Lecture 27: Regulatory Functions of RNA, Posttranscriptional regulation of protein levels, DNA editing

Question 1

RNA World hypothesis

Answer 1

• Nucleotides formed

• Dinucleotides
• Polynucleotides
• Catalytic RNA
• self replication
• protein synthesis

• DNA as storage for RNA

Question 2

rRNA processing examples

Answer 2

• eg psuedouridine
• eg 2’-O-ribose methylation

Question 3

pseudourine modification

(RNA modification)

Answer 3

• don’t know exact function… but important cumulatively
• are concentrated near active sites (domain II, IV, V)
  
  • domain V: P site of ribosome
• found in rRNA and splicing snRNAs
• RNA to be modified leaves one unpaired base pair: target for pseudourine modification
  
  • guided by Box H/ACA snoRNAs
• Dyskerin - pseudouridine synthase in H/ACA complex
  
  • disease: X-linked Dyskeratosis Congenita
    
    • affects rapidly growing cells (dysfunctional rRNA)

Question 4

2’-O-ribose methylation

(RNA modification)

Answer 4

• important for correct function of ribosomes
• found in rRNA and splicing snRNAs
• guided by Box C/D snoRNAs
  
  • box D: guide to select nucleotide to be methylated.

Question 5

RNA Editing examples

Answer 5

• C to U editing
• A to I editing

Question 6

C to U ediitng

(RNA editing)

Answer 6

• cytidine deaminase: changes C to U
• eg apolipoprotein B
  
  • enzyme: APOBEC/ACF - generates stop codon
    
    • ssRNA template
    • single strand template
  • in liver: no editing - creates LDL receptor domain
  • in intestine: site specific deamination - creates stop codon - different length protein

Question 7

A to I editing

(RNA editing)

Answer 7

• Adenosine deaminase (ADAR): adenosine to Inosine
  
  • presplicing
  • require dsRNA template
• A to I editing in mRNA - change codon
  
  • I is read as G
• examples
  
  • ADAR2 self-editing: autoregulation
    
    • by making early splice site
  • AA change
    
    • eg AMPA-type Glutamate receptors
      
      • regulate gating behavior
      • modification of channel kinetics
      • control of receptor trafficking
    • eg 5HT_2C Serotonin receptor
      
      • regulation of G-protein coupling efficiency
  • 3’ UTR editing
    
    • Tankyrase, NADH reductase: modulation of mRNA stability and transport
      
      • by affecting binding of proteins that confer stability

Question 8

Glutamate receptor

(RNA editing - A to I editing)

Answer 8

• eg AMPA-type Glutamate receptors
  
  • regulate gating behavior
  • modification of channel kinetics
  • control of receptor trafficking
• ​can make a membrane protein impermeable to Calcium 2+ ion

Question 9

Serotonin receptor

(RNA editin - A to I editing)

Answer 9

• can reduce activity of serotonin receptors

Question 10

Protein synthesis regulation examples

Answer 10

• eg Riboswitches
• eg miRNA - inhibit translation
• eg siRNA - mRNA degradation

Question 11

Riboswitches

(Protein Synthesis Regulation)

Answer 11

• control bacterial translation
• types

1. thermoswitches (RNA thermometers)
   
   • stable at 37C and melts at 40C
   • sigma32 mRNA activity activated by heat shock - opens hairpin - reveals S-D box and AUG - ribosomes can bind
2. Chemical regulators (metabolite sensors)
   
   • metabolite forces S-D base pairing - turns translation off
   • feedback inhibition
   • eg S-adenosylmethionine
     
     • translation on when unbasepaired.
     • SAM binds and forces basepairing - translation off

Question 12

miRNA (example of RNAi)

Answer 12

• Process

1. miRNA that comes from the nucleus has a hairpin
2. the dicer cuts off the hairpin structure and forms a short dsRNA molecule
3. these RNA mole. split and become ss
4. the ssRNA join RISC (RNA-induced Silencing complex) protein complexes
5. RISC binds to target mRNA
6. RISC either represses translation or cleaves mRNA
   
   • repress translation
     
     • short complementary segments in 3’UTR - base pairing mismatches
     • inhibits translation
     • regulatory
     • eg Lin-4 miRNA inhibits lin-14 trnaslation in C. elegans L1 to L2

• miRNA can repress translation or cleave mRNA based on base pairing

Question 13

siRNA

Answer 13

• natural and artificial pathway for down-regulating protein expression
• extensive complementarity in ORF or 3’ UTR (complete base-pairing)
• only degrades mRNA
• process:
  
  1. Dicer binds dsRNA to siRNA, forms siRNA duplex
  2. unwinds duplex
  3. siRNA binds to RISC (RNA-induced Silencing duplex)
  4. RISC binds to target mRNA
  5. mRNA cleavage

Question 14

purpose of RNAi

Answer 14

• defense vs.
  
  • dsRNA viruses
  • retroposons
• RNA-induced transcriptional silencing (RITS)
  
  • Histone methylation
  • DNA methylation
  • Chromatin condensation

Question 15

Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)

Answer 15

• adaptive immunity in bacteria and archaea
• If it encounters bacteriophage once, it will be immune after
• cleave viral DNA
  
  • proteins from operon incorporates viral genome into host genome (into CRIPSR array)
  • CRISPR array is transcribed and forms pre-crRNA
  • this pre-crRNA is cut and forms crRNA hairpin loops that are hybridized into Cas proteins for recognition
  • Cas proteins then can recognize viral DNA and cut them by endonuclease activity
    
    • wont digest its own DNA bc only cleaves after a specific GG nt sequence
  • consequence
    
    • nonhomolous end joining - gene disruption
    • homologous directed recombination - gene replacement
      
      • gene therapy