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N 6 -methyladenosine (m 6 A) is an abundant post-transcriptional modification that can impact RNA fate via interactions with m 6 A-specific RNA binding proteins. Despite accumulating evidence that m 6 A plays an important role in modulating pluripotency, the influence of m 6 A reader proteins in pluripotency is less clear. Here, we report that YTHDF2, an m 6 A reader associated with mRNA degradation, is highly expressed in induced pluripotent stem cells (iPSCs) and down-regulated during neural differentiation. Through RNA sequencing, we identified a group of m 6 A-modified transcripts associated with neural development that are directly regulated by YTDHF2. Depletion of YTHDF2 in iPSCs leads to stabilization of these transcripts, loss of pluripotency, and induction of neural-specific gene expression. Collectively, our results suggest YTHDF2 functions to restrain expression of neural-specific mRNAs in iPSCs and facilitate their rapid and coordinated up-regulation during neural induction. These effects are both achieved by destabilization of the targeted transcripts., (© 2020 Heck et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

RNA decay plays a major role in regulating gene expression and is tightly networked with other aspects of gene expression to effectively coordinate post-transcriptional regulation. The goal of this work is to provide an overview of the major factors and pathways of general messenger RNA (mRNA) decay in eukaryotic cells, and then discuss the effective interplay of this cytoplasmic process with the protein synthesis machinery. Given the transcript-specific and fluid nature of mRNA stability in response to changing cellular conditions, understanding the fundamental networking between RNA decay and translation will provide a foundation for a complete mechanistic understanding of this important aspect of cell biology., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)

Production of multiple functional RNAs from a single primary transcript is an extremely efficient use of genetic information, although it complicates the ability of the cell to independently regulate the production of each RNA. For the case of small nucleolar RNAs (snoRNAs) encoded within introns of mRNA genes, Lykke-Andersen and colleagues (pp. 2498-2517) demonstrated that alternative splicing and the SMG6 endonuclease of the nonsense-mediated RNA decay pathway are key regulators that control which RNAs accumulate., (© 2014 Wilusz and Wilusz; Published by Cold Spring Harbor Laboratory Press.)

All arthropod-borne flaviviruses generate a short noncoding RNA (sfRNA) from the viral 3' untranslated region during infection due to stalling of the cellular 5'-to-3' exonuclease XRN1. We show here that formation of sfRNA also inhibits XRN1 activity. Cells infected with Dengue or Kunjin viruses accumulate uncapped mRNAs, decay intermediates normally targeted by XRN1. XRN1 repression also resulted in the increased overall stability of cellular mRNAs in flavivirus-infected cells. Importantly, a mutant Kunjin virus that cannot form sfRNA but replicates to normal levels failed to affect host mRNA stability or XRN1 activity. Expression of sfRNA in the absence of viral infection demonstrated that sfRNA formation was directly responsible for the stabilization of cellular mRNAs. Finally, numerous cellular mRNAs were differentially expressed in an sfRNA-dependent fashion in a Kunjin virus infection. We conclude that flaviviruses incapacitate XRN1 during infection and dysregulate host mRNA stability as a result of sfRNA formation.

Pluripotency is a unique state in which cells can self-renew indefinitely but also retain the ability to differentiate into other cell types upon receipt of extracellular cues. Although it is clear that stem cells have a distinct transcriptional program, little is known about how alterations in post-transcriptional mechanisms, such as mRNA turnover, contribute to the achievement and maintenance of pluripotency. Here we have assessed the rates of decay for the majority of mRNAs expressed in induced pluripotent stem (iPS) cells and the fully differentiated human foreskin fibroblasts (HFFs) they were derived from. Comparison of decay rates in the two cell types led to the discovery of three independent regulatory mechanisms that allow coordinated turnover of specific groups of mRNAs. One mechanism results in increased stability of many histone mRNAs in iPS cells. A second pathway stabilizes a large set of zinc finger protein mRNAs, potentially through reduced levels of miRNAs that target them. Finally, a group of transcripts bearing 3' UTR C-rich sequence elements, many of which encode transcription factors, are significantly less stable in iPS cells. Intriguingly, two poly(C)-binding proteins that recognize this type of element are reciprocally expressed in iPS and HFF cells. Overall, our results highlight the importance of post-transcriptional control in pluripotent cells and identify miRNAs and RNA-binding proteins whose activity may coordinately control expression of a wide range of genes in iPS cells.

We have previously demonstrated that PM-Scl-75, a component of the human exosome complex involved in RNA maturation and mRNA decay, can specifically interact with RNAs containing an AU-rich instability element. Through the analysis of a series of deletion mutants, we have now shown that a 266 amino acid fragment representing the RNase PH domain is responsible for the sequence-specific binding to AU-rich elements. Furthermore, we found that the RNase PH domains from two other exosomal components, OIP2 and RRP41, as well as from Escherichia coli polynucleotide phosphorylase, are all capable of specifically interacting with RNAs containing an AU-rich element with similar affinities. Finally, we demonstrate that the interaction of the RNase PH domain of PM-Scl-75 is readily competed by poly(U), but only inefficiently using other homopolymeric RNAs. These data demonstrate that RNase PH domains in general have an affinity for U- and AU-rich sequences, and broaden the potential role in RNA biology of proteins containing these domains.

CUG-BP is the human homolog of the Xenopus EDEN-BP, which was shown previously to bind to mRNAs, such as c-mos, that exhibit rapid deadenylation following fertilization of the oocyte. While several studies have focused on roles of CUG-BP as a splicing or translation regulator in mammalian cells, its role in mRNA decay has not been examined in detail. Here, we have used an in vitro deadenylation assay to dissect the function of CUG-BP in the decay of two ARE-containing mRNAs: c-fos and TNFalpha. CUG-BP binds specifically to both of these RNAs and stimulates poly(A) shortening by PARN. Moreover, CUG-BP interacts with PARN in extracts by coimmunoprecipitation, and this interaction can be recapitulated using recombinant proteins. CUG-BP, therefore, is the first RNA-binding protein shown to directly recruit a deadenylase to an RNA substrate.

Polyadenylation is an essential step for the maturation of almost all cellular mRNAs in eukaryotes. In human cells, most poly(A) sites are flanked by the upstream AAUAAA hexamer or a close variant, and downstream U/GU-rich elements. In yeast and plants, additional cis elements have been found to be located upstream of the poly(A) site, including UGUA, UAUA, and U-rich elements. In this study, we have developed a computer program named PROBE (Polyadenylation-Related Oligonucleotide Bidimensional Enrichment) to identify cis elements that may play regulatory roles in mRNA polyadenylation. By comparing human genomic sequences surrounding frequently used poly(A) sites with those surrounding less frequently used ones, we found that cis elements occurring in yeast and plants also exist in human poly(A) regions, including the upstream U-rich elements, and UAUA and UGUA elements. In addition, several novel elements were found to be associated with human poly(A) sites, including several G-rich elements. Thus, we suggest that many cis elements are evolutionarily conserved among eukaryotes, and human poly(A) sites have an additional set of cis elements that may be involved in the regulation of mRNA polyadenylation.

The stability of mRNAs is an important point in the regulation of gene expression in eukaryotes. The mRNA turnover pathways have been identified in yeast and mammals. However, mRNA turnover pathways in trypanosomes have not been widely studied. Deadenylation is the first step in the major mRNA turnover pathways of yeast and mammals. To better understand mRNA degradation processes in these organisms, we have developed an in vitro mRNA turnover system that is functional for deadenylation. In this system, addition of poly(A) homopolymer activates the deadenylation of poly(A) tails. The trypanosomal deadenylase activity is a 3'-->5' exonuclease specific for adenylate residues, generates 5'-AMP as a product, is magnesium dependent, and is inhibited by neomycin B sulfate. These characteristics suggest similarity with other eukaryotic deadenylases. Furthermore, this activity is cap independent, indicating a potential difference between the trypanosomal activity and PARN, but suggesting similarity to Ccr4p/Pop2p activities. Extracts immunodepleted of Pab1p required the addition of poly(A) competition to activate deadenylation. Trypanosomal Pab1p functions as an inhibitor of the activity under in vitro conditions. Pab1p appears to be one of several mRNA stability proteins in trypanosomal extracts.

The majority of mRNA turnover is mediated either by mRNA decapping/5'-to-3' decay or exosome-mediated 3'-to-5' exonucleolytic decay. Current assays to assess mRNA decapping in vitro using cap-labeled RNA substrates rely on one-dimensional thin layer chromatography. This approach does not, however, resolve free phosphate from 7meGDP, the product of Dcp1p-mediated mRNA decapping. This can result in misinterpretation of the levels of mRNA decapping due to the generation of free phosphate following the action of the unrelated scavenger decapping activity on the products of exosome-mediated decay. In this report, we describe a simple denaturing acrylamide gel-based assay that faithfully resolves all of the possible products that can be generated from cap-labeled RNA substrates by turnover enzymes present in cell extracts. This approach allows a one-step assay to quantitatively assess the contributions of the exosome and DCP-1-type decapping on turnover of an RNA substrate in vitro. We have applied this assay to recalculate the effect of competition of cap-binding proteins on decapping in yeast. In addition, we have used the assay to confirm observations made on regulated mRNA decapping in mammalian extracts that contain much higher levels of exosome activity than yeast extracts.

The pathway of mRNA degradation has been extensively studied in the yeast, Saccharomyces cerevisiae, and it is now clear that many mRNAs decay by a deadenylation-dependent mechanism. Although several of the factors required for mRNA decay have been identified, the regulation and precise roles of many of the proteins involved remains unclear. We have developed an in vitro system that recapitulates both the deadenylation and the decapping steps of mRNA decay. Furthermore, both deadenylation and decapping are inhibited by poly(A) binding proteins in our assay. Our system has allowed us to separate the decay process from translation and we have shown that the poly(A) tail is capable of inhibiting decapping in an eIF4E-independent manner. Our in vitro system should prove invaluable in dissecting the mechanisms of mRNA turnover.

We have developed an in vitro mRNA stability system using HeLa cell cytoplasmic S100 extracts and exogenous polyadenylated RNA substrates that reproduces regulated aspects of mRNA decay. The addition of cold poly(A) competitor RNA activated both a sequence-specific deadenylase activity in the extracts as well as a potent, ATP-dependent ribonucleolytic activity. The rates of both deadenylation and degradation were up-regulated by the presence of a variety of AU-rich elements in the body of substrate RNAs. Competition analyses demonstrated that trans-acting factors were required for RNA destabilization by AU-rich elements. The approximately 30-kD ELAV protein HuR specifically bound to RNAs containing an AU-rich element derived from the TNF-alpha mRNA in the in vitro system. Interaction of HuR with AU-rich elements, however, was not associated with RNA destabilization. Interestingly, recombinant ELAV proteins specifically stabilized deadenylated intermediates generated from the turnover of AU-rich element-containing substrate RNAs. These data suggest that mammalian ELAV proteins play a role in regulating mRNA stability by influencing the access of degradative enzymes to RNA substrates.

We have purified and characterized a factor required for accurate polyadenylation of mammalian pre-mRNAs in vitro. This factor, called cleavage-stimulation factor (CstF), is composed of three distinct polypeptide subunits of 77, 64, and 50 kD. Using monoclonal antibodies directed against the 64- and 50-kD subunits, we show that CstF is required for efficient cleavage of polyadenylation substrates. Furthermore, CstF present in unfractionated nuclear extracts interacts with pre-mRNAs containing the signal sequence AAUAAA, but not AAGAAA, in such a manner that the 64-kD subunit can be cross-linked to the RNA by UV light. This polypeptide is thus identical to the previously described 64-kD nuclear protein that binds to AAUAAA-containing RNAs. Finally, indirect immunofluorescence of fixed cells indicates that CstF is distributed diffusely throughout the nucleus in a granular pattern distinct from the "speckled" pattern displayed by factors involved in pre-mRNA splicing, but similar to that of heterogeneous nuclear ribonucleoproteins. A model is presented in which CstF binds specifically to nascent RNA polymerase II transcripts and, by interacting with other factors, results in a rapid initiation of 3'-end processing of pre-mRNAs.