mRNA Processing

Question 1

3’ tailing/polyadenylation

Answer 1

• poly(A) tail is added to the 3’ end of RNA at the end of transcription
• signalled by AAUAAA consensus sequence in 3’ UTR
• recognized by CstF and CPSF proteins
• RNA cleaved from RNA polymerase II
• poly-A polymerase (PAP) adds ~200-250 adenine nucleotides to the cleaved 3’ end of RNA
• poly-A binding proteins attach to determine final length and keep RNA linear

Question 2

Where does mRNA processing occur in the cell?

Answer 2

in the nucleus before translation

Question 3

5’ capping

Answer 3

• 5’ cap consists of 7-methylguanosine forming 5’ to 5’ triphosphate bridge with the 5’ end of the primary transcript
  1) phosphatase removes a phosphate from the 5’ end of the new/nascent RNA
  2) guanyl transferase cleaves triphosphate group of GTP and adds GMP in reverse linkage (5’ to 5’)
  3) methyl transferase adds methyl to N7 of guanosine
• mRNA must have 5’ cap in order to be exported out of the nucleus and be translated !!

Question 4

RNA splicing

Answer 4

• process of removing introns
• performed by snRNPs (RNA molecules)
• occurs after capping and before tailing
  1) U1snRNP base pairs with 5’ splice site
  2) BBP + U2AF recognize branch point site (A residue)
  3) U2snRNP displaces BBP + U2AF
  4) U4/U6•U5 triple snRNP comes in and distorts structure of RNA
  5) U4/U6 break apart and U6 displaces U1 at 5’ splice site, creating the first phosphoryltransferase catalyzing active site leading to 5’ splice site cleavage and lariat formation
  6) -OH of 3’ end of exon 1 cleaves 3’ splice site, joining 2 exons and excising lariat intron

Question 5

exon/intron junction conserved nucleotides

Answer 5

• 5’ splice site: GU
• branch point: A
• 3’ splice site: AG

Question 6

snRNPs

Answer 6

• small nuclear ribonucleoproteins
• proteins + snRNAs
• function in identifying intron/exon junctions, cleaving phosphodiester bonds and ligating them

Question 7

spliceosome

Answer 7

a complex of snRNPs

Question 8

introns

Answer 8

• regulatory non-coding regions on the DNA

- spliced out; are not translated

Question 9

exons

Answer 9

• coding regions on the DNA that are spliced together after introns are removed
• become a part of the final mRNA

Question 10

alternative splicing

Answer 10

• when exons join in different patterns
• allows one gene to encode many different proteins
  (e. g. alpha-tropomyosin; the same gene can be alternatively spliced in different cell types)

Question 11

NPCs

Answer 11

• nuclear pore complexes
• aqueous channels in the nuclear membrane connecting the nucleus and cytoplasm, through which mRNA will pass though
• mRNA passes through 5’ cap first, from nucleus to cytoplasm

Question 12

CstF and CPSF stand for…?

Answer 12

CstF = cleavage stimulation factor
CPSF = cleavage and polyadenylation specificity factor