RNA Metabolism: RNA Processing Flashcards
Processing of eukaryotic Pre-mRNA
1) 5’ end (capped) 2) internal (splicing introns out) 3) 3’end (polyadenylation)
What is capping?
capping occurs when ser 5 is phosphorylated; nucleotide is added in wrong direction ( connected via 5 prime end with 3’OH on end); 7 methyl guanine (methylation allows for binding to protective proteins); tethered to ctd tail
What is the purpose of capping?
1) protects from exonuclease activity (exonuclease can’t work because of incorrectly placed nucleotide) 2) RNA transport into cytoplasm 3) Translation
3’ Polyadenylation
1) cleavage of RNA 10-30n downstream of 3’ UTR 2) addition of A’s (80-250) to generate a poly A tail
what are the only mRNAs that have poly A tails?
only those who are produced by RNAPII
CPSF
Cleavage/polyadenylation specificity factor (3 subunit protein) recognizes AAUAAA; recruits CtsF (cleavage factors); Recruits PAP (poly A polymerase).
AAUAAA
sequence recognized in RNA 3’ UTR that CPSF binds to.
What are the two different models of termination in eukaryotes?
Torpedo and allosteric termination.
Torpedo Termination
rat1/hxrn2 (exonuclease) tracks along with RNAP,. Pulls RNA out of RNAP. Once it pulls RNA out, RNAP disassociates.
Allosteric Termination
Proteins alter conformation of RNAP causing termination
Why is it more advantageous to have more introns?
1) Diversity 2) regulation via splicing produces different types of mRNAs and subsequently different proteins 3)
Group I/II splicing
Self-splicing; do not require specific factors or ATP; requires two transesterification reactions
Group I
self-splicer; uses guanine nucleoside cofactor; occurs in rRNAs of tetrahymena and mitochondrial regions; 1st cleavage performed by external cofactor guanine
Group II
self-splicer; uses an A residue within intron= forming a branched lariat structure; found in mitochondria and chloroplast pre-mRNAs; 1st cleavage is carried out by 2’OH of A within intron.
Group III
Eukaryotic; spliceosomal introns; the largest class;
Group IV
requires for some tRNAs- requires ATP and an endonuclease.
Group III mRNA mechanism
Step I: cleavage at the 5’ splice site (GUintron; AGexon) and joining of the 5’ end of the introns to the branch point A within the intron, producing a lariat-like intermediate 2) Cleavage at the 3’ (AGintron; G exon) splice site and simultaneous ligation of the exons, resulting in excision of the intron as a lariat- like structure
SnRNP
small nuclear ribonucleoprotein particles; involved in splicing;
What makes up a spliceosome?
SnRNPs and pre-mRNA
Model of snRNP mediated splicing of pre-mRNA
1) U1 finds 5’ GU exon-intron junction (recruited by ASF/SF2 a SR- Ser-Arg-rich region) 2) U2AF sits on pyrimidine track sits between A and 3’ GU of intron 3) U2AF recruits BBP 4) BBP replaced by U2 loop outed 5) U4 (masks U6’s ability to react with U2)/6 (catalytic activity binds to U2)/5; 5) tripothide interacts with U2 and U1. U1 exits, U4 exits, U6 can now interact with U2. 7) Adenine attacks junction forming lariat
How does the spliceosome find the splice site reliably?
1) Coupling with RNAP. Spliceosomal machinery is present at high concentration at poly A signals 2) Associating splicing factors with CTD
Exonic splicing enhancers
ESEs; binding sites for SR proteins; SR proteins interact with each other and recruit the splicing machinery.
U5
helps in finding other splice sites; helps keep U4 and U6 together.
U1
U1SnRNP binds to the 5’ splice junction
