Eukaryotic RNA processing (L14)

Question 1

What is the main type of eukaryotic RNAP and what does it transcribe for?

Answer 1

RNAP II and transcribes for mRNA and snRNA

Question 2

Which eukaryotic RNAP is located in the nucleolus?

Where are the other 2 located?

Answer 2

RNAP I (transribes rRNA);

Nucleoplasm

Question 3

Describe RNAP II initiation in eukaryotes.

Answer 3

1. TFIID complex recognizes TATA box
2. TFIIA promotes TBP binding to TATA
3. TFIIB acts as a bridge b/w TBP and RNAPII
4. TFIIE complex melts DNA at promoter
5. TFIIF weakens interactions b/w RNAPII and nonspecific DNA binding

Question 4

What does alpha-Amanitin do?

Answer 4

It’s a cellular toxin derived from a specific mushroom that binds and inhibits RNAP II, halting mRNA synthesis and ultimately protein synthesis.

Liver, GI tract will be severely effected, death results potentially

Question 5

What does Actinomycin D do?

Answer 5

antibiotic compound that binds to DNA and inhibits ELONGATION of RNA transcription by RNAP.

Toxic but used in pediatric cancers

Question 6

What are the 3 main mRNA processing events that occur?

Answer 6

1. 5’ capping
2. Splicing (only occurs 93% of the time)
3. Cleavage and addition of poly-A tail
   (4. )RNA editing happens rarely

Question 7

Describe the 5’ capping process to protect mRNA from exonucleases in eukaryotes

Answer 7

Starting from pppN1N2N3…:

1. 5’ terminal triphosphate hydrolyzed to diphosphate (ppN1N2…)
2. GTP condenses w/ 5’ end releasing pp_i (G(5’)pppN1)
3. SAM methylates G(5’), mRNA is transported to cytoplasm, and SAM methylates N1 & N2 at 2’ OH (m⁷G(5’)pppN1mN2mN3…)

Question 8

Describe the splicing process

Answer 8

1. 2’OH branch site on A attacks the 5’ splice site AFTER G of the 5’ splice site AG (creates lariate intermediate and separate upstream exon)
2. 3’OH on that G attacks 3’ spice site BEFORE G of the 3’ splice site uniting both exons and separating the lariat form of the intron

Question 9

Which small nuclear RNAs (snRNPs) are involved in splicing and how?

Answer 9

U1 snRNP binds 5’ splice site and U2 snRNP binds branch point (A-(2’OH))

U4,5,6 tri-snRNP then joins assembly to help.

Question 10

What is systemic Lupus Erythematosus (Lupus for short)?

Answer 10

An autoimmune disease where several antibodies attack cellular components, one of them which happens to be snRNPs

Question 11

Describe Cleavage and poly adenylation

Answer 11

1. The cleavage signal (aka polyA site (PAS)) emerges in RNA and is recognized by PAS binding machinery and cleavage occurs 15 nt downstream.
2. After cleavage polyA polymerase adds ~200 A’s at end of mRNA

Question 12

Describe termination of transcription in eukaryotes

Answer 12

After polyA tail added, Xrn2 5’>3’ exonuclease degrades elongating mRNA and catches up to RNAPII (like a “torpedo”) and knocks it off causing termination

Question 13

In nonmetastatic and normal prostate cells, what growth factor pre-mRNA exons are used?

What about cancerous, metastatic cells?

This is an example of what?

Answer 13

FGFR IIIa and IIIb (exons 7 and 8 respectively) for norm/non metastatic;

FGFR IIIa and IIIc (exons 7 and 9 respectively) for metastatic cells;

This is an example of alternate splicing. The IIIb and IIIc exons are mutually exclusive (meaning they won’t be on a processed mRNA together) and affect different ligand binding properties for FGFR mRNA.

Question 14

What are the genetic causes of Spinal Muscular Atrophy and the molecular consequences?

Answer 14

Genetic causes: Deletion/inactivation of SMN1 gene causes reliance on SMN2 gene which is slightly mutated causing significant exon skipping and thus reduced levels of SMN protein. (SMN makes snRNPs)

Molecular consequences: Reduced lvls of SMN cause a defect in snRNP assembly, AND affects NeuroMuscular Junctions in motor neurons

Question 15

Describe how alternate splicing affects IgG formation.

Answer 15

Initially, lymphocytes produce a long transcript that creates a membrane-bound antibody. Once this Ab recognizes an antigen, the cells produce a SHORTER transcript (due to alternate splicing) by using a different cleavage and polyadenylation site, which leads to the antibody without the hydrophobic membrane domain so it can be secreted from the cell into the blood stream.

Question 16

Describe how RNA editing affects apoprotein B (ApoB)

Answer 16

RNA editing will change a single AA in the Apoprotein B gene which makes that codon a stop codon in the intestine.

In the liver, the full ApoB protein is there but thru RNA editing, the intestines has a smaller ApoB protein.

Question 17

What is different about tRNA in eukaryotes than in prokaryotes?

Answer 17

Eukaryotic tRNA has an intron that MUST be removed for the anticodon to function.

Question 18

What does it mean that HIV is a “retrovirus”?

Answer 18

It enters the cell as RNA but is then converted into dsDNA by their own reverse transcriptase.

Question 19

What do the Tat and Rev proteins do for HIV?

Answer 19

Tat stimulates transcription of the viral DNA, and Rev causes the viral RNA transcripts (which have introns) to leave the nucleus unspliced.

The reason for Rev is b/c if the transcript was left inside the nucleus, it would be spliced by the host mechanisms, so Rev moves the unspliced transcript to the cytoplasm where all of it can be translated.

Question 20

How does zidovudine (ZDV) work against HIV?

Answer 20

It is an analog of thymine that lacks a 3’OH for the addition of the next nt so it inhibits the viral DNAP.

Question 21

Why is it recommended that AIDS patients take a number of drugs, including more than one reverse transcriptase inhibitor?

Answer 21

The HIV virus mutates very rapidly due to its reverse transcriptase lacking 3’>5’ exonuclease (proofreading) activity so it frequently develops resistance to these drugs. So more than one is taken to reduce resistance effects.