Identification of hundreds of conserved and nonconserved human microRNAs

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Identification of hundreds of conserved and
nonconserved human microRNAs
Isaac Bentwich, Amir Avniel, Yael Karov, Ranit
Aharonov, Shlomit Gilad et.al Nature
genetics Volume 37 Number 7 JULY 2005
Presented by Weiran
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Abstract

• MicroRNAs are noncoding RNAs of 22
  nucleotids that suppress translation of target
  genes by binding to their mRNA and thus have a
  central role in gene regulation in health and
  disease.
• To date, 222 human microRNAs have been
  identified, 86 by random cloning and sequencing,
  43 by computational approaches and the rest as
  putative microRNAs homologous to microRNAs in
  other species.

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• To prove our hypothesis that the total number of
  microRNAs may be much larger and that several
  have emerged only in primates, we developed an
  integrative approach combining bioinformatic
  predictions with microarray analysis and
  sequence-directed cloning.
• Here we report the use of this approach to clone
  and sequence 89 new human microRNAs (nearly
  doubling the current number of sequenced human
  microRNAs), 53 of which are not conserved beyond
  primates. These findings suggest that the total
  number of human microRNAs is at least 800.

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Method?

• Indentifing and scoring candidate microRNA
  precursors.
• Step 1 Extracting hairpin structures from
  the entire genome.
• All hairpin structures that have at least six
  base pairs, are at least 55 nucleotides long and
  have a loop not longer than 20 nucleotides were
  extracted from the minimum free energy fold of
  the window (excluding overlapping hairpins).

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• Step 2 Assigning each hairpin a stability score.
  
• Indicated by the similarity of the minimum
  free energy graph and the partition functiongraph
  provided by the Vienna package for that hairpin.
• Step 3 Scoring hairpin.
• Structural features hairpin length, loop
  length, stability score, free energy
  pernucleotide, number of matching base pairs and
  bulge size. Sequence features sequence
  repetitiveness, regular and inverted internal
  repeats and free energy per nucleotide
  composition.

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• Determining hairpin conservation.
• A measure of evolutionary conservation for
  each nucleotide in the human genome against the
  genomes of chimpanzee,mouse, rat, dog, chicken,
  pufferfish and zebrafish.
• Microarray high-throughput validation.
• They carried out microarray experiments
  designed to detect expression of mature
  microRNAs. The microarray contains two probes per
  candidate microRNA gene,one for each predicted
  mature microRNA.
• MicroRNA sequence-directed cloning and sequencing.

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• Determining cluster homology.
• They compared microRNA precursors with all
  assembled genomes to determine the homologs of a
  microRNA cluster in the University of California
  Santa Cruz genome browser (BlastZ analysis).
• They looked for homologs of individual
  microRNA cluster members using Blast analysis
  against the wholegenome sequence data in the
  National Center for Biotechnology Information
  Trace databases.

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Methods?

• MicroRNA discovery tools

• (1) computationally scanning the entire human
  genome for hairpin structures
• (2) annotating all hairpins for conserved,
  repetitive and protein-coding regions
• (3) scoring hairpins by thermodynamic
  stability and structural features, using a method
  (PalGrade) that detects a large percentage of
  known microRNAs while selecting a relatively
  small portion of all genome hairpins

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• (4) determining the expression of
  computationally predicted microRNAs by a
  high-throughput microRNA microarray in several
  tissues (placenta, testis, thymus, brain and
  prostate)
• (5) validating the sequence of predicted
  microRNAs that gave high signals on the
  microarray using a new sequence directed cloning
  and sequencing method.

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The microRNA sequence directed cloning methods
2 min
uMACS Streptavidin kit column
Figure1
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Method ?
Figure1
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• Scanning the entire human genome identified 11
  million hairpins.
• Of all hairpins, 434,239 passed a minimal hairpin
  score threshold and were not located on
  repetitive elements or protein-coding regions.
  This smaller group is the initial candidate
  group.
• From the initial candidate group they selected
  5,300 predicted microRNA sequences for
  high-throughput expression validation by
  microarray. (conserved, nonconserved clustered
  and nonconserved nonclustered hairpins)

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• Microarray experiments in placenta, testis,
  thymus, brain and prostate resulted in 886
  candidate microRNAs with significant signals.
• They subjected 359 of these 886 candidate
  microRNAs to sequence validation using our new
  sequence-directed cloning and sequencing method.
• They also carried out sequence validation on 69
  bioinformatically predicted microRNAs, which were
  not present on the microarray but are located
  adjacent to microRNAs (called adjacent
  microRNAs).

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Results?

• They successfully cloned and sequenced 89 human
  microRNA genes that do not appear in the microRNA
  registry
• (1) 56 were found through the methods main
  pipeline (They were part of orginal samples).
• (2) 33 are either similar or adjacent
  microRNAs.
• (3) Only 1 of the 89 emerged from the large
  control group, supporting the distinction between
  the initial candidate group and the remaining.

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Results ?

• Thirty-two of the 36 conserved microRNAs
  discovered and sequenced by they approach score
  highly.
• Fifty three of the new microRNAs that they
  found and sequenced are located in two large
  nonconserved clusters (24 of these were in the
  original sample and 29 were found by searching
  for adjacent micro-RNAs).

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• (1) One of the clusters, located on chromosome
  19 and expressed only in placenta, is the largest
  microRNA cluster ever reported and comprises 54
  new predicted microRNAs,43 of which they cloned
  and sequenced.

None conserved clusters
Figure 3a
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• Although they are highly similar, they
  generate 16 distinct seeds ( nucleotides 28 of
  the mature microRNA).

Figure 4a
None conserved clusters
black, 100 dark gray, 8099 light
gray, 6079.
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• Homology analysis showed that the cluster as a
  whole is conserved only in chimpanzee and
  possibly rhesus Monkey and that none of its
  microRNA members show any homology to nonprimate
  genomes.

None conserved clusters
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• (2)The second cluster is located on chromosome
  X near the gene FMR1 and includes ten microRNAs,
  which are expressed only in testis.

None conserved clusters
Figure 3b
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• These ten microRNAs also form a family of
  related sequences, which generate seven distinct
  seeds .The cluster as a whole is conserved only
  in chimpanzee and possibly rhesus monkey. Seven
  and four of its members are conserved and
  clustered in dog and mouse or rat.

None conserved clusters
Figure 4b
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Conclusion

• They report a new class of microRNAs,
  nonconserved clustered microRNAs, that are not
  detected using other microRNA detection
  approaches.
• The 89 cloned and sequenced microRNAs bring the
  total number of human microRNAs to 311, well
  above a previously stated upper bound of 255.
• The method allows an estimation of the total
  number of both conserved and nonconserved
  microRNA classes. (Conserved 442, nonconserved
  159 )

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Discussion

• The world of human microRNAs is larger than
  initially believed and is not limited to
  conserved sequences.
• These findings support the notion that microRNAs
  have a central role in the regulation of protein
  translation throughout the human genome.

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• The results further indicate that a substantial
  portion of microRNAs are primate-specific.
  Therefore, microRNAs may have a key role in the
  evolutionary process and in the evolved
  complexity of higher mammals.

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• Thank you !