Ribozyme discovery

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Ribozyme discovery?????

• Presented by Yi Zhang

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Definition

• Ribozymes are specialized ribonucleic acid (RNA)
  molecules with enzyme-like properties.
• A ribozyme, or RNA enzyme, is an RNA molecule
  that can catalyze a chemical reaction.

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History (1)

• Before the discovery of ribozymes, only proteins
  were known to have catalytic activity.
• In 1967, Carl Woese, Francis Crick, and Leslie
  Orgel were the first to suggest that RNA could
  act as a catalyst based upon findings that it can
  form complex secondary structures.

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History (2)

• The first ribozyme was discovered in the 1980s by
  Thomas R. Cech , who was studying RNA splicing in
  the ciliated protozoan
• Tetrahymena thermophila.
• This ribozyme was found
• in the intron of a RNA transcript and removed
  itself from the transcript.

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Thomas R. Cech Autobiography

• born in Chicago in 1947
• 1966 entered Grinnell College
• 1970 University of California at Berkeley for
  Ph.D. study. Thesis advisor, John Hearst had an
  enthusiasm for chromosome structure and function
  that proved infectious
• 1975 obtained Ph.D. and moved to postdoctoral
  positions in M.I.T at Cambridge, Massachusetts.
  Mary Lou Pardue's laboratory
• 1978 the faculty position in the University of
  Colorado, Boulder.

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Self-splicing RNA autoexcision and
autocylcization of the ribosomal RNA intervening
sequence of Tetrahymena

• Kelly Kruger, Paula J. Grabowski, Arthur J. Zaug,
  Julie Sands, Daniel Gottschling and Thomas R.
  Cech
• Department of Chemistry, University of Colorado,
  Boulder, Colorado 80309

Cell 31147-157, 1982
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Summary

• In the macronulcear rRNA genes of Tetrahymena
  thermophila, a 413 bp intervening sequence (IVS)
  interrupts the 26S rRNA-coding region.
  (Background)
• A restriction fragment of the rDNA containing the
  IVS and portions of the adjacent rRNA sequences
  (exons) was inserted downstream from the lac UV5
  promoter in a recombinant plasmid. (method 1)
• Transcription of this template by purified
  Escherichia coli RNA polymerase in vitro produced
  a shortened version of the pre-rRNA, which was
  then deproteinized. (method 2)

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• When incubated with monovalent and divalent
  cations and a guanosine factor, this RNA
  underwent splicing. (result 1)
• The reactions that were characterized included
  the precise excision of the IVS, attachment of
  guanosine to the 5 end of the IVS, covalent
  cyclization of the IVS and ligation of the exons.
  (result 2)

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• We conclude that splicing activity is intrinsic
  to the structure of the RNA, and that enzymes,
  small nuclear RNAs and folding of the pre-rRNA
  into an RNP are unnecessary for these reactions.
  (conclusion 1)
• We propose that the IVS portion of the RNA has
  several enzyme-like properties that enable it to
  break and reform phosphodiester bonds.
  (conclusion 2)
• The finding of autocatalytic rearrangments of RNA
  molecules has implications for the mechanism and
  the evolution of other reactions that involve
  RNA. (Implication)

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Introduction (1)

• All copies of the rDNA of some species of
  Tetrahymena are interrupted by a 0.4 kb IVS
  (intron). The IVS is removed in the nucleus by a
  cleavage-ligation process termed RNA splicing.

18S
5.8S
26S
ETS1
ITS1
ITS2
ETS2
Cleavage processing and Splicing
IVS
18S
5.8S
26S
IVS
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Introduction (2)

• The excised linear IVS RNA has a 5-terminal
  guanosine residue that is not encoded by the DNA
• The linear IVS is subsequently converted to a
  circular form.
• A phosphoester transfer mechanism is suggested
  for pre-rRNA splicing.

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Introduction (3)

• The nature of the activity responsible for
  splicing remained perplexing.
• We were able to purify an active form of the
  pre-rRNA that excised and cyclized its IVS
  without the addition of any protein (Cech et al.,
  1981).
• 1. This RNA was called a splicing intermediate
  because of one explanation of tightly association
  of an enzyme with the RNA. The proposed enzyme
  should be unusually stable to survive SDS-phenol
  extraction and boiling in SDS and protease
  treatment

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• 2. Alternatively, the reaction could be mediated
  by the folded RNA molecule itself.
• More recently, radiochemical labeling or density
  shift of the RNA in Cs2SO4 gradient showed no
  evidence of for any protein attached to the RNA
  (B. Bass, A. Zaug and T. Cech, unpublished data).
  
• S1 nuclease mapping experiments showed that the
  splicing intermediate can form a continuous
  RNA-DNA hybrid through both splice junctions

5 exon
3 exon
5 splice site (junction)
3 splice site (junction)
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Introduction (4)

• These results lent credence to the possibility
  that splicing activity was intrinsic to the
  structure of the RNA
• This article is to test this hypothesis by
  synthesizing a portion of the pre-rRNA in a
  completely defined plasimid transcription system
  (AIM).

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Results (1)

• Construction of plasmids for in vitro
  transcription
• In vitro transcription and splicing

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Lac UV5 promoter-operator region
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• In vitro transcription of the plasmid DNA with E.
  coli RNA pol. The template DNA pIVS13 (lane 1)
  and pIVS11 (lane 3) cleaved by Eco RI,
  supercoiled pIVS11 (lane 5).
• Deproteinize RNA preparations, then incubated in
  solution containing GTP, MgCl2 and salt (lanes 4
  and 6)

Lane 2 control
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Results (2)

• Separation of transcription and splicing

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(-) Transcription at condition where splicing is
inhibited (polyamines) () deproteinize the RNA
preparations and splice in the presence of GTP,
MgCl2 and salt
30 min
30 min
30 min
60 min
60 min
60 min
MARKER
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(1) Gel purification of bands a, b, c and d (2)
- no further treatment treated under
splicing conditoin
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Results (3)

• Autoexcision of the IVS shown by exogenous
  GTP-labeling.

Conclusion Autoexcision of the the IVS is
precise at the 5 splice junction. The terminal G
residue is linked to the IVS by a normal 3 ?5
phosphodiester bond.
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RNA transcription from the plasmid ?
deproteinization ? remove residual nucleotides ?
incubate under the splicing condition in the
presence of a32P-GTP
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RNA sequencing of the spliced IVS
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Results (4)

• Autocyclization of the IVS

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Uniformly labeled (a32P-GTP) linear IVS was gel
purified ? incubation in the Mg2-containing
buffer
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Results (5)

• Exon ligation

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Discussion

• Self-splicing RNA
• Enzymatic RNA
• Other ribozymes, past and present

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Cechs contribution-breed a new field
39 Post-doc
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31 graduates