processing of mRNA transcripts

Question 1

what are the two elements, added post-transcription, to eukaryotic mRNA

Answer 1

m7G Cap and poly A tails

Question 2

are m7G Cap and poly Z tails encoded in the genome

Answer 2

No

Question 3

what are the primary transcript processing methods

Answer 3

Capping, splicing, polyadenylation and editing

Question 4

How is primary transcript processing coupled to transcription

Answer 4

RNA pol II CTD

Question 5

What does RNA initially contain at 5’ end

Answer 5

triphosphate

Question 6

what are the two steps in capping

Answer 6

1) GpppN structure
2) methylation

Question 7

what process alters chemical behaviour of bases

Answer 7

methylation

Question 8

what is the functions of m7G cap (4)

Answer 8

• protects mRNA from degradation by 5’-3’ nucleases
• Facilitates splicing
• Facilitates export from the nucleus
• Critical for translation of most mRNA

Question 9

How are the m7G cap functions mediated

Answer 9

Through protein binding
CBP80/CBP20 in the nucleus and elF4 complex in cytoplasm

Question 10

what does elF4 complex mediated in relation to m7G cap function

Answer 10

translation

Question 11

what does CBP80/CBP20 mediated in relation to m7G cap function

Answer 11

processing/export

Question 12

what is the order in which post-transcriptional modification

Answer 12

1) transcription and capping
2) Splicing and polyadenylation

Question 13

which is generally longer in mammals, introns or exons

Answer 13

introns

Question 14

Intron and exon boundaries contain conserved sequences, what and where are they ?

Answer 14

5’ splice site - exon 1
3’ splice site - exon 2
branch site - intron

Question 15

what are the conserved sequences in exon/introns required for

Answer 15

• define limits of exon and intron
• Sequences recruit the splicing machinery required to remove the
  intron and join the exons

Question 16

what type of reaction are the two steps of splicing of introns

Answer 16

2 - trans-esterification

Question 17

what occurs in the two steps of intron splicing

Answer 17

1) cut at 5’ splice site & creation of bond between 5’ end of intron and branch site
2) cut at 3’ splice site to release intron lariat & ligation of two exons

Question 18

what are spliceosomes

Answer 18

enzymatic complex that catalyses the removal of introns

Question 19

what do spliceosomes require

Answer 19

ATP

Question 20

what proteins make up spliceosome

Answer 20

• large complexs containing RNA-binding proteins, ATPase, GTPase and several snRNPs

Question 21

what are snRNPs

Answer 21

Small nuclear ribonucleoprotein particles

Question 22

what do RNA do in terms of splicing

Answer 22

RNAs base-pair with conserved sequences in the intron (5’ splice site, branch point)
*Splicing is catalysed by the snRNAs

Question 23

what are the two types of snRNPs

Answer 23

Sm proteins and snRNAs

Question 24

what reacts against Sm proteins

Answer 24

Anti-Sm antibodies

Question 25

when would a person have anti-Sm antibodies

Answer 25

very rare unless a person has autoimmune disease systemic lupus erythematosus (SLE)

Question 26

what percentage of patients with SLE have anti-Sm antibodies

Answer 26

20%

Question 27

what catalyses the splicing mechanism

Answer 27

RNA

Question 28

explain Step 1 of the splicing mechanism using RNA

Answer 28

1) U1 & U2 binds to the 5' and binding site respectively 2) U4,U6+U5 is then recruited, then U1+U4 is release 3) A bond is created between 5' end of intron and the branch site 4) U6,U5 and U2 holds the free exon 1 together to the looped intron (still connected to exon2)

Question 29

what is the E complex

Answer 29

U1 at the 5' splice site of the intron

Question 30

what is the A complex

Answer 30

U1 at the 5' splice site of the intron U2 is at the branch site

Question 31

What is the B complex

Answer 31

all RNA (U1,2,4,5,6) are joined along mRNA

Question 32

what is the B* complex

Answer 32

The B complex after U1+U4 is released

Question 33

What is the C complex

Answer 33

mRNA with U2,U5,U6 joined after intron lariat being formed and exon one being freed

Question 34

explain Step 2 of the splicing mechanism using RNA

Answer 34

The spliceosome cuts the mRNA at 3', U2,6,5 hold the 2 exons together and allows them to bind - lariat is released

Question 35

why is the number of proteins greater than genes in the genome

Answer 35

because there can be different organisations/arrangements of the exons after splicing

Question 36

what are the 3 conditions caused by mutations causing defects in splicing

Answer 36

- Spinal muscular atrophy - Retinitis Pigmentosa - Myotonic Dystrophy

Question 37

how does polyadenylation occur

Answer 37

- Endonuclease cleavage releases the mRNA from the DNA template when the a specific sequence is recognised - Addition of As by PolyA polymerase

Question 38

what is the recognised specific sequences which activates endonuclease cleavage

Answer 38

- AAUAAA (conserved), 10-35 nucleotides upstream of polyA site

Question 39

what is downstream of poly(A) site

Answer 39

G/U or U rich tract

Question 40

what is upstream of AAUAAA

Answer 40

U-rich element 'USE'

Question 41

what are the proteins required for polyadenylation, and where do they bind to

Answer 41

- Cleavage and polyadenylation specificity factor (CPSF) = binds AAUAAA - Cleavage stimulatory factor (CstF) = binds G/U -Poly(A) polymerase

Question 42

what are the functional significance of the polyA tail

Answer 42

* Enhances export of RNA * Stabilises the 3’ end of the mRNA * Enhances translation of mRNA

Question 43

give an example of a protein which doesnt have a poly A tail

Answer 43

histones