RNA Processing

Question 1

Differentiate prokaryotic vs eukaryotic RNA processing

Answer 1

Prokaryotes:
- mRNA not modified

Eukaryotes:
- modified extensively in nucleus

NOTE: tRNA and rRNA are modified in BOTH

Question 2

Why is mRNA Processing evolved in eukaryotes and NOT in prokaryotes ?

Answer 2

• contains exons and introns (spliced out)
• transcription occurs in nucleus (compartmentalized organelles)
• modification protects mRNA before its export into cytoplasm (for translation); prevents degradation of mRNA

Question 3

What 3 modifications occur during RNA processing ?

Answer 3

1. 5’-cap
2. Poly-A tail at 3’ end
3. Splicing of introns

Question 4

Function of 5’-cap ?

Answer 4

• protects mRNA and transports from nucleus to cytoplasm
• binds to specific cap-binding complex; ensures mRNA stays attached to RNAP II during transcription
• involved in binding ribosomes to initiate translation

Question 5

How is 5’ cap attached to mRNA ?

Answer 5

1. 7-METHYguanosine attached to EXTRA 5’ nucleotide
2. Capping enzymes bind to RNAPII’s phosphorylated CTD
3. 5’ - 5’ triphosphate connection between extra nucleotide and 5’ of first nucleotide of mRNA
4. 2’-hydroxyls of first and second nucleotides in mRNA are also methylated

NOTE: Cap is attached early in transcription

Question 6

Function of Poly-A tail

Answer 6

• protein binding site at 3’-end
• protects mRNA from degradation

Question 7

T or F: Poly-A is encoded in the genome

Answer 7

FALSE; Adenosines are added/ NOT encoded in the genome

Question 8

Describe addition of Poly-A tail

Answer 8

1. recognize sequence (AAUAAA) upstream of where poly A is to be added
2. endonuclease CLEAVES transcript
3. addition of Adenosines to free 3’-OH (no template required)

Question 9

Which code for proteins: exons or introns ?

Answer 9

EXONS code for proteins; introns are spliced out

Question 10

What does it mean when mRNA hybridizes ?

Answer 10

Ovalbumin template DNA hybridizes with mature mRNA transcript
- DNA includes introns = large loops bubble out because introns in mRNA has been spliced out

Question 11

Which intron groups are self-splicing/ autocatalytic ?

Answer 11

Group I and Group II

Question 12

Group I and II introns undergo “transesterification.” What does this mean ?

Answer 12

• do not require ATP; instead, trades one bond for another bond
• self-splicing/ autocatalytic

Question 13

Splicing mechanism of Group I introns

Answer 13

• the 3’-OH of GUANOSINE is a nucleophile (electron donor) and ATTACKS phosphate of 5’ intron
• now the 3’-OH of first exon becomes the second nucleophile ATTACK = releases introns, and connects both exons together

NOTE: 2 phosphodiester bonds formed; 2 are broken; 2 transesterification reactions

Question 14

Splicing mechanism of Group II introns

Answer 14

• 2-OH of Adenosine nucleotide in INTRON ATTACKS 3’-end of first exon
• 3’-end of first exon is the second nucleophile and ATTACKS 5’-end of second exon = branched lariat intron is released and connects exons together

Question 15

How does sRNA work ?

Answer 15

ATP consumed at each step:
1. U1 recognizes (exon) AG/GU (intron) sequence
2. U2 recognizes (intron) AG/GU (exon)
— mRNA contains A residue that is excluded from base pairing = “bulge” for later nucleophile attack
3. U4, U5, U6 join spliceosome
4. U1 and U4 dissociate
5. U2, U5, U6 leave with intron lariat; exons are attached
— nucleophile attacks from extra A residue and exon 3’-OH

NOTE: AG/GU is known as “consensus” sequence
sRNA - spliceosomal RNA

Question 16

T or F: intron splicing does not have directionality

Answer 16

FALSE; intron splicing DOES have directionality
- 5’ splice sites join with 3’ splice sites

Question 17

What is differential RNA processing ?

Answer 17

• transcriptome = one primary transcript can produce multiple different mRNAs
• due to different cleavage/ polyadenylation sites and/ or splice patterns

Question 18

When is the 5’-cap added ?

Answer 18

Early in transcription

Question 19

Name the enzyme responsible for mRNA tailing (polyA)

Answer 19

Polyadenylate polymerase