Principles of mRNA control by human PUM proteins elucidated from multimodal experiments and integrative data analysis

Abstract

The human PUF-family proteins, PUM1 and PUM2, post-transcriptionally regulate gene expression by binding to a PUM recognition element (PRE) in the 3' UTR of target mRNAs. Hundreds of PUM1/2 targets have been identified from changes in steady state RNA levels; however, prior studies could not differentiate between the contributions of changes in transcription and RNA decay rates. We applied metabolic labeling to measure changes in RNA turnover in response to depletion of PUM1/2, showing that human PUM proteins regulate expression almost exclusively by changing RNA stability. We also applied an in vitro selection workflow to precisely identify the binding preferences of PUM1 and PUM2. By integrating our results with prior knowledge, we developed a 'rulebook' of key contextual features that differentiate functional vs. non-functional PREs, allowing us to train machine learning models that accurately predict the functional regulation of RNA targets by the human PUM proteins.

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

“…Emerging evidence indicates that PUM1&2 can alternatively promote expression of certain genes (Naudin et al 2017;Bohn et al 2018;Wolfe et al 2020), referred to as PUM-mediated activation (Goldstrohm, Hall, and McKenney 2018). Here, we identified 62 genes that were downregulated by depletion of PUM1&2, contain PREs, and are bound by PUMs ( Figure S2F, Table S3), providing additional support for direct PUM-mediated activation.…”

“…Compositional CNOT requirements for PUM-mediated repression. We and others have shown that human PUMs can accelerate degradation of PRE-containing mRNAs (Morris, Mukherjee, and Keene 2008;Bohn et al 2018;Goldstrohm, Hall, and McKenney 2018;Wolfe et al 2020) and implicated the CNOT deadenylase complex in this mechanism (Van Etten et al 2012). However, the functional requirement for CNOT and the molecular basis of the PUM1&2mediated repression remained unknown.…”

“…The most significantly enriched GO terms include positive regulation of RNA polymerase II transcription, MAPK signaling, regulation of cell migration, endocytosis, Wnt signaling, and post-embryonic development, as well as cancer terms (Table S3). Many of these are direct targets of PUM1&2, based on the presence of PRE sites, evidence of binding by PUMs, and supported by comparative analyses(Bohn et al 2018;Goldstrohm, Hall, and McKenney 2018;Wolfe et al 2020). Combining these PAC-Seq results from HCT116 cells with data from HEK293 cells(Bohn et al 2018;Wolfe et al 2020) increases the number of identified PUM1&2-repressed mRNAs to 1476.…”

“…Given that all vertebrates have two Pumilio paralogs (Goldstrohm, Hall, and McKenney 2018), do PUM1&2 have redundant, overlapping, or unique regulatory functions? In vitro RNA-binding studies showed that PUM1&2 display highly correlated specificities (Cheong and Hall 2006;Lu and Hall 2011;Van Etten et al 2012;Jarmoskaite et al 2019;Wolfe et al 2020). RNA coimmunoprecipitation analysis demonstrated significant overlap of bound mRNAs but also provided evidence for unique subsets (Morris, Mukherjee, and Keene 2008;Galgano et al 2008;Hafner et al 2010;Yamada et al 2020).…”

“…Wang et al 2002). They regulate PRE-containing mRNAs from diverse genes, predominantly acting as repressors that cause degradation of target mRNAs and reduce protein production (Morris, Mukherjee, and Keene 2008;Van Etten et al 2012;Bohn et al 2018;Goldstrohm, Hall, and McKenney 2018;Wolfe et al 2020;Yamada et al 2020).…”