The SSUP-72/PINN-1 module is required for efficient 3’end processing and coordinates developmental gene expression during exit from diapause in C. elegans [RNAseq-suppression]

During the exit from the developmental diapause of Caenorhabditis elegans (C. elegans), a network of growth and developmental genes is activated. These genes are organized into operons, where transcriptional termination is uncoupled from mRNA 3’-end processing. Although the Pol II CTD-S2P mark deposited by CDK-12 is unnecessary during embryogenesis, it plays a critical role in the timely expression of most operonic genes by enhancing SL2 trans-splicing at position 2 and above. This process protects mRNA from degradation and prevents termination. Here, a genetic screen has identified the SSUP-72/PINN-1 module as an effective suppressor of CDK-12-induced defects. Our data show that the SSUP-72/PINN-1 module regulates intra-operon termination and affects 3’ pausing genome-wide, coordinating growth and developmental gene expression during post-embryonic development in C. elegans. To investigate the phenotypical suppression of cdk-12as inhibition by ssup-72[E22K]; we conducted RNA-seq on synchronyzed L1 worms (N2, cdk-12as, ssup-72[E22K], cdk-12as;ssup-72[E22K]) grown in presence or absence of 3MB-PP1 (3MB-PP1). Worms were L1 synchornized and grown for 4H with the inhibitor or DMSO.

秀丽隐杆线虫（Caenorhabditis elegans，C. elegans）的发育滞育解除过程中，生长与发育相关基因的调控网络被激活。这些基因以操纵子（operon）的形式组织，其转录终止与mRNA 3’端加工过程解偶联。尽管由CDK-12沉积的RNA聚合酶II（Pol II）羧基末端结构域S2磷酸化修饰（CTD-S2P）在胚胎发生过程中并非必需，但它可通过增强位置2及以上的SL2反式剪接，对大多数操纵子基因的适时表达发挥关键调控作用。该过程可保护mRNA免于降解，并阻止异常转录终止。本研究通过遗传筛选，发现SSUP-72/PINN-1复合物是CDK-12诱导缺陷的有效抑制因子。我们的研究数据表明，SSUP-72/PINN-1复合物可调控操纵子内部的转录终止，并在全基因组范围内影响3’端暂停，进而在秀丽隐杆线虫的胚胎后发育过程中协调生长与发育相关基因的表达。为探究ssup-72[E22K]对cdk-12as抑制作用的表型抑制效应，我们将线虫同步化至L1期，随后在添加3MB-PP1或二甲基亚砜（DMSO）的培养基中培养4小时，进而对同步化的L1期线虫（包括野生型N2、cdk-12as突变体、ssup-72[E22K]突变体以及cdk-12as;ssup-72[E22K]双突变体）开展RNA测序（RNA-seq）。