Uncovering a mammalian neural-specific poly(A) binding protein with unique properties.

The mRNA 3' poly(A) tail plays a critical role in regulating both mRNA translation and turnover. It is bound by the cytoplasmic poly(A) binding protein (PABPC), an evolutionarily conserved protein that can interact with translation factors and mRNA decay machineries to regulate gene expression. Mammalian PABPC1, the prototypical PABPC, is expressed in most tissues and interacts with eukaryotic translation initiation factor 4G (eIF4G) to stimulate translation in specific contexts. In this study, we uncovered a new mammalian PABPC, which we named neural PABP (neuPABP), as it is predominantly expressed in the brain. neuPABP maintains a unique architecture as compared with other PABPCs, containing only two RNA recognition motifs (RRMs) and maintaining a unique N-terminal domain of unknown function. neuPABP expression is activated in neurons as they mature during synaptogenesis, where neuPABP localizes to the soma and postsynaptic densities. neuPABP interacts with the noncoding RNA BC1, as well as mRNAs coding for ribosomal and mitochondrial proteins. However, in contrast to PABPC1, neuPABP does not associate with actively translating mRNAs in the brain. In keeping with this, we show that neuPABP has evolved such that it does not bind eIF4G and as a result fails to support protein synthesis in vitro. Taken together, these results indicate that mammals have expanded their PABPC repertoire in the brain and propose that neuPABP may support the translational repression of select mRNAs.

The EDC4-XRN1 interaction controls P-body dynamics to link mRNA decapping with decay.

Deadenylation-dependent mRNA decapping and decay is the major cytoplasmic mRNA turnover pathway in eukaryotes. Many mRNA decapping and decay factors are associated with each other via protein-protein interaction motifs. For example, the decapping enzyme DCP2 and the 5'-3' exonuclease XRN1 interact with the enhancer of mRNA-decapping protein 4 (EDC4), a large scaffold that has been reported to stimulate mRNA decapping. mRNA decapping and decay factors are also found in processing bodies (P-bodies), evolutionarily conserved ribonucleoprotein granules that are often enriched with mRNAs targeted for decay, yet paradoxically are not required for mRNA decay to occur. Here, we show that disrupting the EDC4-XRN1 interaction or altering their stoichiometry inhibits mRNA decapping, with microRNA-targeted mRNAs being stabilized in a translationally repressed state. Importantly, we demonstrate that this concomitantly leads to larger P-bodies that are responsible for preventing mRNA decapping. Finally, we demonstrate that P-bodies support cell viability and prevent stress granule formation when XRN1 is limiting. Taken together, these data demonstrate that the interaction between XRN1 and EDC4 regulates P-body dynamics to properly coordinate mRNA decapping with 5'-3' decay in human cells.

PABP prevents the untimely decay of select mRNA populations in human cells.

Gene expression is tightly regulated at the levels of both mRNA translation and stability. The poly(A)-binding protein (PABP) is thought to play a role in regulating these processes by binding the mRNA 3' poly(A) tail and interacting with both the translation and mRNA deadenylation machineries. In this study, we directly investigate the impact of PABP on translation and stability of endogenous mRNAs in human cells. Remarkably, our transcriptome-wide analysis only detects marginal mRNA translation changes in PABP-depleted cells. In contrast, rapidly depleting PABP alters mRNA abundance and stability, albeit non-uniformly. Otherwise stable transcripts, including those encoding proteins with constitutive functions, are destabilized in PABP-depleted cells. In contrast, many unstable mRNAs, including those encoding proteins with regulatory functions, decay at similar rates in presence or absence of PABP. Moreover, PABP depletion-induced cell death can partially be suppressed by disrupting the mRNA decapping and 5'-3' decay machinery. Finally, we provide evidence that the LSM1-7 complex promotes decay of "stable" mRNAs in PABP-depleted cells. Taken together, these findings suggest that PABP plays an important role in preventing the untimely decay of select mRNA populations.

P-bodies directly regulate MARF1-mediated mRNA decay in human cells.

Processing bodies (P-bodies) are ribonucleoprotein granules that contain mRNAs, RNA-binding proteins and effectors of mRNA turnover. While P-bodies have been reported to contain translationally repressed mRNAs, a causative role for P-bodies in regulating mRNA decay has yet to be established. Enhancer of decapping protein 4 (EDC4) is a core P-body component that interacts with multiple mRNA decay factors, including the mRNA decapping (DCP2) and decay (XRN1) enzymes. EDC4 also associates with the RNA endonuclease MARF1, an interaction that antagonizes the decay of MARF1-targeted mRNAs. How EDC4 interacts with MARF1 and how it represses MARF1 activity is unclear. In this study, we show that human MARF1 and XRN1 interact with EDC4 using analogous conserved short linear motifs in a mutually exclusive manner. While the EDC4-MARF1 interaction is required for EDC4 to inhibit MARF1 activity, our data indicate that the interaction with EDC4 alone is not sufficient. Importantly, we show that P-body architecture plays a critical role in antagonizing MARF1-mediated mRNA decay. Taken together, our study suggests that P-bodies can directly regulate mRNA turnover by sequestering an mRNA decay enzyme and preventing it from interfacing with and degrading targeted mRNAs.

PP2ACdc55 dephosphorylates Pds1 and inhibits spindle elongation in S. cerevisiae.

PP2ACdc55 (the form of protein phosphatase 2A containing Cdc55) regulates cell cycle progression by reversing cyclin-dependent kinase (CDK)- and polo-like kinase (Cdc5)-dependent phosphorylation events. In S. cerevisiae, Cdk1 phosphorylates securin (Pds1), which facilitates Pds1 binding and inhibits separase (Esp1). During anaphase, Esp1 cleaves the cohesin subunit Scc1 and promotes spindle elongation. Here, we show that PP2ACdc55 directly dephosphorylates Pds1 both in vivo and in vitro Pds1 hyperphosphorylation in a cdc55 deletion mutant enhanced the Pds1-Esp1 interaction, which played a positive role in Pds1 nuclear accumulation and in spindle elongation. We also show that nuclear PP2ACdc55 plays a role during replication stress to inhibit spindle elongation. This pathway acted independently of the known Mec1, Swe1 or spindle assembly checkpoint (SAC) checkpoint pathways. We propose a model where Pds1 dephosphorylation by PP2ACdc55 disrupts the Pds1-Esp1 protein interaction and inhibits Pds1 nuclear accumulation, which prevents spindle elongation, a process that is elevated during replication stress.

A Screen for Candidate Targets of Lysine Polyphosphorylation Uncovers a Conserved Network Implicated in Ribosome Biogenesis.

Polyphosphates (polyP) are chains of inorganic phosphates found in all cells. Previous work has implicated these chains in diverse functions, but the mechanism of action is unclear. A recent study reports that polyP can be non-enzymatically and covalently attached to lysine residues on yeast proteins Nsr1 and Top1. One question emerging from this work is whether so-called "polyphosphorylation" is unique to these proteins or instead functions as a global regulator akin to other lysine post-translational modifications. Here, we present the results of a screen for polyphosphorylated proteins in yeast. We uncovered 15 targets including a conserved network of proteins functioning in ribosome biogenesis. Multiple genes contribute to polyphosphorylation of targets by regulating polyP synthesis, and disruption of this synthesis results in translation defects as measured by polysome profiling. Finally, we identify 6 human proteins that can be modified by polyP, highlighting the therapeutic potential of manipulating polyphosphorylation in vivo.