Aliens? Oddities? Or misunderstood?

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Aliens? Oddities? Or misunderstood?

• Transposons and miRNAs

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Genome sizes (haploid)
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Number of genes in different organisms
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What is a transposon?

• Contiguous piece of DNA of varying length (300 bp
  to 6.5kb or so)
• Repeated with minor variations throughout the
  host genome
• Can replicate itself by cut and paste or copy and
  paste mechanisms (can move around!)
• No known function most synthetic genome
  projects aim to remove them
• Structural and functional analogies to viruses
• Much of the terminology reflects this

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• Barbara McClintock, 1940s
• Discovered transposons and characterized their
  effects on their hosts
• She was ostracized for her ideas but won the
  Nobel Prize in 1983.

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Types of transposons

• Cut and paste
• DNA transposons
• Copy and paste
• Autonomous retrotransposons
• ERVs possibly active in human genome
• L1 relatives active in human genome
• Nonautonomous retrotransposons
• SINEs (Alu) active in human genome
• SVA active in human genome
• Composite element (SINE, VNTR, Alu)
• Processed pseudogenes

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Transposons comprise 45 of the human genome

• DNA transposons 3
• Autonomous retrotransposons
• ERVs
• L1 18 (500,000 copies)
• L2 3
• L3 relatives 1
• Nonautonomous retrotransposons
• SINEs (Alu) 15 (1 million copies)
• SVA (3000 copies)
• Processed pseudogenes (gt8000)
• (Simple repeats occupy almost another 10)

LTR retrotransposons
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Junk DNA?

• What do transposons do?
• Make more of themselves
• Move genes around
• Serve as reservoirs of new sequence
• Cause genetic instability (repeats stimulate
  translocation L1 causes chromosome breakage)
• Can contribute to genes and gene expression
• 5 of alternatively spliced internal human exons
  come from Alus
• 80 of genes have some L1 sequence in noncoding
  portion
• 1-4 of coding sequence is L1-derived
• Act as methylation centers

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Importance in genomics

• Transposons are a source of human variability
• Roughly 5 of people have a transposon not found
  in either parent (not due to nonpaternity!)
• Overall polymorphism variable but remarkable
  (40-50 of youngest elements are polymorphic)
• Transposons can be useful in medicine
• Occasionally cause disease (de novo insertion in
  factor VIII clotting gene led to L1 discovery in
  1980s)
• May often be linked to disease loci

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Importance in genomics

• Transposons in introns may disrupt gene
  expression
• Mechanism depends on whether they are on the
  sense or antisense strand
• () strand orientation transcription stalling
• (-) strand orientation premature
  polyadenylation, gene splitting

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Importance in genomics

• Can have huge effects, through chromosomal
  translocation, inversion, breakage

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Transposon domestication

• Overly active transposons will kill a cell (and
  then the organism)
• Transposons have tempered
• active almost exclusively in germ line
• also in cancer cells and neuronal cell precursors

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Transposon domestication

• Host cells use many mechanisms to control
  transposons
• Methylation (original role?)
• miRNA defense
• Sequestered in stress granules
• Nucleic acid editing
• APOBEC family of proteins edits cytosines to
  uracils
• ADARs edit dsRNA adenosine to inosine

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What to do with transposons?

• Study them
• Work around them (be aware)
• RepeatMasker (Smit Jurka)
• Problem each element is at least in part unique,
  and RepeatMasker will mask that too

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Another old element, new to sciencemicroRNAs

• RNA world hypothesis
• First organism was a strand of RNA that could
  somehow replicate itself.
• Eventually RNA used DNA as a more stable storage
  for genetic material.
• 1982 Tom Cech reported self-splicing RNAs

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microRNA

• 21-25 nucleotide small RNAs
• Discovered in a C. elegans screen
• Alter gene expression at the post-transcriptional
  level (precise mechanism unknown)
• Tend to be high-level regulators (gt100 targets
  each)
• Percentage of human genes under miRNA control is
  unknown but possibly 20-30
• Often are developmental or cell state switches

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miRNA
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Two mechanisms Perfect match to target leads to
mRNA cleavage or Imperfect match leads to
translational repression Neither is
well-understood, but likely involve the dsRNA
recognition system
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Another role?

• Under conditions of cell stress, a miRNA may be
  activating instead, as responding regulatory
  proteins interpret the signal differently

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Seems odd . . .

• Why would a cell use this sort of mechanism? Its
  making an mRNA and then degrading it. Should be
  easier to just not make it . . .
• But what if the cell is not in control of that
  RNA, for example if its coming from an invasive
  nucleic acid species under its own promoter?
• Transposon control!!!
• piRNA (piwi RNA) are a whole class of small RNAs
  that control transposons
• Invasive RNA was a big problem in the RNA world!

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Occams razor

• All other things being equal, the simplest
  solution is the best
• My alternative If a biological principle is
  simple, its probably wrong.
• Evolution tends to higher complexity, as old
  mechanisms are reused and theres little
  incentive to clean up.

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Looking for new miRNAs

• Often found within stem-loop precursor structures
  (hairpins)
• Associated (in the cell) with polysomes and other
  structures
• Bioinformatics unexpected sequence conservation
  in noncoding region, or homology to miRNA in a
  closely related species (works less often than
  you would think)
• Identify candidate miRNA targets (TargetScan, by
  Chris Burges group)
• A target protein usually has multiple target sites

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Problems with miRNAs

• Small! Unstable, hard to get large quantities
• Binding is degenerate, noncontiguous, and
  includes not only mismatches but bulges
• Actual sequence recognition only 15 or so
  nucleotides (noncontiguous), varies by target
• Essential seed element not well characterized
• Sequences not well conserved across species
• miRNA microarrays statistics problematic because
  there are so few spots