RNA processing Flashcards
What does it mean that RNA processing is tightly coupled with transcription?
RNA processing happens at the same time as transcription (rather than one after another)
what are the RNA processing that occurs in eukaryotes?
- RNA capping
- polyadenylation
- RNA splicing
what is RNA capping?
- process of adding a methylated guanine nucleotide at the 5’ end of mRNA
what is the function of the 5’ cap?
- protection in the nucleus against exonucleases
- ribosome binding site
what is polyadenylation?
- post translational
- addition of a series of repeated adenine (A) nucleotides to the 3’ tail of mRNA
How does polyadenylation occur?
- cleavage at consensus sequence
- addition of repeated adenines (A), generally a few hundred nts long
what cleaves mRNA at consensus sequence during polyadenylation?
CPSF and CStF binds consensus sequence and terminates transcription by cleaving off the end of mRNA
- CPSF = Cleavage and polyadenylation specificity factor
- CStF = Cleavage stimulation factor
what adds repeated adenines to the 3’ cut end?
PAP = polyadenosine polymerase
what is the function of polyadenylation?
- terminate transcription
- slows mRNA degradation in cytoplasm against deadenylating nuclease
- aid export through nuclear pores
- the targeting singal of mRNA
what is ribonucleoprotein formation of mRNA?
- performed by PABP (polyadenosine binding protein)
- PABPs recognises poly-A-tail and binds to it, making it a ribonucleoprotein => a complex of protein and RNA
Steps of mRNA degradation?
- de-adenylation
- degradation
what performs mRNA de-adenylation?
- DAN = deadenylating nuclease
- can play tug of war with cytoplasmic PAP (polyadenosine polymerase) => as DAN de-adenylates, PAP adenylates => slows degradation of mRNA
how does degradation of mRNA occur after deadenylation?
- deadenylation allows the recognition and destruction of mRNA by exosome
- exosome cut mRNA into small pieces before recycling
what do the 5’ cap and poly-A-tail do as a whole?
identify the molecule as a complete mRNA molecule
what is histone mRNA?
- mRNA that provides the histone proteins necessary for packaging newly replicated DNA
- tightly regulated, present in high levels only in S-phase
- lacks poly-A-tail
- form stem loops
what is a stem loop?
- Stem-loop intramolecular base pairing is a pattern that can occur in ssRNA
- an RNA secondary structure
- bound by stem loop binding protein (SLBP)
sequences at 3’ UTR (just before the poly-A-tail can be modified to target eukaryotic mRNA to specific locations in the cytoplasm.
3’ UTR = 3’ untranslated regions
- PABP (polyadenosine binding protein) bind to those sequences and cytoskeletal motors
- cytoskeletal motors then move RNA via actin and tubulin
what is the purpose of moving mRNA?
some mRNA encodes for proteins that functions in certain locations of the cell, moving the mRNA to that location means production/translation of those proteins will occur at their sites of function
what is the benefit of moving the mRNA instead of the whole protein?
mRNA is much more easier to move => more efficient
what is the purpose of RNA splicing?
remove introns (intragenic noncoding regions) and ligate exons (coding regions) together
what is precursor-mRNA (or pre-mRNA)?
mRNA before splicing has occured
what catalyeses splicing?
spliceosome: a group of snRNPs
- snRNP: small nuclear riboproteins = snRNA + protein(s)
- snRNA = small nuclear RNA
there are 3 blocks of specific consensus sequences in the intron which allows it to be cut out, where and what are those sequences?
- GU at 5’ start of the intron (invariable)
- AG at 3’ end of the intron (invariable)
- YURAC in between the start and end of intron act as the bridge, with A being the branch point (Y = pyrimidine, R = purine). Variable although the indicated nucleotides are prefered
- these sequences allow the intron to be cut out as a lariat structure
How is the lariat structure formed?
- A (branch point) nucleophillically attack 5’ splice site
- breaks sugar phosphate backbone between exon and intron
- cut 5’ end of intron becomes covalently linked to A at branch point
- the released free 3’ end of exon has -OH group that reacts with the start of next exon => joined together and release the intron in the form of a lariat
How does spliceosome catalyse splicing?
- snRNPs of spliceosome are called U particles
- U particles bind to different sites of intron to facillate looping and splicing:
- U1 base pairs with 5’ splice site
- U2 base pairs branch point
- U4/U6•U5 triple snRNP enters reaction, in this triple, U4/U6 are tightly base paired
- subsequent rearrangements create active site of spliceosome and positions the appropriate portions of pre-mRNA substrate for first phosphoryl-transferase reaction => cuts 5’ splice site
- further RNA-RNA reactions break the base pairing between U4/U6
- U4 released from reaction
- U6 allowed to displace U1 at 5’ splice site to form active site for second phosphoryl-transferase reaction => cuts 3’ splice site
- completion of splice
how arre capping (adding 5’ cap), tailing (adding As) and splicing factors associated with CTD (C-terminal domain) of RNAP-II
- phoshorylation of CTD by TFIIH enables binding of CBC (cap binding complex) and splicesome as soon as mRNA is formed
- CTD interacts with spliceosome, act as a staging post for splicesome so the supply of it never stops
- CTD also interacts with tailing factors, terminates transcription as soon as necessary
How does spliceosome ligate the right exons together when there are so many exons?
timing:
- splicing occurs as soon as mRNA is made
- splicesome pair exon with next exon available while later exons still haven’t been transcribed
what is alternative splicing
- alternative splicing is when:
- an exon is skipped
- an intron is included
- etc.
- can be used to produce variant proteins from a single gene
