hnRNA Processing

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mRNA PROCESSING, hnRNPs AND snRNPs

• M.Prasad Naidu
• MSc Medical Biochemistry,
• Ph.D.Research Scholar

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mRNA PROCESSING, hnRNPs AND snRNPs

• Processing of mRNA
• hnRNP
• snRNP particles
• 5Capping
• 3Cleavage and polyadenylation
• Splicing
• Pre-mRNA methylation

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Processing of mRNA prokaryotes

• There is essentially no processing of prokaryotic
  mRNA, it can start to be translated before it has
  finished being transcribed.
• Prokaryotic mRNA is degraded rapidly from the 5
  end

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Processing of mRNA in eukaryotes

• In eukaryotes, mRNA is synthesized by RNA Pol II
  as longer precursors (pre-mRNA), the population
  of different RNA Pol II transcripts are called
  heterogeneous nuclear RNA (hnRNA).
• Among hnRNA, those processed to give mature mRNAs
  are called pre-mRNAs

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• Pre-mRNA molecules are processed to mature mRNAs
  by 5-capping, 3-cleavage and polyadenylation,
  splicing and methylation.

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Eukaryotic mRNA processing overview
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hnRNP hnRNA proteins

• The hnRNA synthesized by RNA Pol II is mainly
  pre-mRNA and rapidly becomes covered by proteins
  to form heterogeneous nuclear ribonucleoprotein
  (hnRNP)
• The hnRNP proteins are though to help keep the
  hnRNA in a single-stranded form and to assist in
  the various RNA processing reactions

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snRNP particles snRNA proteins

1. snRNAs are rich in the base uracil, which complex
   with specific proteins to form snRNPs.
2. The most abundant snRNP are involved in pre-mRNA
   splicing, U1,U2,U4,U5 and U6.
3. A large number of snRNP define methylation sites
   in pre-rRNA.

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snRNP Particles

• snRNAs are synthesized in the nucleus by RNA Pol
  II and have a normal 5-cap.
• Exported to the cytoplasm where they associate
  with the common core proteins and with other
  specific proteins.
• Their 5-cap gains two methyl groups and then
  imported back into the nucleus where they
  function in splicing.

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5 Capping

• Very soon after RNA Pol II starts making a
  transcript, and before the RNA chain is more then
  20 -30 nt long, the 5-end is chemically
  modified.
• 7-methylguanosine is covalently to the 5 end of
  pre-mRNA.
• Linked 5 ? 5
• Occurs shortly after initiation

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7-methylguanosine (m7G)
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Function of 5cap

• Protection from degradation
• Increased translational efficiency
• Transport to cytoplasm
• Splicing of first exon

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3 Cleavage and polyadenylation

• In most pre-mRNAs, the mature 3-end of the
  molecule is generated by cleavage followed by the
  addition of a run, or tail, of A residues which
  is called the poly(A) tail.

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• RNA polymerase II does not usually terminate at
  distinct site
• Pre-mRNA is cleaved 20 nucleotides downstream of
  polyadenylation signal (AAUAAA)
• 250 AMPs are then added to the 3 end
• Almost all mRNAs have poly(A) tail

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Function of poly(A) tail

• Increased mRNA stability
• Increased translational efficiency
• Splicing of last intron

AAAAAA
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Splicing

• the process of cutting the pre-mRNA to remove the
  introns and joining together of the exons is
  called splicing.
• it takes place in the nucleus before the mature
  mRNA can be exported to the cytoplasm.

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• Introns non-coding sequences
• Exons coding sequences
• RNA splicing removal of introns and joining of
  exons
• Splicing mechanism must be precise to maintain
  open reading frame
• Catalyzed by spliceosome (RNA protein)

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Biochemical steps of pre-mRNA splicing
Step 1 a cut is made at the 5'splice site,
separating the left exon and the right
intron-exon molecule. The right intron-exon
molecule forms a lariat, in which the 5'terminus
of the intron becomes linked by a 5'-2' bond to a
base within the intron. The target base is an A
in a sequence that is called the branch site
Step 2 cutting at the 3' splice site releases
the free intron in lariat form, while the right
exon is ligated (spliced) to the left exon.
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Lariat
C U R A Y
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Nuclear splicing occurs by two transesterification
reactions in which a free OH end attacks a
phosphodiester bond.
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Spliceosome

• Catalyzes pre-mRNA splicing in nucleus
• Composed of five snRNPs (U1, U2, U4, U5 and U6),
  other splicing factors, and the pre-mRNA being
  assembled
• U1 binds to the 5 splice site, then U2 to the
  branchpoint, then the tri-snRNP complex of U4, U5
  and U6. As a result, the intron is looped out and
  the 5- and 3 exon are brought into close
  proximity.
• U2 and U6 snRNA are able to catalyze the splicing
  reaction.

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(No Transcript)
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(No Transcript)
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Splicing cycle
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Pre-mRNA methylation

• The final modification or processing event that
  many pre-mRNAs undergo is specific methylation of
  certain bases.
• The methylations seem to be largely conserved in
  the mature mRNA.

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(No Transcript)
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ALTERNATIVE mRNA PROCESSING

• Alternative processing
• Alternative poly(A) sites
• Alternative splicing
• RNA editing

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Alternative processing

• Alternative mRNA processing is the conversion of
  pre-mRNA species into more than one type of
  mature mRNA.
• Types of alternative RNA processing include
  alternative (or differential) splicing and
  alternative (or differential) poly(A) processing.

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Alternative poly(A) sites

• Some pre-mRNAs contain more than one poly(A) site
  and these may be used under different
  circumstances to generate different mature mRNAs.
• In one cell the stronger poly(A) site is used by
  default, but in other cell a factor may prevent
  stronger site from being used.

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Alternative splicing

• The generation of different mature mRNAs from a
  particular type of gene transcript can occur by
  varying the use of 5- and 3- splice sites in
  four ways
• By using different promoters
• By using different poly(A) sites
• By retaining certain introns
• By retaining or removing certain exons

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Alternative splicing
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Alternative splicing
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(A) A cassette exon can be either included in the
mRNA or excluded.
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(B) Mutually exclusive exons occur when two or
more adjacent cassette exons are spliced such
that only one exon in the group is included at a
time.
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(C, D) Alternative 5 and 3 splice sites allow
the lengthening or shortening of a particular
exon.
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(E, F) Alternative promoters and alternative
poly(A) sites switch the 59- or 39-most exons of
a transcript.
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(G) A retained intron can be excised from the
pre-mRNA or can be retained in the translated
mRNA.
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(H) A single pre-mRNA can exhibit multiple sites
of alternative splicing using different patterns
of inclusion.