RNA Processing Flashcards
RNA processing overview
addition of 5’ cap
poly A tail at 3’ end, 80-250 adenosine residues
RNA splicing to remove designated exons
protection of RNA
catalyzed by ribozymes (catalytic RNA)
5’ cap structure and function
added before primary transcript synthesis is complete
7-methylguanosine residue added to 5’ terminal residue of mRNA via unusual 5’-5’ phosphate linkage
occurs early, after 20-30 bases
function: protection from ribonucleases, binds to specific cap-binding complexes of proteins and helps bind mRNA to ribosome to initiate translation
5’ capping mechanism
4 enzymes tethered to CTD on Pol II synthesize 5’ cap
CBC (cap-binding complex) keeps cap tethered to CTD
intron/exon info
exons = coding regions for mRNA transcript, <1000bp
introns = non-coding regions, up to 20,000 bp
exception - histone genes do not have introns
more introns than exons by a lot (200k to 20k)
splicing: introns removed, exons spliced together to form continuous sequence that specifies a functional polypeptide
classes of introns
- I and II are self-splicing (no enzymes involved, no ATP)
- Spliceosome introns are removed by ribonucleoproteins (RNPs) AKA spliceosomes
- Protein catalyzed introns removed by enzymes
Group I splicing introns
Self splicing
1. 3’ OH of FREE guanosine acts as nucleophile and attacks phosphodiester bond of U-A intron-exon junction
2. OH (now attached to exon) attacks adjacent 5’ phosphodiester bond of intron-exon junction
3. this rejoins the exon pieces releasing the intron and free guanosine residue
Spliceosome splicing structure and types of RNA
most common introns in eukaryotes
spliceosome structure: made up of small nuclear ribonucleoproteins (100-200 snRNPs) and other proteins (100)
5 snRNAs involved and act as catalyst: U1, U2, U4, U5, U6
spliceosome and group IV introns require ATP
U1 Donor site is complementary to intron 5’, U2 acceptor site (3’)
U4-6 snRNAs make it a spliceosome
Spliceosome sequence beginning/end markers
Where U1/U2 binds
timing of all this shit
beginning of intron marked by GU 5’
end of intron marked by AG 3’ (end of splicing site)
U1 binds to 5’ GU, U2 binds to A at branching site
in tandem with transcription, some components of spliceosome are tethered to CTD
Spliceosome mechanism
- U1 contains regions complementary to 5’ splice site (Donor site) and binds
- U2 binds near 3’ end of intron (branching site), creates bulge which makes an adenosine a better nucleophile form 2’,5’ phosphodiester bond (lariat loop as a result)
- Addition of U4,5,6 snRNAs and protein friends leads to formation of spliceosome (requires ATP)
- U1 and U4 dissociate and cleavage occurs
- U2, U5 and U6 expelled with intron attached to U6
Poly A tail attachment mechanism
A tail added to 3’ end of most eukaryotic mRNA that undergo translation
function: serves as specific protein binding site and for protection against degradation
1. endonuclease cleaves RNA past cleavage signal sequence, 10-30 nucleotides 3’ downstream of conserved sequence
2. polyadenylate polymerase synthesizes poly A tail at beginning of cleavage site
proteins tethered to CTD for poly A tail attachment
polyadelylate polymerase
endonucleases
proteins involved in sequence recognition and stimulation of cleavage
ALL tethered to CTD
alternative splicing
mRNA transcripts that can be processed in different ways to produce different polypeptide products (ex. some exons may be excluded or included)
95% of human genes are alternatively spliced
processing factors promote particular path of primary transcript processes (all paths have molecular signals in transcript)
example alternative splicing of calcitonin
Primary transcript of calcitonin has 2 poly A tail sites
Thyroid vs brain, different exons removed, producing different polypeptide products
