Section O

1
Section O RNA processing and RNPs
2
Contents

• O1 rRNA processing and ribosomes
• Types of RNA processing, rRNA processing in
  prokaryote, rRNA processing in eukaryotes, RNPs
  and their study, Prokaryotic ribosomes,
  Eukaryotic ribosomes
• O2 tRNA processing, RNase P and ribozymes
• tRNA processing in prokaryotes, tRNA
  processing in eukaryotes, RNase P, Ribozymes
• O3 mRNA processing, hnRNPs and snRNPs
• Processing of mRNA, hnRNP, snRNP particles,
  5 Capping, 3 Cleavage and polyadenylation,
  Splicing, Pre-mRNA methylation
• O4 Alternative mRNA processing
• Alternative processing, Alternative poly(A)
  site, Alternative splicing, RNA editing

3
O1 rRNA processing and ribosomes
Types of RNA processing

• Very few RNA molecules are transcribed directly
  into the final mature RNA. Most newly transcribed
  RNA molecules (primary transcripts) undergo
  various alterations to yield the mature product.
  RNA processing is the collective term used to
  describe the molecular events allowing the
  primary transcripts to become the mature RNA.

4
Cytoplasm
Nucleus or Nucleolus
primary transcript
RNA processing
Romoval of nucleotides
addition of nucleotides to the 5- or 3- ends
modification of certain nucleotides
mature RNA.
5

• (1) Removal of nucleotides by both endonucleases
  and exonucleases
• (2) Addition of nucleotides to 5-or 3-ends of
  the primary transcripts or their cleavage
  products.
• (3) Modification of certain nucleotides on either
  the base or the sugar moiety.

6
O1 rRNA processing and ribosomes rRNA
processing in prokaryote

1. There are 7 different operons for rRNA that are
   dispersed throughout the genome.
2. Each operon contains one copy of each of the
   5S,the 16S and the 23S rRNA sequences. About 14
   coding sequences for tRNA molecules are also
   present in these rRNA operons.
3. The initial transcript has a sedimentation
   coefficient of 30s (6000 nt) and is normally
   quite short-lived.

rRNA operon
7
Step 1 Following or during the primary
transcription, the RNA folds up into a number of
stem-loop structures by base pairing between
complementary sequences
RNA folding
8

• Step 2 The formation of this secondary
  structure of stems and loops allows some proteins
  to bind to form a RNP complex which remain
  attached to the RNA and become part of the
  ribosome

RNP complex formation
9

• Step 3 After the binding of proteins, nucleotide
  modifications take place.
• Example methylation of adenine by methylating
  agent S-Adenosylmethonine (SAM)
• Step 4 RNA cleavage

10
rRNA operon
11
O1 rRNA processing and ribosomes rRNA
processing in eukaryotes

• rRNA in eukaryotes is also generated from a
  single, long precursor molecule by specific
  modification and cleavage steps
• The processes are not so well understood

12

1. The rRNA genes are present in a tandemly repeated
   cluster containing 100 or more copies of the
   transcription unit, and are transcribed in
   nucleolus by RNA Pol I
2. Precursor sizes are different among organisms
   (yeast 7000 nt mammalian 13500 nt), and
   pre-mRNA processing is also slightly different
   among organism.

13

• 3. The precursor contains
• one copy of the 18S coding region and
• one copy each of the 5.8S and 28S coding regions,
  which together are the equivalent of the 23S rRNA
  in prokaryote
• 4. The large precursor RNA undergoes a number of
  cleavages to yield mature RNA and ribosome.

14

• 5. The eukaryotic 5S rRNA
• is transcribed by RNA Pol III from unlinked genes
  to give a 121nt transcript
• the transcript undergoes little or no processing

15
18S
5.8S
28S
47S
ETS1
ITS1
ITS2
ETS2
45S
41S
20S and 32S
Mature rRNAs
18S rRNA
5.8S rRNA
28S rRNA
Mammalian pre-rRNA processing
Indicates RNase cleavage
16

• The 5.8S region must base-pair to the 28S rRNA
  before the mature molecules are produced.
• Mature rRNAs complex with protein to form RNPs
  (nucleolus)
• Methylation occurs at over 100 sites to give
  2-O-methylribose, which is known to be carried
  out by snRNPs (nucleolus)

17

• Introns (group I) in rRNA genes of some lower
  eukarytes (Tetrahymena thermophila) must be
  spliced out to generate mature rRNAs.
• Many group I introns are found to catalyze the
  splicing reaction by itself in vitro, therefore
  called ribozyme

18
(No Transcript)
19
O1 rRNA processing and ribosomes
RNPs and their study

• Cells contain a variety of RNA-protein complexes(
  RNPs).
• These can be studied using techniques that help
  to clarify their structure and function.
• These include dissociation, re-assembly, electron
  microscopy, use of antibodies, RNase protection,
  RNA binding, cross-linking and neutron and X-ray
  diffraction.
• The structure and function of some RNPs are quite
  well characterized.

20
O1 rRNA processing and ribosomes
Prokaryotic ribosomes

• Protein biosynthetic machinery
• Made of 2 subunits (bacterial 30S and 50S,
  Eukaryotes 40S and 60S)

• Intact ribosome referred to as 70S ribosome in
  Prokaryotes and 80S ribosome in Eukaryotes
• In bacteria, 20,000 ribosomes per cell, 25 of
  cell's mass.

21
(No Transcript)
22
Features of the E.coli ribosome
Cleft
Platform
Central protuberance
Stalk
Small
23
Ribosome Structure (1)
24
Ribosome Structure (2)

• mRNA is associated with the 30S subunit
• Two tRNA binding sites (P and A sites) are
  located in the cavity formed by the association
  of the 2 subunits.
• The growing peptide chain threads through a
  tunnel that passes through the 30S subunit.

25
O1 rRNA processing and ribosomes
Eukaryotic ribosomes

• larger and more complex than prokaryotic
  ribosomes, but with similar structural and
  functional properties

26
(No Transcript)
27
O2 tRNA processing, RNase P and ribozymes
tRNA processing in prokaryotes

• Mature tRNAs are generated by processing longer
  pre-tRNA transcripts, which involves
• specific exo- and endonucleolytic cleavage by
  RNases D, E, F and P (general) followed by
• base modifications which are unique to each
  particular tRNA type.

28
(No Transcript)
29
O2 tRNA processing, RNase P and ribozymes
tRNA processing in eukaryotes

• The pre-tRNA is synthesized with a 16 nt
  5-leader a 14 nt intron and two extra
  3-nucleotides.

30

1. Primary transcripts forms secondary structures
   recognized by endonucleases
2. 5 leader and 3 extra nucleotide removal
3. tRNA nucleptidyl transferase adds 5-CCA-3 to
   the 3-end to generate the mature 3-end
4. Intron removal

31
O2 tRNA processing, RNase P and ribozymes
RNase P

• Ribonuclease P (RNase P) is an enzyme involved in
  tRNA processing that removes the 5' leader
  sequences from tRNA precursors
• RNase P enzymes are found in both prokaryotes and
  eukaryotes, being located in the nucleus of the
  latter where they are therefore small nuclear
  RNPs (snRNPs)
• In E. coli, the endonuclease is composed of a 377
  nt RNA and a small basic protein of 13.7kDa.
• RNA component can catalyze pre-tRNA in vitro in
  the absence of protein. Thus RNase P RNA is a
  catalytic RNA, or ribozyme.

32
O2 tRNA processing, RNase P and ribozymes
Ribozymes

• Ribozymes are catalytic RNA molecules that can
  catalyze particular biochemical reactions.
• RNase P RNA is a ribozyme.
• RNase P RNA from bacteria is more catalytically
  active in vitro than those from eukaryotic and
  archaebacterial cells. All RNase P RNAs share
  common sequences and structures.
• Self-splicing introns the intervening RNA that
  catalyze the splicing of themselves from their
  precursor RNA, and the joining of the exon
  sequences
• Group I introns, such as Tetrahymena intron
• Group II introns.

33

• Self-cleaving RNA encoded by viral genome to
  resolve the concatameric molecules of the viral
  genomic RNA
• HDV ribozyme
• Hairpin ribozyme
• Hammer head ribozyme
• Ribozymes can be used as therapeutic agents in
• correcting mutant mRNA in human cells
• inhibiting unwanted gene expression
• Kill cancer cells
• Prevent virus replication

34
O3 mRNA processing, hnRNPs and snRNPs
Processing of mRNA

• 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
• 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

35
Eukaryotic mRNA processing overview
36
O3 mRNA processing, hnRNPs and snRNPs
hnRNP

• 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

37
O3 mRNA processing, hnRNPs and snRNPs
snRNP particles

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

38
O3 mRNA processing, hnRNPs and snRNPs
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

39
7-methylguanosine (m7G)
40
Function of 5cap

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

41
(No Transcript)
42
O3 mRNA processing, hnRNPs and snRNPs
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.
• 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

43
(No Transcript)
44
Function of poly(A) tail

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

45
O3 mRNA processing, hnRNPs and snRNPs
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.
• 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)

46
Biochemical steps of pre-mRNA splicing
47
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.
48
Lariat
C U R A Y
49
Nuclear splicing occurs by two transesterification
reactions in which a free OH end attacks a
phosphodiester bond.
50
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.

51
(No Transcript)
52
(No Transcript)
53
Splicing cycle
54
O3 mRNA processing, hnRNPs and snRNPs
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.

55
O4 Alternative mRNA processing
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.

56
O4 Alternative mRNA processing
Alternative poly(A) site

• 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.

57
O4 Alternative mRNA processing
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

58
Alternative splicing
59
(A) A cassette exon can be either included in the
mRNA or excluded.
60
(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.
61
(C, D) Alternative 5 and 3 splice sites allow
the lengthening or shortening of a particular
exon.
62
(E, F) Alternative promoters and alternative
poly(A) sites switch the 59- or 39-most exons of
a transcript.
63
(G) A retained intron can be excised from the
pre-mRNA or can be retained in the translated
mRNA.
64
(H) A single pre-mRNA can exhibit multiple sites
of alternative splicing using different patterns
of inclusion.
65
Alternative splicing
66
O4 Alternative mRNA processing RNA editing

• An unusual form of RNA processing in which the
  sequence of the primary transcript is altered is
  called RNA editing.
• Changing RNA sequence (after transcription)

67

• RNA editing is known to occur in two different
  situations, with different causes.
• In mammalian cells there are cases in which a
  substitution occurs in an individual base in
  mRNA, causing a change in the sequence of the
  protein that is coded. (Base modificationA or C
  deamination)
• In trypanosome mitochondria, more widespread
  changes occur in transcripts of several genes,
  when bases are systematically added or deleted.
  (Base U insertion and deletion)

68
(No Transcript)
69
Multiple choice questions

• 1. Which of the following terms correctly
  describe parts of the E. coli large (50S)
  subunit?
• A stalk central protuberance valley and cleft.
• B upper third lower third valley and stalk.
• C cleft valley stalk and small protuberance.
• D stalk polypeptide exit site valley and
  central protuberance.
• 2. Which ribonucleases are involved in producing
  mature tRNA in E. coli?
• A RNases A, D, E and F.
• B RNases D, E, F and H.
• C RNases D, E, F and P.
• D RNases A, D, H and P.

70

• 3. Most eukaryotic pre-mRNAs are matured by
  which of the following modifications to their
  ends?
• A capping at the 3-end cleavage and
  polyadenylation at the 5'-end.
• B addition of a GMP to the 5'-end,cleavage and
  polyadenylation to create the 3'-end.
• C addition of a guanine residue to the 5'-end
  cleavage and polyadenylation to create the
  3'-end.
• D addition of a GMP to the 5'-end,polyadenylatio
  n,then cleavage to create the 3'-end.
• 4. Which one of the following statements
  correctly describes the splicing process
  undergone by most eukaryotic pre-mRNAs?
• A in a two-step reaction, the spliceosome
  removes the exon as a lariat and joins the two
  introns together.
• B splicing requires conserved sequences which
  are the 5?splice site,the 3' -splice site the
  branch-point and the polypurine tract.
• C the U1 snRNP initially binds to the 5'-splice
  site,U2 to the branchpoint sequence and then the
  tri-snRNP, U4, US and U6 can bind.
• D in the first step of splicing the G at the
  3'-end of the intron is joined to the 2-hydroxyl
  group of the A residue of the branchpoint
  sequence to create a lariat.

71
THANK YOU !