processing of mRNA transcripts Flashcards
what are the two elements, added post-transcription, to eukaryotic mRNA
m7G Cap and poly A tails
are m7G Cap and poly Z tails encoded in the genome
No
what are the primary transcript processing methods
Capping, splicing, polyadenylation and editing
How is primary transcript processing coupled to transcription
RNA pol II CTD
What does RNA initially contain at 5’ end
triphosphate
what are the two steps in capping
1) GpppN structure
2) methylation
what process alters chemical behaviour of bases
methylation
what is the functions of m7G cap (4)
- protects mRNA from degradation by 5’-3’ nucleases
- Facilitates splicing
- Facilitates export from the nucleus
- Critical for translation of most mRNA
How are the m7G cap functions mediated
Through protein binding
CBP80/CBP20 in the nucleus and elF4 complex in cytoplasm
what does elF4 complex mediated in relation to m7G cap function
translation
what does CBP80/CBP20 mediated in relation to m7G cap function
processing/export
what is the order in which post-transcriptional modification
1) transcription and capping
2) Splicing and polyadenylation
which is generally longer in mammals, introns or exons
introns
Intron and exon boundaries contain conserved sequences, what and where are they ?
5’ splice site - exon 1
3’ splice site - exon 2
branch site - intron
what are the conserved sequences in exon/introns required for
- define limits of exon and intron
- Sequences recruit the splicing machinery required to remove the
intron and join the exons
what type of reaction are the two steps of splicing of introns
2 - trans-esterification
what occurs in the two steps of intron splicing
1) cut at 5’ splice site & creation of bond between 5’ end of intron and branch site
2) cut at 3’ splice site to release intron lariat & ligation of two exons
what are spliceosomes
enzymatic complex that catalyses the removal of introns
what do spliceosomes require
ATP
what proteins make up spliceosome
- large complexs containing RNA-binding proteins, ATPase, GTPase and several snRNPs
what are snRNPs
Small nuclear ribonucleoprotein particles
what do RNA do in terms of splicing
RNAs base-pair with conserved sequences in the intron (5’ splice site, branch point)
*Splicing is catalysed by the snRNAs
what are the two types of snRNPs
Sm proteins and snRNAs
what reacts against Sm proteins
Anti-Sm antibodies
when would a person have anti-Sm antibodies
very rare unless a person has autoimmune disease systemic lupus erythematosus (SLE)
what percentage of patients with SLE have anti-Sm antibodies
20%
what catalyses the splicing mechanism
RNA
explain Step 1 of the splicing mechanism using RNA
1) U1 & U2 binds to the 5’ and binding site respectively
2) U4,U6+U5 is then recruited, then U1+U4 is release
3) A bond is created between 5’ end of intron and the branch site
4) U6,U5 and U2 holds the free exon 1 together to the looped intron (still connected to exon2)
what is the E complex
U1 at the 5’ splice site of the intron
what is the A complex
U1 at the 5’ splice site of the intron
U2 is at the branch site
What is the B complex
all RNA (U1,2,4,5,6) are joined along mRNA
what is the B* complex
The B complex after U1+U4 is released
What is the C complex
mRNA with U2,U5,U6 joined after intron lariat being formed and exon one being freed
explain Step 2 of the splicing mechanism using RNA
The spliceosome cuts the mRNA at 3’, U2,6,5 hold the 2 exons together and allows them to bind
- lariat is released
why is the number of proteins greater than genes in the genome
because there can be different organisations/arrangements of the exons after splicing
what are the 3 conditions caused by mutations causing defects in splicing
- Spinal muscular atrophy
- Retinitis Pigmentosa
- Myotonic Dystrophy
how does polyadenylation occur
- Endonuclease cleavage releases the mRNA from the DNA template when the a specific sequence is recognised
- Addition of As by PolyA polymerase
what is the recognised specific sequences which activates endonuclease cleavage
- AAUAAA (conserved), 10-35 nucleotides upstream of polyA site
what is downstream of poly(A) site
G/U or U rich tract
what is upstream of AAUAAA
U-rich element ‘USE’
what are the proteins required for polyadenylation, and where do they bind to
- Cleavage and polyadenylation specificity factor (CPSF) = binds AAUAAA
- Cleavage stimulatory factor (CstF) = binds G/U
-Poly(A) polymerase
what are the functional significance of the polyA tail
- Enhances export of RNA
- Stabilises the 3’ end of the mRNA
- Enhances translation of mRNA
give an example of a protein which doesnt have a poly A tail
histones
