Eukaryotic RNA Processing Flashcards
Actinomycin D
Inhibitor of Transcription
- slips into the major groove between the DNA strands (intercalates), preventing the unwinding of the DNA template strands
- inhibits initiation and elongation
- effective in both prokaryotes and eukaryotes
In bacterial mRNA, the 5’ end would have the structure:
5’ PPP
b/c mRNA (bacterial) has no processing and it means the 1st base is a triphosphate and it won’t be cleaves
What is hnRNA?
heterogenous nuclear RNA –> precursor of mRNA in eukaryotic cells
Post-transcriptional modification in mRNA (eukaryotes)
5’ cap
3’ poly A tail
Splicing to remove introns
Where does post-transcriptional modification happen in eukaryotes?
Nucleus, to allow for transport into cytoplasm
mRNA cap
GTP added backwards to 5’ end to form 5’-5’ triphosphate linkage thru Guanylyl Transferase
How many phosphates released in making the mRNA cap?
Release of gamma phosphate from RNA and release of PPi from GTP
Function of Poly-A tail-3’ end
related to transcription termination
Protects 3’ end from degradation
Stabilization of mRNA
Function of AAUAAA in poly A tail formation
Functions as a signal for dephosphylation and endonuclease to cleave upstream 3’ –> poly A polymerase using ATP adds around 200 A’s –> poly A binding proteins bind for protection of 3’ end and stabilizes mRNA
What does splicing require?
Involves snRNA + proteins= snRNPs
U1,2,4,5,6
2’-5’ phosphodiester bonds at branch point
Consensus Sequences on Exon/Intron borders
All introns begin with GU and end with AG
What is the branch point
An A located in a pyrimidine-rich sequence approx 50 bases from the 3’ end of the intron
Role of snRPs
Involved in mRNA precursor splicing
contains one small snRNA and several proteins
U1
Binds to 5’ splice site (there can be mismatched sequences in this bindings, but there is a conservative sequence at which U1 binds to)
U2
Binds the branch site and forms part of the catalytic center
U5
Binds the 3’ splice site, loops over to the 5’ site
U4
Masks the catalytic activity of U6, until ready to splice
U6
Catalyzes splicing- it is a ribozyme
Why does U1 snRNP bind the 5’ splice site?
Bc it is complimentary to the sequence on the 5’ end
Pathway for Spliceosome Assembly
1) U1 binds to 5’ end of intron
2) U2 binds to branch site
3) U4,5,6 complex comes in and brings exon1/intron (U1 area) junction closer to the branch site (A) thru looping –> creation of spliceosome
- -> structured so that A is close to the 3’ end of exon 1
4) U1/U4 leave
Actual splicing pathway
1) U2 and U6 have complementary sequences and can base pair- they form the catalytic site
U6 catalyzes:
1) attack of 2’ OH on A to the 5’ splice site
2) Newly formed 3’ OH on exon 1 attacks the 3’ splice site on the other exon still attached to the intron
3) Exons are joined
4) Intron is released in the form of a lariat
Key Points in Splicing
Highly regulated process- introns removed in order unique to each mRNA
Alternative splicing
Mutations in splice sites are involved in many diseases processes
RNA binding proteins
Facilitate alternative splicing
Also important as many genes are alternatively spliced in malignant cells
Blocks snRPs from binding so those exons are excluded from specific tissues
SR
Serine/arginine protein (positive-favors splicing)- binds on exons
hnRNPS
heterogeneous nuclear ribonucleoprotein (negative- inhibits splicing)
binds both on exons and introns or both
ESE, ISE
exon splicing enhancer- binds SR
intron splicing enhancer- binds SR
ESS, ISS
exon splicing silencer- binds hnRNP
intron splicing silencer- binds hnRNP
beta-thalassemia major
one base mutation in the beta globin gene (G to A)
causes low beta globin protein and severe anemia
Single point mutation of G –> A in an intron of the beta-globin gene results in mis-splicing..creates a new 3’ splice site –> protein cut short and not functional
Variant exons
V5 forms in many metastatic cancers–> facilitates movement of cancer cells
Inclusion of variant exon
SAM binds ESS site –> hnRNP cannot bind –> SR binds –> U2 binds –> splicing occurs –> exon will be included in that variant protein
Exclusion of variant exon
hnRNP + silencer –> prevents binding of SR proteins and blocks E2 from binding –> prevents splicing of variant exon –> variant exon expressed in final product
