Modulation of Yorkie activity by alternative splicing is required for developmental stability

Abstract

The mechanisms that contribute to developmental stability are barely known. Here we show that alternative splicing of yorkie (yki) is required for developmental stability in Drosophila. Yki encodes the effector of the Hippo pathway that has a central role in controlling organ growth and regeneration. We identify the splicing factor B52 as necessary for inclusion of yki alternative exon 3 that encodes one of the two WW domains of Yki protein. B52 depletion favors expression of Yki1 isoform carrying a single WW domain, and reduces growth in part through modulation of yki alternative splicing. Compared to the canonical Yki2 isoform containing two WW domains, Yki1 isoform has reduced transcriptional and growth-promoting activities, decreased binding to PPxYcontaining partners, and lacks the ability to bridge two proteins containing PPxY motifs. Yet, Yki1 and Yki2 interact similarly with transcription factors and can thus compete in vivo. Strikingly, flies deprived from Yki1 isoform exhibit increased fluctuating wing asymmetry, a signal of increased developmental noise. Our results identify yki alternative splicing as a new level of control of the Hippo pathway and provide the first experimental evidence that alternative splicing participates in developmental robustness.

The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm

“…To produce FLAG-FFL-Aub, Aub-coding sequence was PCR amplified from the p8161 plasmid 66 and cloned into pENTR by directional Topo cloning (Invitrogen). The resulting plasmid was used in gateway cloning to insert Aub sequence into pAct-FLAG-Firefly-RfA 72 (pAFW (DGRC) in which the FFL-coding sequence has been added). To produce HA-RL tagged versions of eIF3b (DGRC, FI08008), eIF3d, 49 eIF3f (DGRC, LD47792), eIF3k (DGRC, LD03569) and eIF4E (from E. Wahle), the coding sequences were amplified by PCR and cloned into pENTR by directional Topo cloning.…”

“…To produce HA-RL tagged versions of eIF3b (DGRC, FI08008), eIF3d, 49 eIF3f (DGRC, LD47792), eIF3k (DGRC, LD03569) and eIF4E (from E. Wahle), the coding sequences were amplified by PCR and cloned into pENTR by directional Topo cloning. The resulting plasmids were used in gateway cloning to insert the coding sequences into pAct-HA-Renilla-RfA 72 (pAHW (DGRC) in which the RL-coding sequence has been added). For the HA-RL tagged versions of PABP (DNASU, DmCD00772781), eIF3g (DNASU, DmCD00766429), eIF3h (DNASU, DmCD00764259) and eIF4a (DNASU, DmCD00764657), plasmids were directly used in gateway cloning to insert the sequence into pAct-HA-Renilla-RfA.…”

“…For the HA-RL tagged versions of PABP (DNASU, DmCD00772781), eIF3g (DNASU, DmCD00766429), eIF3h (DNASU, DmCD00764259) and eIF4a (DNASU, DmCD00764657), plasmids were directly used in gateway cloning to insert the sequence into pAct-HA-Renilla-RfA. FLAG-FFL-Cherry, HA-RL-Cherry and Sd-RL-HA 72 were used as negative controls. To produce GST-PABP clones, the coding sequences of five pAbp domains, RRM1, RRM2, RRM3, RRM4 and PABC were amplified by PCR from a plasmid provided by E. Wahle.…”

The evolution of splicing: transcriptome complexity and transcript distances implemented inTranD

“…Further, observations of conservation of sex (e.g., Dauwalder, et al 1996) and neurological (e.g. Srivastava, et al 2021) splicing factors between human and fly indicate that there are potentially more conserved regulatory mechanisms of splicing, even as genes that are regulated by these factors will almost certainly differ depending on the larger transcriptional regulatory environment.…”