RNA Processing Eukaryotes.

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RNA Processing
M.Prasad Naidu MSc 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 cytoplasm
• RNA capping
• Polyadenylation

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Over all Processes

• 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

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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 hydrolysis
• The 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 cap
• The N-7 nitrogen of the terminal G is then
  methylated by S-adenosyl methionine to form cap0

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(No Transcript)
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Uses of Capping

• Caps 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-Adenylation

• Most Eukaryotic mRNAs contain poly A tail
• Poly A tail is not encoded by DNA
• Some 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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Cleavage site
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Assembly of the cleavage/polyadenylation complex

• 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

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Cleavage site
(PABP)
Cleavage and polyadenylation Specificity Factor
Cleavage Stimualtory Factor
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(i)
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(ii)
CPSF
PAP
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(iii)
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(No Transcript)
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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 Activity

• snRNPs come together and cut out the intron and
  rejoin the ends of the RNA
• U1 snRNP attaches to GU of the 5 intron
• U2 snRNP attaches to the branch site
• U4, U5 and U6 snRNPs form a complex bringing
  together both U1 and U2 snRNPs
• First 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)
Exception RIBOZYME
Nobel prize in 1989
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Self-splicing of Intron Sequences

• Group I intron sequences bind a free G nucleotide
  to a specific site to initiate splicing
• Group 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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(No Transcript)
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Comparison
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Alternative Splicing Patterns
1, 2B, 3
1, 2A, 3
1, 2A, 2B, 3
1, 3
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Two predominant Poly(A) sites in Rats
Cell type specific RNA splicing
(Calcitonin-gene related protein)
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Processing of pre-rRNA transcripts
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Benefits of Splicing

• Allows 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
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Conclusions

• 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