RNA Processing

Question 1

How many RNA polymerases are there in eukaryotes and what do they transcribe?

Answer 1

3
Pol I - rRNA (main)
Pol II - mRNA (main), snRNA, miRNA
Pol III - tRNA (main), 5S rRNA, other small RNAs

Question 2

What are the different types of RNA processing?

Answer 2

Cleavage, Splicing, 5’ capping, polyadenlyation, base insertion, deletion, modification

Question 3

What are 2 characteristics of rRNA genes in the genome?

Answer 3

Polycistronic messages and found repeated in all genomes.

Question 4

How are rRNAs primarily processed?

Answer 4

Through endo and exonucleolitic activities.

Question 5

List the 5 ways tRNAs are processed post-transcriptionally in eukaryotes?

Answer 5

1. 5’ cleavage by RNase P
2. 3’ cleavage by tRNase Z
3. CCA is added to 3’ end by enzyme
4. Post-transcriptional modifications of bases
5. Intron excision

Question 6

What are two common post-transcriptional modifications of rRNA in eukaryotes and which snoRNAs mediate those modifications?

Answer 6

1. 2’ O methylation by box C/D snoRNA

2. Pseudouridylation by H/ACA snoRNA

Question 7

How do snoRNAs facilitate modification of nucleotides?

Answer 7

In a template driven manner. snoRNAs bind to complementary sequence and position nucleotide for modification within active site.

Question 8

What is odd about the 5’ cap found on mRNAs?

Answer 8

The guanosine is attached to the first base in a 5’ -> 5’ linkage

Question 9

What are the 3 steps involved in 5’ capping?

Answer 9

1) Removal of terminal phosphate from nascent RNA
2) Addition of GMP to the end, forming a 5’ -> 5’ linkage
3) Methylation of guanine

Question 10

What binds to the poly A tail?

Answer 10

Poly A Binding Protein (PABP)

Question 11

What can be used to enrich mRNA transcripts from lysates and why is this significant?

Answer 11

Use Poly-A tail by pulling down with Poly-T oligos. only 3% of total cellular RNA is mRNA. Majority is rRNA

Question 12

Which mRNAs do not have poly A tails and how do they get around this?

Answer 12

Histone mRNAs. Have a 3’ end stem loop generated by U7 snRNP-mediated interaction. Stem loop is bound by its own protein instead of PABP

Question 13

5 roles for 5’ cap and poly A tail

Answer 13

1. Help downstream maturation including splicing and transcription termination
2. mRNA stability
3. Facilitate transport to cytoplasm
4. Important for translation initiation
5. Alternative polyA/capping can be regulatory

Question 14

What consensus sequences are recognized by the splicing machinery?

Answer 14

5’ splice site GU
3’ splice site AG
Branch point A
Purine Rich Sequence downstream branch point

Question 15

Describe or draw the 3 steps of pre-mRNA splicing?

Answer 15

1) 2’ OH of branch point Adenosine attacks 5’ splice site
2) Free 3’ OH of exon attacks 3’ splice site.
3) Get lariat (2’5’) intermediate that’s degraded

Note: not energetically demanding

Question 16

What is the main part of snRNPs that catalyze reactions? What is the theme associated with the spliceosome?

Answer 16

RNAs. The spliceosome isn’t a large machine that works all at once. It consists of smaller RNPs that dynamically lead to splicing.

Question 17

How do the mechanisms of Group I and Group II introns differ?

Answer 17

Group I introns use an exogenous guanosine to initiate attack 5’ splice site. Group II introns use branch point adenosine, similar to how introns are spliced in eukaryotes.

Question 18

What happens if you mutate the 5’ splice site of an intron in higher eukaryotes?

Answer 18

You get exon exclusion. Exons are defned in higher eukaryotes.

Question 19

What happens if you mutate the 5’ splice site of an intron in lower eukaryotes?

Answer 19

You get intron inclusion. Introns are “defined” in lower eukaryotes.

Question 20

How are the strengths of splice sites modulated in different tissues?

Answer 20

Differential expression of RNA binding proteins (such as intronic silencers) can change the splice site that will be used. This can cause proteins to be functional or non-functional trough alternative splicing.

Question 21

What is an example of RNA editing?

Answer 21

Nucleotide addition and deamination

Question 22

How is RNA canonically destroyed in eukaryotes?

Answer 22

PAPB disassociates and a 3’-5’ exonuclease / exosome cleaves one end.
Another enzyme decaps the mRNA and Xrn1 cleaves 5’->3’ with exonuclease.