BCH3031 Lecture Notes - Lecture 9: Eukaryotic Initiation Factor, Open Reading Frame, Guanosine
25 May 2018
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The Life Cycle of Eukaryotic mRNAs
Lecture 9 – RNA transport and localisation
• DNA (transcription) → mRNA (translation)→ protein
• Origin of life – RNA world
• Active RNAs involved in
o RNA splicing
▪ Cell splicing extrons
o Protein synthesis
o Expression regulation
▪ miroRNAs
▪ siRNA
▪ Riboswitches (sensor molecule)
mRNA Synthesis
• Before mRNA can be exported from nucleus – has to go through series of
steps in order to be translated in cytoplasm
1) Capping
• Modified nucleotide on 5’ RNA (protect 5’ from degradation)
• Difficult to degrade – essential for mRNA stability
• Transport translation
• 3 phosphate groups linking – impermeable to enzyme
• Adding the cap
o Cleave 5’ triphosphate of primary transcript
o Add guanosine residue via 5’-5’ linkage
o Methylate cap guanosine
o Methylate 3’ hydroxyls of first several bases
o Provide directionality for RNA
• Cap binding complex (CBC) – nuclear protein with two subunits: 20kD
(CBP20) subunit binds Cap 80kD (CBP80)
o Required to guide splicing
o RNA stabilization
o Transport into the cytoplasm where mRNA exchanges CBC for
eukaryotic initiation factor 4 (eIF4E)
2) Splicing
• Removal of introns
• Pre-mRNA (hnRNA) is cleaved at specific intron/extron
boudnaries
• Splicosome – cleaver
o 5 snRNAs
o Contains over 100 proteins
o Splices most protein encoding mRNAs
o rRNAs, mitochondrial RNAs self splice
• A is where 5’of both ends is joined
• Get conformational changes that help bring mRNA exons
together
• Key output: open reading frame
• How to know where intron starts and where it stops:
o Conserved frequencies found in exon and intron
boundaries
find more resources at oneclass.com
find more resources at oneclass.com
o 90% of 5’ exon 1 start with AG
o Base pairing is important to identify
o System driven by noncoding small RNA
▪ U1 – snrnps (small nuclear RNA): helps recruit proteins on
RNA complex (costs energy to unwind RNA)
▪ Can have replacement of U1 with U6
o Also involves U2, U4/U6 and U5
o Exon junction complex: important where it sits*
▪ Joins 5’ of joining of two exons – makes sure RNA is properly
formed
• Humans/worms/fly all have exons around 200 base pairs long (peak)
o Ranges 0-1000 bp
• Introns range: 0-30,000
• SR proteins: rich in syrinine and argines
o Bind with elements in exon
o Help recruit U1 and U2 – sit in close proximity
• Alternative splicing
o Most mammalian genes encode for more than one protein through
having different exons joined together
o Genome size doesn’t correlate with organismal complexity
▪ Increased alterative splicing appears to correlate with
organismal complexity
o Consequences of alternative splicing: skipping (regulated process)
▪ ORFs with different exons – increase proteome diversity: gene
X; 5 exons
▪ U2AF binds to exon 1 – induces stop condon → truncated
protein
▪ Sxl competes with U2AF → displaces U2AF binding →
splicing machinery can’t get access → U2AF will bind further
down and will cap out the stop codon → make functional full
length protein that drives phenol development in fly
• Sex determination of fly is based on this competition
between Sxl and U2AF
▪ ORFs with different 3’ UTR
▪ Regulation of gene expression:
• Localisation
• mRNA stability
• Translational efficiency
3) Cleavage of Polyadneylation → co-transcriptional processing
• Recognition of target sequences on RNA by cleavage factors
• Cleavage and polyadenylation specificity factor (CPSF)
o Recognises AAUAAA in 3’ UTR → recruits PolyA cap
o 3’end cleavage and polyadenylation occur
as a single complex
o Cleavage event provides an indirect signal
for transcription termination
o Occurs at 3 end of virtually all eurkaryotes
find more resources at oneclass.com
find more resources at oneclass.com
