Lecture 33: RNA Processessing Flashcards
spliced functional RNA products
generated after cleavage
Processing of mRNA transcripts
5’ capping (protect from degredation)
intron splicing (take out non coding regions)
polyadenylation (protect from degredation on 3’ end)
(also cleavage)
how can multiple protein be derived from 1 gene
alternative intron splcing
use of differential polyadenylation sites
Micro RNAs
short double stranded RNA molecs interferring RNA molecs (RNAi) target mRNA degredation or to interfere w/ translation to decrease expression important for gene regulation
where does RNA processing happen?
in nucleus
after transcription
BEFORE exportation of mRNA to cytoplasm for translation
what is nascent RNA
pre RNA
pre rna has…
both exons (coding) and introns(non-coding) need cleavage and religation to get mature RNA product
rRNA and tRNA modifcations
modification at ribonucleotide bases
to produce a variety of methylated products
structural roles
modified bases required for function
unlike mRNA!!! its just a sequence, a message to go to a protein
S
spedbergs
sedimentation properties
the larger the number, the more sedimentary it will be when ceterfuged
rRNA processing
in nucleolus
make mature 60S (large subunit) and 40S (small subunit) ribosomal complexes before exportation to cytoplasm
modification of rRNA and tRNA
bases modified at specific sites BEFORE processing
produces variety of methylated products
needed for function
45S rRNA contains…
28S, 18S, and 5.8S genes
portions that eventually encode the mature rRNA
rRNA modfication happens in nucleolus
tRNA processing
remove intron and add 3’CCA sequence
remove 5’ and 3’ sequences and an intron near anticodon sequence
base modification
adopts cloverleaf structure
3D: L structure
all have basically same shape
to get amino acid attachment by a tRNA synthase enzyme we need…
prescence of specific modifed bases
anticodon sequence
addition of CCA to 3’
review figures on slide 6
review figures on slide 6
processing of mRNA transcript
5’ gets gaunine cap
introns removed
add 80-250 adenine residues to 3’ end
Ovalbumin gene: mRNA processessing
77000 basepairs
one of first genes studied for processing
primary transcript is huge, extra RNA at 3’ end
add cap, splice, cleave, polyadenylate
end up w/ mature RNA of 1800 nucleotides long
Proccessing seems wasteful: Why do it??
so that we can get many different proteins out of just one gene
differential splicing
mRNA Processing: CTD
Carboxy terminal domain of RNA Pol II has 50 repeats of serine rich amino acid sequence that some become phosphorylated (after initiation of RNA synth)
phosphorylated CTD region….
binding site for enzymes needed for 5' capping intron splicing 3' polyadenylation so things can happen right away
What is the advantage of mRNA processing factors binding to the phosphorylated CTD?
they’re ready to work as its being made
makes things a lot faster
5’ Capping: how
cap synthing complex binds to CTD
catalyzes addition of GTP to 5’ NTP (4 step rxn; methylation of guanine base and ribose sugar of 5’ NTP (Adds cap)
5’ cap synth complex dissociates and is replaced by the cap binding complex
this dissociates when RNA Pol II disengages from DNA templaet
why cap?
to stop other enzymes from degrading the mRNA
Self Splicing reactions
intron has sequence that lets it adopt a structure that lets it catalyze the reaction to splice
what enzyme catalyzes RNA splicing?
ribozymes
What is splicing?
removal of introns
group 1 introns
a few particular sequence must be conserved in order for the right orientation to facilitate this reactio
intron is removed in rxn catalyzed by GTP mediated nucleophilic attack on RNA sequence in the intron
bind to intron
new 3’ end, make new attack, cleave
exons covalently joined again
splice junctions
junctions between exons and introns
things need to be here for splicing
Group 2 introns
a few particular sequence must be conserved in order for the right orientation to facilitate this reaction
fungi
nucleophile for reaction is hydroxyl group fron adenosine in the intron attacks
lariat structure formed (HOWS ITS DIFFERNFT FROM GROUP 1)
formation of 2’-5’ phosphodiester linkage
lariat structure gets released at the end
happens only once per mRNA
true or false: most introns are released by self splicing
False
most introns are removed by…
action of small nuclear RNA (snRNA) within small ribonucleoproteins (snRNPs)
5 snRNAs contained in snRNPs
U1 U2 U4 U5 U6
snRNP happens when snRNAs associate with proteins
What do snRNAs do?
mediate splicing reaction using sequences at the 5’ and 3’ splice junctions and w/in the intron
U1
binds to 5’ splice site
U2
binds to branch site and forms part of catalytic center
U4
binds to 5’ splice site and then 3’ splice site
U5
masks catalytic activity of U6
U6
catalyzes splicing
what recognizes 5’ splice junction?
U1 snRNP
what recognizes intron branch site?
U2 snRNP
what binds to U1 and U2 to form almost active splicesome complex?
U4/U6 snRNP and U5 snRPN
what activates the inactive splicesome complex?
Then what happens?
ATP mediated conformational changes
this results in formation of active U2, U5, U6 splicesosome (U1 and U4 leave)
lariat is formed, cleavage occurs, the intron gets released
What are RNA catalyzed cleavage steps and lariat formation similar to?
mechanism for self cleavage by group II intronts
Final step of splicing
splicesome and intron complex dissociate
5’ and 3’ junctions of 2 adjacent exons are ligated together
How do snRNPs know the difference between an exon and intron sequence? i.e. what makes splicing so precise?
Base pairing! (recognition by U1 and U2)
there IS structire to mRNA
Polyadenylation
cleavage signal at 3’ end of gene is transcribed, this marks transcript for cleavage by endonuclease
transcript is released from DNA template
polyA tail added by enzyme poly(A) polymerase
what is splicesome associated with?
CTD of RNA Pol II
so they are ready to act on intron as soon as it has been synthed
this all hapens while the RNA is synthed
why would the cell do all these modifications?
we have a lot more proteins than we have genes
we can do alternate RNA processing to get this diversity
Ways to get diversity
alternate RNA processing
a little bit from things like antibodies and alternate start site
2 primary mechanisms of alternate RNA processing
differential RNA splicing alternate Poly (A) sites
how to get alternate RNA processing
skip splice sites
combine splice sites
regulatory splicesomes ignroe splice sites (tissue specific expression)
when we also consider alternative poly (A) sites, there are huge numbers of possible mRNA transcripts
could be random or due to the fact that different tissues recognize/use different things
Defective spliceing and human disease
point mutation in intron
of beta globin gene has been linked to decreaed hemoglobin (thalassemia) due to degredation of an abnromaly spliced mRNA
what kind of mutations cause defects in alternative splicing
point mutations
deletions
micro RNAs
noncoding RNAs in cells that are complementary to regions of “target” mRNA transcripts
22 nucleotides long and double stranded
partially processed in nucleus and then cleaved by Dicer enzyme in cytoplasm
Micro RNAs and function
interferring RNAs
incorporated into RISC protein complexs
to mediate mRNA cleavage or translational repression
How might miRNA be useful to eukaryotic cells? (Hint: protection and regulation)
degrade RNA viruses and modulate expression of regulatory proteins
down regulate mRNAs
how MiRNAs work
make premiRNA in nucleus, export recognized by DIcer Direcer makes duplex from hairpin unwind duplex with helicase load with RISK, its MATURE! target either near perfect or partial complementarity
partial complementarity of miRNA and RNA
repress translation from the RNA
near perfect complementarity of miRNA and RNA
cleave the targeted RNA
