Lecture 6

Question 1

What is the binding of RNA processing proteins regulated by?

Answer 1

• Phosphorylation of RNA polymerase tail

Question 2

What is the c-terminal domain (CTD)?

Answer 2

• Binds RNA processing proteins and transfers them to RNA at the appropriate time
• In humans: 7 AA’s repeated 52 times

Question 3

What are the 3 markers of mature mRNA before export?

Answer 3

• Cap binding complex (CBC): binds to 5’ cap
• Exon junction complexes (EJC): binds splice sites where introns are removed
• Poly-A-binding proteins: binds to 3’ end

Question 4

Where do mature mRNA’s travel to?

Answer 4

• From nucleus to the cytosol

Question 5

What happens to immature mRNA markers?

Answer 5

• They must be lost for export
• Ex. proteins involved in RNA splicing (snRNPs)

Question 6

What happens to improperly processed mRNA’s?

Answer 6

• Degraded in the nucleus by the exosome

Question 7

Eukaryotic initiation factors (eIF’s) in translation initiation machinery

Answer 7

• Required for start of translation
• 5’ cap bound by eIF4E
• Poly-A binding protein bound by eIF4G

Question 8

What occurs during normal splicing in eukaryotes?

Answer 8

• Ribosome binds mRNA as it emerges from the nuclear pore (looping)
• EJC’s are displaced by the moving ribosome
• STOP codon is in the last exon
• No EJC’s remain bound when ribosome reaches STOP codon
• mRNA released in cytosol

Question 8

Nonsense-mediated mRNA decay in eukaryotes

Answer 8

• Happens when mRNA is entering the cytosol
• Surveys for nonsense (STOP) codons in the wrong place (immature)
• Indicator of improper splicing
• Triggers mRNA degradation

Question 9

What occurs during abnormal splicing in eukaryotes?

Answer 9

• Ribosome binds mRNA as it emerges from the nuclear pore (looping)
• EJC’s not displaced from the moving ribosome
• STOP codon is premature
• EJC’s remain on mRNA when the ribosome reaches the STOP codon
• mRNA degraded by Upf proteins

Question 10

mRNA quality control in prokaryotes

Answer 10

• Ribosomes stall on broken or incomplete mRNA’s and do not release
• Special RNA (tmRNA) recruited to the A site, carries an alanine, acts as both tRNA and mRNA
• Broken mRNA is released
• Alanine added onto polypeptide from tmRNA (no anticodon-codon binding)
• Ribosome translates 10 codons from tmRNA that acts like mRNA, 11 AA tag is recognized by proteases that degrade the entire protein

Question 11

In prokaryotes, what do exonucleases do?

Answer 11

• Rapidly degrade most mRNA’s from both ends
• When mRNA reaches the cytoplasm (timer of mRNA lifetime)

Question 12

2 degradation mechanisms when the poly-A tail reaches a critical length (humans = 25 nucleotides)

Answer 12

• Decapping: remove 5’ cap so exonuclease can degrade from that end
• No decapping: exonuclease degrades from 3’ to 5’

Question 13

Transferrin receptor with iron in the cell (mRNA stability)

Answer 13

• Iron starvation: mRNA stabilized by cytosolic aconitase, binds 3’ UTR of mRNA, blocks poly-A so it isn’t degraded as fast, mRNA is stable and translated because transferrin receptor is made
• Excess iron: aconitase binds iron and conformational change, mRNA released from aconitase, exposes 3’ UTR endonucleolytic cleavage site (poly-A removed), mRNA is degraded because no tranferrin receptor is made

Question 14

mRNA stability competition between mRNA translation and degradation

Answer 14

• Degradation: deadenylase shortens the poly-A tail, binds to 5’ cap (like eIF’s)
• Translation: 5’ cap occupied by eIF4E, eIF4G binds to eIF4E, poly-A binding protein attached to eIF4G (actively transcribed RNA molecules are not as easily degraded)

Question 15

mRNA stability with miRNA’s

Answer 15

• microRNA’s (non-coding RNA’s) regulate mRNA stability by base-pairing with specific mRNA’s
• Synthesized by RNA polymerase 2 & get a 5’ cap and poly-A tail
• Once entered the cytosol, cleavage by the enzyme ‘dicer’, one strand degraded
• After special processing, miRNA strand associates with a protein complex called an RNA-induced silencing complex (RISC)

Question 16

What does RISC do?

Answer 16

• Seeks mRNA with complementary nucleotide sequences
• Protein of RISC called argonaute helps base-pair miRNA with mRNA
• 2 possible outcomes: rapid mRNA degradation (extensive match, argonaute slices, no cap or tail) or eventual mRNA degradation (less extensive match, blocks ribosome so no translation)

Question 17

RNA interference (RNAi)

Answer 17

• Double-stranded RNA’s that end up suppressing the gene expression of other RNA’s in a sequence-specific manner
• Proteins used in miRNA regulatory mechanisms also serve as defence mechanism against foreign RNA molecules
• RNAi destroys double stranded RNA, initiated by dicer protein complex, dicer cuts RNA into siRNA’s (small interfering RNA)

Question 18

siRNA’s

Answer 18

• Can interact with argonaute and RISC proteins & follow miRNA route to destroy double-stranded RNA or interact with argonaute and RITS to regulate transcripton

Question 19

siRNA with argonaute and RISC

Answer 19

• RNA degradation

Question 20

siRNA with argonaute and RITS

Answer 20

• Transcriptional silencing
• RITS interacts with newly transcribed RNA, recruits chromatin modifying enzymes to make it more compact so no more transcription of viral DNA

Question 21

Prokaryotic immunity: CRISPR-Cas immunity

Answer 21

• Short fragments of viral DNA integrate into the CRISPR region of genome & become templates to produce crRNA’s (CRISPR RNA’s)
• CRISPR region is DNA sequences from all the previous viral infections
• Viral DNA’s complementary to CRISPR regions are directed for degradation by Cas (CRISPR-associated) proteins: use RNA to destroy DNA
• RNA bound to Cas protein (crRNA’s), so now if it is infected by the same virus again it has memory and chops that DNA