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 2-ASER 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.