Unique repression domains of Pumilio utilize deadenylation and decapping factors to accelerate destruction of target mRNAs

Abstract

Pumilio is an RNA-binding protein that represses a network of mRNAs to control embryogenesis, stem cell fate, fertility and neurological functions in Drosophila. We sought to identify the mechanism of Pumilio-mediated repression and find that it accelerates degradation of target mRNAs, mediated by three N-terminal Repression Domains (RDs), which are unique to Pumilio orthologs. We show that the repressive activities of the Pumilio RDs depend on specific subunits of the Ccr4–Not (CNOT) deadenylase complex. Depletion of Pop2, Not1, Not2, or Not3 subunits alleviates Pumilio RD-mediated repression of protein expression and mRNA decay, whereas depletion of other CNOT components had little or no effect. Moreover, the catalytic activity of Pop2 deadenylase is important for Pumilio RD activity. Further, we show that the Pumilio RDs directly bind to the CNOT complex. We also report that the decapping enzyme, Dcp2, participates in repression by the N-terminus of Pumilio. These results support a model wherein Pumilio utilizes CNOT deadenylase and decapping complexes to accelerate destruction of target mRNAs. Because the N-terminal RDs are conserved in mammalian Pumilio orthologs, the results of this work broadly enhance our understanding of Pumilio function and roles in diseases including cancer, neurodegeneration and epilepsy.

Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome

“…Accumulating evidence indicates that the repression mechanism of PUF proteins is conserved. In addition to the data reported here, evidence from worms (Suh et al 2009) , fruit flies (Kadyrova et al 2007; Weidmann et al 2014; Arvola et al 2020) , fission yeast (Webster, Stowell, and Passmore 2019), and budding yeast (Goldstrohm et al 2006) support the central role of CNOT in PUF-mediated repression. The interaction of the highly conserved RBD of PUF proteins with CNOT appears to be universal (Goldstrohm et al 2006; Hook et al 2007; Kadyrova et al 2007; Suh et al 2009; D.…”

“…In contrast, the N-terminal repression domains that bind to CNOT, including RD3, are found in PUF proteins from organisms ranging from insects to vertebrates, but no homologous domains were detected in lower eukaryotes (Weidmann and Goldstrohm 2012; Goldstrohm, Hall, and McKenney 2018). In Drosophila, the sole Pumilio protein has three distinct N-terminal repression domains that can recruit CNOT to elicit repression (Weidmann and Goldstrohm 2012; Arvola et al 2020), whilst an unrelated, unique region of the S. pombe PUF3 protein also binds CNOT (Webster, Stowell, and Passmore 2019). In addition to deadenylation, the involvement of the decapping factors in PUF-mediated repression is also conserved in humans (this study), Drosophila (Arvola et al 2020) , and S. cerevisiae (Olivas and Parker 2000; Goldstrohm et al 2006; Blewett and Goldstrohm 2012).…”

“…In Drosophila, the sole Pumilio protein has three distinct N-terminal repression domains that can recruit CNOT to elicit repression (Weidmann and Goldstrohm 2012; Arvola et al 2020), whilst an unrelated, unique region of the S. pombe PUF3 protein also binds CNOT (Webster, Stowell, and Passmore 2019). In addition to deadenylation, the involvement of the decapping factors in PUF-mediated repression is also conserved in humans (this study), Drosophila (Arvola et al 2020) , and S. cerevisiae (Olivas and Parker 2000; Goldstrohm et al 2006; Blewett and Goldstrohm 2012). Collectively, this research highlights the deep evolutionary conservation of the PUF regulatory mechanisms that dynamically control transcriptomes.…”

“…For reporter assays measuring the activity of endogenous PUMs via PREs, fold change values were calculated from the RRR values for the PUM-regulated Nluc 3xPRE reporter relative to the unregulated mutant PRE reporter, Nluc 3xPRE mt. For RNAi experiments that tested the role of putative co-repressors, the effect of each RNAi condition on PRE/PUM-mediated repression was measured within that same RNAi condition, as previously established (Arvola et al 2020). In this manner, the specific effect of co-repressor depletion on PRE/PUM activity is determined.…”

“…Next, the RNA was purified using a RNA Clean and Concentrator-25 spin-column (Zymo), eluted in 25 μl of water, and again quantitated. Reverse transcription was then performed as previously described using GoScript reverse transcriptase (Promega) with random hexamer primers (Van Etten et al 2012; Arvola et al 2020).…”