Telomerase RNA

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Telomerase RNA
from sequence to structure
Elhanan Elboher Dr. Dudy Tzfati, Dept. of
Genetics, HUJI
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The Telomerase RNP complex adds short DNA repeats
to the end of the chromosome
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The Telomerase RNP complex adds short DNA repeats
to the end of the chromosome
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The Telomerase RNP complex adds short DNA repeats
to the end of the chromosome
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The Telomerase RNP complex adds short DNA repeats
to the end of the chromosome
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The Telomerase RNP complex adds short DNA repeats
to the end of the chromosome
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Telomerase RNA (TER) - Functions

• Contains the template for the telomeric repeats
• Regulates the length of the transcribed sequence
  (by a template boundary element)
• Scaffold that the elements of the complex (TERT
  and other factors) are bound to
• Regulates the activity of the whole complex, by a
  switch between different states

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Telomerase RNA (TER) Sequential and
Structural Characteristics

• Divergent length (Vertebrates 450 nt, Ciliates
  150 nt, Yeast 1000 nt)
• Low conservation of the sequence, even among
  closed species
• Higher conservation of short subsequences which
  are involved in formation of functional
  structural elements
• General structure in Yeast species three long
  arms and a central catalytic core, contains an
  essential pseudoknot element

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Saccharomyces
Secondary Structure Models of Two
Yeast TER Groups
Kluyveromyces
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Conserved Sequences in the Kluyveromyces Group
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My Project - Objectives

• Focus on a third yeasts group, Candida
• Finding the exact start end points of the
  folded sequence
• Structural elements which are known as essential
  from other species (template boundary, protein
  binding domains, ?-knot etc.)
• Novel structural elements
• Contribute to the general model of the secondary
  and tertiary structure of the yeast telomerase RNA

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My Project - Objectives

• Focus on a third yeasts group, Candida
• Finding the exact start end points of the
  folded sequence
• Structural elements which are known as essential
  from other species (template boundary, protein
  binding domains, ?-knot etc.)
• Novel structural elements
• Contribute to the general model of the secondary
  and tertiary structure of the yeast telomerase RNA

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Delimitation of the Gene

• Gussinova and Tomaska (2007) found the locus of
  TLC1 in six species
• This locus contains an expected template
  sequence which fits the telomeric repeat
• The exact limits of the folded sequences were
  determined by multiple alignment against the
  known sequence of C. albicans
• The Sm protein binding site was found during the
  alignment

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Secondary Structure Prediction Classical Tools
and Specific Problems of Yeast TER

• Two basic programs
• Mfold / RNAfold folds a single sequence to the
  secondary structure with the minimal free energy
  (M. Tzuker)
• RNAalifold folds a group of aligned sequences.
  Takes into consideration energy, conservation and
  co-variation (Vienna RNA Package)
• These tools are based on a dynamic programming
  algorithm.
• The Problems in the case of yeast TER
• Very long sequences (1500-1800 nt)
  many different
  reasonable optional foldings
• Ignoring previous biological data (conserved
  sub-sequences and functional structural elements)
• These tools cannot predict pseudoknots

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Most of the RNA structural elements obey the
brackets rule
( ( D ) ( A ) ( T ) )
ACGUGCCACGAUUCAACGUGGCACAG ((((((((
))))))))
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Pseudoknot is a RNA structural element which
breaks the brackets rule
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j
j
i
i
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These two stems cross each other. Formally, i j Dynamic programming algorithms cannot predict
such structures!
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CS3 and CS4 form a special structure of a
pseudoknot which contains a triple helix
Lets convert it to formal constraints
CS3
CS4 (
( ( ( ( ( ( ( ( ( )
) ) ) ) ) ) ) ) )
stem 1 UUU UUU stem 1
AAA
( ( (
( ( (
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Searching for the Pseudoknot

• Generate a base pairs matrix for each sequence
• Represent all optional stable stems (parameters
  length, H. bonds, bulges)
• Find all optional structures which hold the
  pseudoknots constraints
• Look for conservation of the pattern at closed
  locations
• Will the program find the PKs of the known
  species?

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Pseudoknot - Results

• The program found the CS3 of Kluyveromyces and
  Saccharomyces as the most likely option.
• There was found a homologous sequence for the
  Candida species, in an appropriate place within
  the whole sequence.
• From the relevant candidates for CS4, there is
  one with high similarity to S.cerevisiae, both in
  sequence and structure.

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Evolution of the CS3 Loop
The stem of CS3 is not conserved at all among the
different groups.
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Models of the Pseudoknot Saccharomyces,
Kluyveromyces, Candida
K. lactis
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Est1p Binding Site
obtained by a manipulation of a motif finding
program (MEME), Mfold, RNAalifold, and previous
knowledge
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An Essential Three-Way Junction

• Approaches
• Pattern Location
• Parsing tree of a probabilistic CFG

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Future Directions

• Find all known essential elements (TWJ, template
  boundary etc.)
• Demonstrate a full secondary structure model
• Identify additional tertiary interactions
• Generalize the results to a model that will
  enable genomic searching

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Acknowledgments

• Dudy Tzfati
• Lubo Tomaska, Josef Nosek
• Nickolay Ulyanov
• Yogev Brown
• Hanah Margalit
• Tommy Kaplan