MicroRNAs act sequentially and asymmetrically to control chemosensory laterality in the nematode

1. 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

2. 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.

3. The bilaterally symmetrical chemosensory neurons ASE left (ASEL) andASE 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.

4. 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

6. 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

7. Die-1 expression is strongly biased towards ASEL

9. 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

10. Die-1acts through lsy-6 to repress cog-1 expression

11. 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

13. 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.

14. Bilateral expression of mir-273 disrupts die-1 expression

15. 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.