Download MicroRNAs act sequentially and asymmetrically to

MicroRNAs act sequentially and
asymmetrically to control
chemosensory laterality in the
nematode
Sarah Chang, Robert J. Johnston JR,
Christian Frokjaer-Jensen, Shawn
Lockery and Oliver Hobert
MicroRNAs
• Small RNAs that regulate expression of
complementary messenger RNA
• Found in diverse groups of animals, and
many of these microRNAs are
phylogenetically conserved
• Animal microRNAs prevent the expression
of specific messenger RNAs by binding to
their 3´ untranslated region.
The bilaterally symmetrical chemosensory
neurons ASE left (ASEL) and ASE right
(ASER) display left/right asymmetrical gene
expression patterns
• Guanylyl cyclase
receptor genes gcy-6
and gcy-7 are only
expressed in ASEL,
whereas gcy-5 is only
expressed in ASER
• The chemosensory
capacities of these two
neurons is also
asymmetrical.
The microRNA lsy-6 is required for the
left/right asymmetrical expression of the (gcy)
genes in ASEL and ASER, but the regulatory
pathway is poorly understood
• An essential component of ASEL/R laterality is
the restriction of lsy-6 expression to the ASEL
neuron
• Conducted genetic screens for mutants that show
defects in asymmetric expression of ASE specific
chemoreceptors.
• Ot26 showed 100% lsy phenotype: both ASE cells
expressed the normally ASER specific gcy-5 gene,
and concomitantly lost the expression of the
normally ASEL specific gcy-7
Ot26 is an allelle of the die-1
gene
• The die-1 gene encodes a C2H2 zinc-finger
transcription factor
• die-1 expression is present in both ASEL
and ASER, but expression is strongly biased
towards ASEL
Die-1 expression is strongly
biased towards ASEL
Die-1 (ot26) mutant animals
exhibit a complete loss of lim-6
homeobox gene expression
• Correct lim-6 expression requires a regulated
balance of the ceh-36 activator homeobox gene
and the cog-1 repressor gene.
• Loss of lim-6 could either mean an increase in
expression of the ceh-36 activator or a decrease in
expression of the cog-1 repressor.
• loss of die-1 had no effect on ceh-36 expression
Die-1acts through lsy-6 to repress
cog-1 expression
How is die-1 and hence lsy-6
activation spatially biased
towards ASEL
• Previously shown that cog-1 expression is
controlled by the miRNA lsy-6 binding to
the cog-1 3´ UTR.
• Is die-1 expression also controlled by its 3´
UTR.
• constructed ‘sensor genes’, in which gfp
constructs were produced under the control
of the ceh-36 promoter in both ASEL and
ASER
Mir-273 a microRNA controls
die-1 by binding to its 3´UTR
• die-1 3´UTR contains sequences that are
complementary to mir-273
• Expression of mir-273 is significantly higher in
ASER than in ASEL
• Forced symmetric expression of mir-273 represses
die-1 expression, and hence disrupts ASE laterality.
Transgenic animals that express mir-273 from the
bilateral ceh-36 promoter exhibit downregulation of
the die-1resc::gfp expression and also show the 2ASER chemoreceptor profile characteristic of the
die-1 mutant phenotype.
Bilateral expression of mir-273
disrupts die-1 expression
• The asymmetric expression of gcy-7 and
gcy-5 is specified by differential expression
of upstream transcription factors including
die-1, cog-1, and lim-6.
• Die-1 is translationally repressed in ASER
by the mir-273 miRNA, and cog-1 is
translationally repressed in ASEL by lsy-6
miRNA.