RNA Processing Flashcards
5’ cap [7-methyl-guanosine] synthesis steps
The first nucleotide added to the RNA does not lose its phosphate group. This is taken advantage of in this mechanism
1) Removal of one phosphate from first nucleotide in mRNA (results in ppNp)
2) GTP loses inorganic pyrophosphate (-ppi) and binds to ppNP forming (GpppNp)
3) S-adenosyl-methionine (SAM) adds methyl groups to N-7 of the guanosine and sometimes the 2’-OH of first two sugars (riboses) in the RNA.
m7GpppNp
1 phosphate from GTP and 2 from the original nucleotide.
poly A tail
There will be heterogeneity because it contains cellular and viral mRNA
1) PolII extends past site where Poly(A) tail needs to be added
2) Complex of proteins (associated w CTD of polII) bidns to the desired location of the polyA tail
- Two sequences demarkate the cleavage site. (AAUAA ~30 nucleotides to the 5’ end of cleavage site and (less conserved) sequence ~20-40 nts downstream from RNA cleavage site
3) Endonuclease in complex cleaves at desired site using the sequences as a guide
4) Resulting 3’-OH group accepts A residues from enzyme polyadenylate polymerase. Each addition of A kicks out an inorganic pyrophosphate
Group I Intron Splicing Mechanism
Self-splicing mechanism does not need any NTP or proteins in the process. Requires an external nucleophile (Guanosine).
Two Transesterification Reactions
1) An external nucleophile the 3’-OH of the guanosine carries out a nucleophilic attack on the 5’ splice site.
2) The 3’-OH of the 5’ exon carries out a nucleophilic attack on the 3’ splice site.
End result: we have linear exons joined together and an intron released.
Group II Intron Splicing Mechanism
Self-Splicing, does not need any NTP or proteins in the process.
Two Transesterification Reactions
1) The 2’-OH of a specific adenosine found within the intron carries out a nucleophilic attack on the 5’ splice site.
2) The 3’-OH of the 5’ exon carries out a nucleophilic attack on the 3’ splice site.
End result: release of a lariat introns and exons are joined together.
Splicesome [Assembly]
1) U1snRNP and U2snRNP match 5’ splice site and branch point A, respectively (needs ATP to mark sites
2) U4/U6 +U5 are assembled in between the markers (inactive splicesome, also needs ATP)
3) U1 released and U6 takes its place (U5 forms specific base pairing interactions with 5’ exon)
4) U4 released and (U6 and U2 catalyze the reaction) [U2 stays put throughout]
Splicesome [Actual chemistry]
1) 2’-OH on adenosine attacks 5’ splice site
2) 3’-OH of 5’ splice site attacks 3’ splice site liberating the lariat with the proteins
Intron removal from tRNA
1)Nuclease removes intron thus leaving 2’,3’ cyclic phosphate on 5’ exon (on left) and 5’ OH on 3’ exon (on right)
2) Cyclic phosphodiesterase opens the 5’ exon (phosphate group now on 2’ and hydroxyl on 3’)
3) 5’OH (exon on right) gets phosphorylated
4) AMP added to phosphate (by ligase adenylated on lysine)
5) 5’ Exon (left) links to 3’ Exon (right) by kicking out AMP (now leaving 2 phosphates between the exons; one on 2’ OH and one between exons)
6) 2’ phosphate on 5’ (left) exon gets removed
RAN cycle (out of nucleus)
1) RNA associates with exportin and RAN-GTP
2) Complex moves to cytoplasm through NPC
3) GTP hydrolyzed. RAN-GDP and exportin fall off. RNA is dropped off