Regulation of MuDR/Mu Transposons of Maize

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Regulation of MuDR/Mu Transposons of Maize
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Developmental Regulation of MuDR/Mu Transposons

• Late timing, in the final cell divisions How do
  plant organs cease cell division?
• Different transposition outcome in somatic and
  pre-germinal cells Lacking a germ line, how
  do cells differentiate for meiosis?

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Late excisions occur in the soma

• High frequency
• Throughout plant
• Somatic insertions

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Possible Mechanisms to Control Timing of MuDR/Mu

• Transcription of MuDR
• RNAi
• microRNAs regulate translation
• Modification of MURA or MURB
• Inhibitor proteins encoded by hMuDRs
• Competition at MURA binding sites

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TIRBGUS and TIRBLuciferase are expressed in
meristems and differentiated cells. There is
very high expression in floral tissues and pollen.
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TIRs contain numerous floral and pollen enhancer
motifs
AGGACA-----CAAACA-GAAA-GTGAC-GCGC-TGGAGCT---TAAAC
Floral
Site I, II
MURA
Methylation gradient during silencing
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Inhibitor proteins encoded by homologs?
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Where did MuDR/Mu come from? With genome
sequencing homologs also found in Arabidopsis
and rice, but only with the mudrA transposase
gene. Any question about origins in maize
should start in Mexico.
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Alphabetical survey of exotic maize lines
identifies Zapalote chico as containing MuDR-like
elements.
Plasmid reconstructions of 1 and 10 copies of MuDR
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Selling totopos is a major source of income in
Zapotec farm families. The resources and income
are managed by women. All high quality totopos
are made from Zapalote chico corn. Totopos now
come in many flavors and are exported from the
Isthmus.
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Locally grown Zapalote chico corn is made into a
masa, holes poked into the flattened discs, and
then cooked.
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Totopo are cooked in a clay oven pit after
placing them on the cooking surface inside.
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Totopo prepared from Zapalote chico corn in Oaxaca
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Innovation in manufacturing. Using a pasta maker
this woman can keep 4 ovens full.
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Uncoupling of RNA and protein abundance. hMuDR
elements encode proteins.
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Zapalote chico MuDR elements contain homologs of
both the mudrA and mudrB genes. The Mutator
system is silenced (or of low activity) but is
reactivated when crossed with non-Zapalote chico
corn. Hybrid dysgenesis and many mutations occur.
This fits a Zapotec legend that if you steal
their corn, the Zapotec corn will be so unhappy
that it will kill your corn.
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Test of Model Big Spots?

• Will mutating bases required for transcription
  factor binding alter excision timing?

Anthocyanin gene
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Spontaneous timing change in bz1-mu1

• Distinguish cis versus trans control
• A2 bz1-mu1/bz1 X a2-mu1/a2 Bz1
• Blind Test purple kernels
• a2 X a2-mu1/A2 bz1-mu1/Bz1
• bz1 X a2-mu1/A2 bz1-mu1/Bz1
• Conclusions
• Trans-regulation
• Single dominant factor
• Model MuDR or host gene mutant

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Anther somatic cells have late excision events,
but pollen and its precursors have germinal
insertions without excisions gt No germinal
revertants!
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Without a germ line, how do plants form gametes?
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Epidermis L1 Endothecium Middle Layer
L2 Tapetum Pollen
Each pollen grain has two sperm
Vegetative cell
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What controls cell fate determination in flowers?
Meiosis -gt gametophyte -gt gametes
Somatic fate
By what mechanism do MuDR/Mu elements switch
behavior during fate determination?
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Epidermis
Epidermis L1 Endothecium Middle Layer
L2 Tapetum Pollen
Endothecium
Middle Layer
Meiocyte
Tapetum
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Evidence for Pre-Meiotic Germinal Insertions

• Recovery of Duplicate Mutations
• Recovery of Multiple RescueMu Plasmids at the
  Same Insertion Site

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When do Mu insertions occur?
Switch in transposition outcome is not at
meiosis
20 in floral sectors gt multiple progeny with
the same mutation
60 during meiosis and in gametophytes gt 1 or 2
progeny with the same mutation
20 in sperm gt embryo and endosperm differ
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Somatic and Germinal Mu Transposition Mechanisms

Somatic Cut Paste
Germinal Replicative
new
old
new
old
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RescueMu deletions occur in new sites at a 5
frequency, evidence for true replicative
transposition.

• Deleted rescued plasmids are being sequenced to
  ask if repeats mark deletion end points.

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Intergenic
Transposase mudrA
Helper mudrB
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Intron 1 alternative splicing and intron 3
retention result in three transcript types
Transposase mudrA
Intergenic
823
736
854
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Excision only transposases MURA823 736
337 intron 428
450 252 start UC/GUGAGU ----UAG/ ATG
Excision and Insertion transposase MURA854
224 333
intron 466 168 start ATG 36 aa
UCCU/GUUCGU----UAG /UUUCAAT
Out-of-frame
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The "slippery sequence" created by splicing
UCCU/UUUCAAT is similar to viral transcript
frameshift sites. Predicted shifted frame
UCC UUU UUC AAT To avoid long string of
Us UCC UUC UUC AAT
Construct in transgenic maize
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Transgenic maize plants with Corrected R or
Frameshift F cDNA 1 to 215 45 bp
224 R TIR A S tag ATG 36
aa UUC UUC In frame F TIR A S
tag ATG 36 aa UUUUC Frameshift
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Reconstruction of developmentally late somatic
excision events
MURA854 F and R with LcRescueMu
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MURA854 F and R support Mu insertion
MURA854 x a1-mum2 germinal
MURA854 x bz2 somatic
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MURA Transposase Functions in Transgenic Maize
823 736 854
Somatic excision Yes Yes Yes
Somatic insertion No No Yes
Germinal insertion No No Yes
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Multiple Levels of Regulation of MURA854

• Transcription site choice
• First intron alternative splicing
• Frameshift translation
• MURB-mediated step (biochemistry unknown)

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RescueMu Gillian Nan Manish Raizada Bret
Schneider Darren Morrow John Fernandes
MURA MURB structure and interaction Akemi Ono
Soo-Hwan Kim Matt Fitzgerald Nathan Manley
MURA Transposase Manish Raizada George Rudenko
Early excision Virginia Walbot
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Helper mudrB
Intergenic
167
Retention of in-frame introns 2 3
191
207
231
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What does MURB do?

• Does not bind DNA or MURA in vitro or in vivo in
  somatic cells
• Localized to the cytoplasm in somatic cells may
  be nuclear in germinal cells
• mudrB deletion mutants and transgenic MURA MURB
  expression data suggest a role in insertion.

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Questions about MuDR/Mu

• Biochemistry of MURAs and MURBs
• Regulation of the switch from somatic to
  germinal programs
• Mechanism of germinal insertion (gap repair or
  true replicative)

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Gap repair cut paste can appear to be
replicative
Replicated chromosomes
Cut paste transposition
old site
new site
Gap repair from sister
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Different Amounts of DNA Synthesis Required for
Transposition
Somatic Cut Paste
Germinal Replicative
Synthesis of transposon strand plus 9 bp at both
the old and new sites
New 9 bp duplications
new
old
Old empty site fill in
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