Gerber L2 RNA Processing Flashcards
mRNA accounts for how much of transcribed RNA
1.5%
what is the start codon
AUG
what is the stop codon
UAA, UGA, UAG
RNA binding proteins (RBPs) comprise what % of protein repertoire
3-11%
RNA binding proteins have what structure
modular structure comprised of RNA-binding motifs
the CARBOXY-TERMINAL domain of RNA Pol ll does what
- acts as a scaffold for assembly and coordination
- recruits RNA processing enzymes:
- 5’ cap formation enzyme
- splicing proteins
- 3’ end processing factors
describe the structure of the 5’ cap
- consists of 7-METHYL GUANOSINE attached to the 5’ most nucleotide through a 5’-P-P-P-5’
- the 2’ OH group of ribose 1 and 2 can be methylated
what is the function of the 5’ cap in eukaryotic mRNAs
- 5’ cap marks RNA molecules as mRNA (other RNAs don’t have cap)
- regulates NUCLEAR EXPORT of mRNAs
- protects mRNAs from RNA digesting enzymes
- promotes translation through interaction with a translation initiation factor (eIF4e, the cap-binding protein)
what is splicing
moval of introns from the pre-mRNA
describe splicing
1) TRANSESTERIFICATION
- 2’OH group of branch-site adenosine (A) attacks the phosphate group at the 5’ splice-site intron
2) 3’OH group at 5’ splice-site attacks phosphate at 3’ splice site
3) Branched lariat is formed and rapidly degraded by nuclear exosome
snRNAs are what
small nuclear RNAs
what is the structure of the spliceosome
5 snRNAs
~170 proteins
how is splice site determined
give examples
CONSENSUS SEQ
base pairing between seq in non-coding RNA (snRNAs) and “splice-site” seq
1) U1 snRNA has a complementary seq to exonintron splice site, O base pairs
2) U2 snRNA : pre-mRNA annealing at branch point
where does U1 snRNP ( small nuclear ribonucleoproteins) assemble
1) U1 snRNP assembles at the 5’ splice site; U2 snRNPat the 3’splice site; Splicing factor 1 (SF1) at the branch-point A
2) trimeric snRNP complex (U4, U5, U6) joins to form the spliceosome
3) Rearrangement of base-pairing interactions to from catalytically active spliceosome. U1/U4 snRNPs released
4) Catalytic core catalyses the first transesterification reaction > intron lariat is formed
5) Further rearrangement joins the two exons in a second transesterification reaction.
6) The excised lariat intron is converted to a linear RNA by a debranching enzymes and degraded by the exosome
how does exon size compare with introns
~10x shorter than introns
- avg length = ~150 bases
- ~10 axons per gene
introns
- avg ~1.5kb length
- largest= 1.1Mb
which additional factors are used to guide snRNPs to splice sites
SR PROTEINS
- serine arginine rich proteins bind preferentially to EXON seq
- Heterogenous nuclear ribonucleoproteins (hnRNPs) preferentially bind intron sequences
what is the purpose of alternative spliicing in euk
generating mRNA variants from the same gene
- alternative exons can gen different transcripts which are translated
- cell/tissue specific splicing
- allows for high diversity
give an extreme example of alternative splicing
DSCAM gene
functions:
- immune syst (pathogen phagocytosis)
- neurons (cell-surface molecule to specify neural connections)
what is alternative splicing controlled by
activator and repressor (simply sits on the splice site and hinders the access of the spliceosome to the splicing reaction)
mutations of splice sites can cause what?
DISEASE :(
1) BETA-thalassemia
- INHERITED blood disorder (autosomal recessive)
- severe anaemia due to low Hb
- mutation in splice sites in beta globin gene
2) MYOTONIC DYSTROPHY
- Neuromuscular disease, 2 types (DM1 and DM2)
- DM1: Depletion of a splicing factor (MBNL) leads to mis-splicing of pre-mRNA targets.
3) CF
4) PARKINSON’S
5) RETINITIS PIGEMENTOSA
6) PREMATURE AGEING
7) CANCER
mechanism of 3’ cleavage
components
req protein complex consisting:
- CPSF(cleavage and polyadenylation specificity factor)
- CstF(cleavage stimulatory factor)
- Two cleavage factors (CFI, CFII)
- Poly(A) polymerase (PAP)
mechanism of 3’ cleavage
1) CPSF binds to the AAUAAA
sequence, CstF to the U-rich
sequence, creating a loop.
2. PAP joins the complex; RNA is cleaved
3. CStF and CFs are released, and PAP
adds ~ 10 As to the new 3’ end.
4. Poly(A) binding protein II (PABPII) binds to the short poly(A) tail and
stimulates further addition of Adenosines
5. The whole poly(A) tail (~200 Adenosines) is
ultimately covered by PABPII.
what is the function of the poly (A) tail in euk
- Required for export of the mRNA from the nucleus to the cytoplasm (binding of PABPII)
- Promotes translation initiation and translation
- Stabilizes the mRNA > shortening of poly(A) tail may lead to reduced translation and eventual decay of the
mRNA
why is regulated cleavage and polyadenylation important
determines whether antibodies are secreted or remain membrane-bound
what are the 5 patterns of alt splicing
1) exon/intron skipping
2) intron retention
3) alternative 5’ splice site
4) alternative 3’ splice site
5) mutually exclusive exons (dep on direction)
how does cleavage and polyadenylation determine whether ABs are secreted or membrane bound
- if resting B cell, low CstF
- poly A tail added
- causes splicing of intron
- membrane domain to keep ABs membrane bound is encoded
- AB attached to surface of membrane
or
- signal comes to activate B lymphocytes
- high CstF
- O weak poly A site
- splicing machinery assembles on weak poly A site
- causes shorter pre-mRNA
- HYDROPHILIC domain encoded
- O ABs can be secreted
how are substances exported out of the nucleus
- small mol and proteins can diffuse through thr membrane
- macromolecules (RNP) need active transport
how is mRNA exported out of the nucleus
- cytoplasmic cap-binding protein (eIF4e)
- nuclear cap-binding protein, nuclear poly A binding protein
- nuclear export factors (NXF1/T1), these are released in cytoplasm and reimported to the nucleus. Req energy
