rna processing final eukaryotes
DESCRIPTION
goodTRANSCRIPT
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RNA Processing
M.Prasad NaiduMSc Medical Biochemistry, Ph.D,.
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Overview of the Eukaryotic mRNA Processing
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Eukaryotic cells process the RNA in the nucleus before it is moved to the cytoplasm for protein synthesis
The RNA that is the direct copy of the DNA is the primary transcript
Two methods are used to process primary transcripts to increase the stability of mRNA for its export to the cytoplasmRNA cappingPolyadenylation
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RNA capping happens at the 5’ end of the
RNA, usually adds a methylgaunosine shortly
after RNA polymerase makes the 5’ end of the
primary transcript
Splicing of introns removes the intervening
sequences in RNA
Polyadenylation modifies the 3’ end of the
primary transcript by the addition of a string
of As
Over all Processes
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Modified guanine nucleotideadded to the 5 end
Protein-coding segment
3 UTRStop codonStart codon
5 Cap 5 UTR
AAUAAA
TRANSCRIPTION
RNA PROCESSING
DNA
Pre-mRNA
mRNA
TRANSLATION
Ribosome
Polypeptide
G P P P
53
a) 5’ Capping of Transcript
Modified GTP is added, backwards, on the 5’ end
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After about 30 nt are added, 5’-P is almost immediately modified
A phosphate (terminal) is released by hydrolysisThe diphosphate 5’ end then attacks the alfa
phosphate of GTP to form a very unusual 5’-5’ triphosphate linkage – this is called condensation
This highly distinctive terminus is called a capThe N-7 nitrogen of the terminal G is then methylated
by S-adenosyl methionine to form cap0
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Uses of CappingCaps are important for subsequent splicing
reactions
They also contribute to the stability of mRNAs by protecting their 5’ ends from phosphatases and nucleases
In addition, caps enhance the translation of mRNA by eukaryotic protein-synthesizing systems
Note: tRNA and rRNA molecules do not have caps
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b) Poly-AdenylationMost Eukaryotic mRNAs contain poly A tailPoly A tail is not encoded by DNASome mRNAs contain an internal AAUAAA (AAU
= Asn, AAA = Lys). This highly conserved sequence is only a part of the cleavage signal, but its context is also important
The cleavage site is 11 to 30 nt away from the AAUAAA site on the 3’ side
After the cleavage by an endonuclease, 50 to 250 A residues are added by Poly adenylate polymerase
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50 to 250 adenine nucleotidesadded to the 3 end
Protein-coding segment Polyadenylation signal
Poly-A tail3 UTRStop codonStart codon
5 Cap 5 UTR
AAUAAA AAA…AAA
TRANSCRIPTION
RNA PROCESSING
DNA
Pre-mRNA
mRNA
TRANSLATION
Ribosome
Polypeptide
G P P P
53
Cleavage site
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Mutating the cleavage sequence in the parent DNA will result in mRNA that is not polyadenylated and not exported to the cytoplasm – instead it is rapidly degraded
A second downstream signal that is a G/U rich sequence is required for efficient cleavage and polyadenylation, and is located ca. 50 nucleotides from the site of cleavage.
The cleavage and polyadenylation specficity factor (CPSF), a large 4-subunit protein (ca. 360 kDa), forms an unstable complex with the AAUAAA sequence that is subsequently stabilized by the addition of at least 4 separate protein complexes that bind to the CPSF-RNA complex.
CstF: Cleavage stimulatory factor interacts with G/U rich sequence CFI: Cleavage factor I and CFII help stabilize protein-RNA complex PAP: Poly(A) polymearse binds to complex before cleavage occurs PABP: Polyadenylate-binding protein binds the Poly (A ) polymerase
Assembly of the cleavage/polyadenylation complex
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Cleavage and polyadenylation Specificity Factor
Cleavage Stimualtory Factor
(PABP)
Cleavage site
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(i)
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(ii)
CPSF
PAP
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(iii)
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TRANSCRIPTION
RNA PROCESSING
DNA
Pre-mRNA
mRNA
TRANSLATION
Ribosome
Polypeptide
5 Cap
Exon Intron
1
5
30 31
Exon Intron
104 105 146
Exon 3
Poly-A tail
Poly-A tail
Introns cut out andexons spliced together
Codingsegment
5 Cap
1 1463 UTR5 UTR
Pre-mRNA
mRNA
c) Splicing out Introns
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RNA splicing is responsible for the removal of the introns to create the mRNA
Introns contain sequences that act as clues for their removal
Carried out by assembly of small nuclear ribonucleoprotein particles (snRNPs) – Spliceosomes
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Spliceosome ActivitysnRNPs come together and cut out the intron
and rejoin the ends of the RNAU1 snRNP attaches to GU of the 5’ intronU2 snRNP attaches to the branch siteU4, U5 and U6 snRNPs form a complex
bringing together both U1 and U2 snRNPsFirst the donor site is cut followed by 3’ splice
site cut
Intron is removed as a lariat – loop of RNA like a cowboy rope
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(U1, U2, U4, U5 and U6)
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Mechanism of Splicing
1. The branch-point A nucleotide in the intron sequence, located close to the 3’ splice site, attacks the 5’ splice site and cleaves it.
The cut 5’ end of the intron sequence becomes covalently linked to this A nucleotide
2. The 3’-OH end of the first exon sequence that was created in the first step adds to the beginning of the second exon sequence, cleaving the RNA molecule at the 3’ splice site, and the two exons are joined
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Thomas Cech (1981)
Nobel prize in 1989
Exception: RIBOZYME
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Self-splicing of Intron SequencesGroup I intron sequences bind a free G
nucleotide to a specific site to initiate splicingGroup II intron sequences use s specially
reactive A nucleotide in the intron sequence itself for the same purpose
Both are normally aided by proteins that speed up the reaction, but the reaction is mediated by the RNA in the intron sequence
The mechanism used by Group II intron sequences forms a lariat and resemble the activity of spliceosomes
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Comparison
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Alternative Splicing Patterns1, 2A, 3 1, 2B, 3
1, 2A, 2B, 3 1, 3
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(Calcitonin-gene related protein)
Two predominant Poly(A) sites in Rats
Cell type specific RNA splicing
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Processing of pre-rRNA transcripts
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Benefits of SplicingAllows for genetic recombination
Link exons from different genes together to create a new mRNA
Also allows for one primary transcript to encode for multiple proteins by rearrangement of the exons
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RNA Editing
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How do mRNAs get to the cytosol?
Why do eukaryotes have DNA within a membrane bound compartment and prokaryotes do not?
Could eukaryotes function without it?
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Correspondence between exons and protein domains
GeneDNA
Exon 1 Intron Exon 2 Intron Exon 3
Transcription
RNA processing
Translation
Domain 3
Domain 1
Domain 2
Polypeptide
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Sequences removed are called Introns
Coding sequences flanking introns are called Exons
Exons are not removed and are in the mRNA
Intron removal is referred to as Splicing
Splicing is mediated by a particle: Spliceosome
A spliceosome is made of snRNA and protein
There are several snRNAs in a spliceosome, U1 to U6 Some introns have self-splicing sequences: Ribozymes
Conclusions