl group to guanosine of GMP

Question 11

Sequences on mRNA needed for splicing

Answer 11

5’ splice site, 3’ splice site, sequence at middle of intron

Question 12

5’ splice site consensus sequence

Answer 12

AG|GURAGU (R is A or G)

Question 13

3’ splice site consensus sequence

Answer 13

CAG|G

Question 14

Consensus sequence in middle of intron (for splicing)

Answer 14

YURAC (Y is C or U - a pyrimidine)

Question 15

Composition of spliceosome

Answer 15

Large assembly of snRNAs (small nuclear RNAs) and snRNPs (small nuclear ribonucleoproteins)

Question 16

Purpose of spliceosome

Answer 16

Perform splicing of pre-mRNA

Question 17

What part of the spliceosome recognizes the 5’ splice junction, branch-point site, and 3’ splice junction? How?

Answer 17

snRNAs and snRNPs recognize the sites through complementary base pairing

Question 18

What is U1 and what does it do?

Answer 18

snRNP that recognizes 5’ splice site

Question 19

What is U6 and what does it do?

Answer 19

snRNP that catalyzes the actual splicing reaction (intron removal) - it replaces U1 at the 5’ splice site after ATP hydrolysis

Question 20

What form to introns take once removed?

Answer 20

Lariat: a “loop” of RNA in which the 5’ splice site is attached to branch-point site, with 3’ splice site “hanging” off

Question 21

What is alternative splicing?

Answer 21

pre-mRNA of a gene can be spliced in different ways, resulting in different mRNA, which code for different proteins

Question 22

Purpose of Poly(A) polymerase

Answer 22

Adds A residues to 3’ end of mRNA

Question 23

In polyadenylation, what part of the mRNA is cleaved and degraded in nucleus? Why?

Answer 23

GU-rich or U-rich sequence, which is important for processing and cleavage recognition

Question 24

Does Poly(A) polymerase need a template?

Answer 24

Nope.

Question 25

How long is a Poly(A) tail?

Answer 25

200-250 adenosine residues

Question 26

Functions of Poly(A) tail (3)

Answer 26

Protect mRNA from premature degradation by 3’ to 5’ exonuclease; length of tail determines half life of mRNA in cytoplasm; maintain stability of RNA

Question 27

What is CPSF?

Answer 27

Cleavage and polyadenylation specificity factor

Question 28

What does CPSF do and where does it bind?

Answer 28

Important for cleavage; binds to AAUAAA, upstream of cleavage site

Question 29

What is CstF?

Answer 29

cleavage stimulation factor

Question 30

What does CstF do and where does it bind?

Answer 30

Leaves once cleavage is done and recruits Poly(A) polymerase; binds to GU-rich element

Question 31

What does polycistronic mean?

Answer 31

One promoter makes one mRNA, which makes multiple polypeptides