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

Abstract

The Hippo signaling pathway is a major regulator of organ growth, which controls the activity of the transcription coactivator Yorkie (Yki) in Drosophila and its homolog YAP in mammals. Both Yki and YAP proteins exist as alternatively spliced isoforms containing either one or two WW domains. The biological importance of this conserved alternative splicing event is unknown. Here, we identify the splicing factor B52 as a regulator of yki alternative splicing in Drosophila and show that B52 modulates growth in part through modulation of yki alternative splicing. Yki isoforms differ by their transcriptional activity as well as their ability to bind and bridge PPxY motifs-containing partners, and can compete in vivo. Strikingly, flies in which yki alternative splicing has been abrogated, thus expressing only Yki2 isoform, exhibit fluctuating wing asymmetry, a signal of developmental instability. Our results identify yki alternative splicing as a new level of modulation of the Hippo pathway, that is required for growth equilibration during development. This study provides the first demonstration that the process of alternative splicing contributes to developmental robustness.

The identification of protein and RNA interactors of the splicing factor Caper in the adult Drosophila nervous system

“…Additionally, Caper interacting proteins were enriched for the GO term “regulation of alternative mRNA splicing” ( Figure 2A ), which aligns with studies that have previously identified a role for Drosophila Caper and its human and mouse ortholog RBM39 in alternative splicing ( Park et al, 2004 ; Dowhan et al, 2005 ; Huang et al, 2012 ; Ashton-Beaucage et al, 2014 ; Brooks et al, 2015 ; Mai et al, 2016 ; Stepanyuk et al, 2016 ; Stegeman et al, 2018 ). Specifically, several interactors of Caper that have been implicated in mRNA splicing include B52 ( Liu and Bossing, 2016 ; Zhang et al, 2018 ; Srivastava et al, 2021 ), Acinus (Acn) ( Rodor et al, 2016 ), and Splicing regulatory protein 54 (Srp54) ( Garcia-Garcia et al, 2017 ). Finally, Caper also coimmunoprecipitated with U2 small nuclear riboprotein auxiliary factor 50 (U2af50), one of the subunits of U2 auxiliary factor (U2af), which is critical for the recruitment of the U2 small nuclear ribonucleoprotein (snRNP) to the 3′ splice site to initiate spliceosomal assembly ( Zamore and Green, 1989 ).…”

Allele-specific alternative splicing of Drosophila Ribosomal protein S21 suppresses a lethal mutation in the Phosphorylated adaptor for RNA export (Phax) gene

“…A prior cross-comparison of published RNA-seq data for local splicing variations revealed a decrease in RpS21 intron 3 retention upon RNAi knockdown of mRNA for the splicing factor B52 in Drosophila S2 cells ( Srivastava et al 2021 ). As expected from these transcriptomic findings, B52 knockdown in S2-DRSC cells here led to a decrease in RpS21 intron 3 retention and a corresponding 30% increase in the percentage of spliced RpS21 transcripts ( Fig.…”

“…Overexpression of B52 has been linked to increased growth and depletion to decreased growth ( Fernando et al 2015 ; Wada et al 2021 ). In addition to RpS21 , third-party analysis of published RNA-seq data of B52 knockdowns in S2 cells uncovered alternative splicing of yorkie transcripts, a transcriptional co-activator in Hippo signaling ( Srivastava et al 2021 ). Hippo signaling is a highly conserved pathway that controls organ size ( Halder and Johnson 2011 ; Hariharan 2015 ; Yu et al 2015 ; Meng et al 2016 ).…”

Mechanical Control of Cell Migration by the Metastasis Suppressor Tetraspanin CD82/KAI1

“…Maybe YAP localization in the nucleus is also to deplete it from the cytoplasm and to regulate its cytoplasmic functions as we recently described for the junctional protein MAG1 [ 59 ]. Additionally, there exists up to eight different YAP1 isoforms due to splicing and some may have different functions [ 60 ]. Clearly, YAP functions and regulations are complex and more studies are needed to understand the full picture of CD82 function in breast epithelial cells.…”