Anti-prion Drugs Targeting the Protein Folding Activity of the Ribosome Reduce PABPN1 Aggregation.

Prion diseases are caused by the propagation of PrPSc, the pathological conformation of the PrPC prion protein. The molecular mechanisms underlying PrPSc propagation are still unsolved and no therapeutic solution is currently available. We thus sought to identify new anti-prion molecules and found that flunarizine inhibited PrPSc propagation in cell culture and significantly prolonged survival of prion-infected mice. Using an in silico therapeutic repositioning approach based on similarities with flunarizine chemical structure, we tested azelastine, duloxetine, ebastine, loperamide and metixene and showed that they all have an anti-prion activity. Like flunarizine, these marketed drugs reduced PrPSc propagation in cell culture and in mouse cerebellum organotypic slice culture, and inhibited the protein folding activity of the ribosome (PFAR). Strikingly, some of these drugs were also able to alleviate phenotypes due to PABPN1 nuclear aggregation in cell and Drosophila models of oculopharyngeal muscular dystrophy (OPMD). These data emphasize the therapeutic potential of anti-PFAR drugs for neurodegenerative and neuromuscular proteinopathies.

Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells.

Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease.

Cytoplasmic poly(A)-binding protein 1 (PABPC1) interacts with the RNA-binding protein hnRNPLL and thereby regulates immunoglobulin secretion in plasma cells.

Increasing evidence indicates that alternative processing of mRNA, including alternative splicing, 3' alternative polyadenylation, and regulation of mRNA stability/translation, represents a major mechanism contributing to protein diversification. For example, in alternative polyadenylation, the 3' end of the immunoglobulin heavy chain mRNA is processed during B cell differentiation, and this processing involves RNA-binding proteins. hnRNPLL (heterogeneous nuclear ribonucleoprotein L-like protein) is an RNA-binding protein expressed in terminally differentiated lymphocytes, such as memory T cells and plasma cells. hnRNPLL regulates various processes of RNA metabolism, including alternative pre-mRNA splicing and RNA stability. In plasma cells, hnRNPLL also regulates the transition from the membrane isoform of the immunoglobulin heavy-chain (mIgH) to the secreted isoform (sIgH), but the precise mechanism remains to be identified. In this study, we report that hnRNPLL specifically associates with cytoplasmic PABPC1 (poly(A)-binding protein 1) in both T cells and plasma cells. We found that although PABPC1 is not required for the alternative splicing of CD45, a primary target of hnRNPLL in lymphocytes, PABPC1 does promote the binding of hnRNPLL to the immunoglobulin mRNA and regulates switching from mIgH to sIgH in plasma cells. Given the recently identified role of PABPC1 in mRNA alternative polyadenylation, our findings suggest that PABPC1 recruits hnRNPLL to the 3'-end of RNA and regulates the transition from membrane Ig to secreted Ig through mRNA alternative polyadenylation. In conclusion, our study has revealed a mechanism that regulates immunoglobulin secretion in B cells via cooperation between a plasma cell-specific RBP (hnRNPLL) and a universally expressed RBP (PABPC1).

Identification of a Nuclear Exosome Decay Pathway for Processed Transcripts.

The RNA exosome is fundamental for the degradation of RNA in eukaryotic nuclei. Substrate targeting is facilitated by its co-factor Mtr4p/hMTR4, which links to RNA-binding protein adaptors. One example is the trimeric human nuclear exosome targeting (NEXT) complex, which is composed of hMTR4, the Zn-finger protein ZCCHC8, and the RNA-binding factor RBM7. NEXT primarily targets early and unprocessed transcripts, which demands a rationale for how the nuclear exosome recognizes processed RNAs. Here, we describe the poly(A) tail exosome targeting (PAXT) connection, which comprises the ZFC3H1 Zn-knuckle protein as a central link between hMTR4 and the nuclear poly(A)-binding protein PABPN1. Individual depletion of ZFC3H1 and PABPN1 results in the accumulation of common transcripts that are generally both longer and more extensively polyadenylated than NEXT substrates. Importantly, ZFC3H1/PABPN1 and ZCCHC8/RBM7 contact hMTR4 in a mutually exclusive manner, revealing that the exosome targets nuclear transcripts of different maturation status by substituting its hMTR4-associating adaptors.

Interaction of PABPC1 with the translation initiation complex is critical to the NMD resistance of AUG-proximal nonsense mutations.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3'-5' linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.

Poly(A)-binding proteins are functionally distinct and have essential roles during vertebrate development.

Translational control of many mRNAs in developing metazoan embryos is achieved by alterations in their poly(A) tail length. A family of cytoplasmic poly(A)-binding proteins (PABPs) bind the poly(A) tail and can regulate mRNA translation and stability. However, despite the extensive biochemical characterization of one family member (PABP1), surprisingly little is known about their in vivo roles or functional relatedness. Because no information is available in vertebrates, we address their biological roles, establishing that each of the cytoplasmic PABPs conserved in Xenopus laevis [PABP1, embryonic PABP (ePABP), and PABP4] is essential for normal development. Morpholino-mediated knockdown of PABP1 or ePABP causes both anterior and posterior phenotypes and embryonic lethality. In contrast, depletion of PABP4 results mainly in anterior defects and lethality at later stages. Unexpectedly, cross-rescue experiments reveal that neither ePABP nor PABP4 can fully rescue PABP1 depletion, establishing that PABPs have distinct functions. Comparative analysis of the uncharacterized PABP4 with PABP1 and ePABP shows that it shares a mechanistically conserved core role in promoting global translation. Consistent with this analysis, each morphant displays protein synthesis defects, suggesting that their roles in mRNA-specific translational regulation and/or mRNA decay, rather than global translation, underlie the functional differences between PABPs. Domain-swap experiments reveal that the basis of the functional specificity is complex, involving multiple domains of PABPs, and is conferred, at least in part, by protein-protein interactions.

Role of GW182 proteins and PABPC1 in the miRNA pathway: a sense of déjà vu.

GW182 proteins have emerged as key components of microRNA (miRNA) silencing complexes in animals. Although the precise molecular function of GW182 proteins is not fully understood, new findings indicate that they act as poly(A)-binding protein (PABP)-interacting proteins (PAIPs) that promote gene silencing, at least in part, by interfering with cytoplasmic PABP1 (PABPC1) function during translation and mRNA stabilization. This recent discovery paves the way for future studies of miRNA silencing mechanisms.