Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5

Abstract

RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables normal cell morphogenesis.DOI: http://dx.doi.org/10.7554/eLife.14216.001

Fission yeast NDR/LATS kinase Orb6 regulates exocytosis via phosphorylation of exocyst complex

“…For simplicity, we will refer to our orb6-as2 cells treated with 3-BrB-PP1 as "Orb6-inhibited" cells. Consistent with previous work 23,25 , Orb6 inhibition in vivo led to an increase in cell width, as well as some ectopic localization of Cdc42-GTP and the actin cytoskeleton to cell sides, and a defect in cell separation during septation ( Fig. 1A,C; Suppl.…”

“…To date, the only reported substrates of Orb6 are Gef1 and Sts5, both of which were phosphorylated in vitro by immunoprecipitates of the Orb6 coactivator Mob2 23,24 . Sts5 is not known to be associated with exocytosis, and our results indicate that decreased exocytosis after Orb6 inhibition does not involve Gef1.…”

“…4). Because Orb6 is normally essential for viability, and viable orb6∆ cells can be recovered only in an sts5∆ background 23 , we used Mob2-GFP immunoprecipitated from sts5∆ cells for the kinase reaction and Mob2-GFP immunoprecipitated from sts5∆ orb6∆ cells as a negative control. Consistent with global phosphoproteomics, we identified phosphorylation of Sec3 serine-201 on GST-Sec3 and of Sec5 serine-50 on GST-Sec5 in kinase reactions prepared from sts5∆ cells but not from sts5∆ orb6∆ cells or buffer-only controls.…”

“…The cell-separation defect of Orb6-inhibited cells is suppressed by deletion of sts5, which encodes an RNA-binding protein 23 . It has been proposed that Orb6 negatively regulates the recruitment of Sts5 into ribonucleoprotein granules and P-bodies, thereby controlling the translational repression of Sts5-associated mRNAs encoding proteins involved in polarized growth and cell separation 23 .…”

“…In fission yeast, temperature-sensitive orb6-25 mutants lose polarity at non-permissive temperature, and cells become round rather than rod-shaped 12 . In vitro, Orb6 can phosphorylate the Ssd1 homolog Sts5 23 , as well as Gef1, a GEF for the Rho-family cell-polarity GTPase Cdc42 24 .…”