Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling.

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA.

Polyadenosine RNA-binding proteins (Pabs) regulate multiple steps in gene expression. This protein family includes the well studied Pabs, PABPN1 and PABPC1, as well as the newly characterized Pab, zinc finger CCCH-type containing protein 14 (ZC3H14). Mutations in ZC3H14 are linked to a form of intellectual disability. To probe the function of ZC3H14, we performed a transcriptome-wide analysis of cells depleted of either ZC3H14 or the control Pab, PABPN1. Depletion of PABPN1 affected ∼17% of expressed transcripts, whereas ZC3H14 affected only ∼1% of expressed transcripts. To assess the function of ZC3H14 in modulating target mRNAs, we selected the gene encoding the ATP synthase F0 subunit C (ATP5G1) transcript. Knockdown of ZC3H14 significantly reduced ATP5G1 steady-state mRNA levels. Consistent with results suggesting that ATP5G1 turnover increases upon depletion of ZC3H14, double knockdown of ZC3H14 and the nonsense-mediated decay factor, UPF1, rescues ATP5G1 transcript levels. Furthermore, fractionation reveals an increase in the amount of ATP5G1 pre-mRNA that reaches the cytoplasm when ZC3H14 is depleted and that ZC3H14 binds to ATP5G1 pre-mRNA in the nucleus. These data support a role for ZC3H14 in ensuring proper nuclear processing and retention of ATP5G1 pre-mRNA. Consistent with the observation that ATP5G1 is a rate-limiting component for ATP synthase activity, knockdown of ZC3H14 decreases cellular ATP levels and causes mitochondrial fragmentation. These data suggest that ZC3H14 modulates pre-mRNA processing of select mRNA transcripts and plays a critical role in regulating cellular energy levels, observations that have broad implications for proper neuronal function.

Post-transcriptional regulation of programmed cell death 4 (PDCD4) mRNA by the RNA-binding proteins human antigen R (HuR) and T-cell intracellular antigen 1 (TIA1).

Post-transcriptional processing of mRNA transcripts plays a critical role in establishing the gene expression profile of a cell. Such processing events are mediated by a host of factors, including RNA-binding proteins and microRNAs. A number of critical cellular pathways are subject to regulation at multiple levels that allow fine-tuning of key biological responses. Programmed cell death 4 (PDCD4) is a tumor suppressor and an important modulator of mRNA translation that is regulated by a number of mechanisms, most notably as a target of the oncomiR, miR-21. Here, we provide evidence for post-transcriptional regulation of PDCD4 by the RNA-binding proteins, HuR and TIA1. Complementary approaches reveal binding of both HuR and TIA1 to the PDCD4 transcript. Consistent with a model where RNA-binding proteins modulate the PDCD4 transcript, knockdown of HuR and/or TIA1 results in a significant decrease in steady-state PDCD4 mRNA and protein levels. However, fractionation experiments suggest that the mode of regulation of the PDCD4 transcript likely differs in the cytoplasm and the nucleus as the pool of PDCD4 mRNA present in the cytoplasm is more stable than the nuclear pool of PDCD4 transcript. We observe a competitive mode of binding between HuR and TIA1 on the PDCD4 transcript in the cytoplasm, suggesting that these two factors dynamically interact with one another as well as the PDCD4 transcript to maintain tight control of PDCD4 levels. Overall, this study reveals an additional set of regulatory interactions that modulate the expression of PDCD4, a key pro-apoptotic factor, and also reveals new insights into how HuR and TIA1 functions are integrated to achieve such regulation.

Poly(A) RNA-binding proteins and polyadenosine RNA: new members and novel functions.

Poly(A) RNA-binding proteins (Pabs) bind with high affinity and specificity to polyadenosine RNA. Textbook models show a nuclear Pab, PABPN1, and a cytoplasmic Pab, PABPC, where the nuclear PABPN1 modulates poly(A) tail length and the cytoplasmic PABPC stabilizes poly(A) RNA in the cytoplasm and also enhances translation. While these conventional roles are critically important, the Pab family has expanded recently both in number and in function. A number of novel roles have emerged for both PAPBPN1 and PABPC that contribute to the fine-tuning of gene expression. Furthermore, as the characterization of the nucleic acid binding properties of RNA-binding proteins advances, additional proteins that show high affinity and specificity for polyadenosine RNA are being discovered. With this expansion of the Pab family comes a concomitant increase in the potential for Pabs to modulate gene expression. Further complication comes from an expansion of the potential binding sites for Pab proteins as revealed by an analysis of templated polyadenosine stretches present within the transcriptome. Thus, Pabs could influence mRNA fate and function not only by binding to the nontemplated poly(A) tail but also to internal stretches of adenosine. Understanding the diverse functions of Pab proteins is not only critical to understand how gene expression is regulated but also to understand the molecular basis for tissue-specific diseases that occur when Pab proteins are altered. Here we describe both conventional and recently emerged functions for PABPN1 and PABPC and then introduce and discuss three new Pab family members, ZC3H14, hnRNP-Q1, and LARP4.