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In eukaryotes, mRNAs with long poly(A) tails are translationally active, but deadenylation and uridylation of these tails generally cause mRNA degradation. However, the fate of uridylated mRNAs that are not degraded quickly remains obscure. Here, using tail-seq and microinjection of the 3′ region of mRNA, we report that some mRNAs in starfish are re-polyadenylated to be translationally active after deadenylation and uridylation. In oocytes, uridylated maternal cyclin B mRNAs are stable without decay, and they are polyadenylated to be translated after hormonal stimulation to resume meiosis, whereas they are deadenylated and re-uridylated at the blastula stage, followed by decay. Similarly, deadenylated and uridylated maternal ribosomal protein mRNAs, Rps29 and Rpl27a, were stable and inactive after hormonal stimulation, but they had been polyadenylated and active before hormonal stimulation. At the morula stage, uridylated maternal ribosomal protein mRNAs were re-polyadenylated, rendering them translationally active. These results indicate that uridylated mRNAs in starfish exist in a poised state, allowing them to be recycled or decayed. [ABSTRACT FROM AUTHOR]

Abstract Background The 5´ untranslated region (5´ UTR) plays a key role in regulating translation efficiency and mRNA stability, making it a favored target in genetic engineering and synthetic biology. A common feature found in the 5´ UTR is the poly-adenine (poly(A)) tract. However, the effect of 5´ UTR poly(A) on protein production remains controversial. Machine-learning models are powerful tools for explaining the complex contributions of features, but models incorporating features of 5´ UTR poly(A) are currently lacking. Thus, our goal is to construct such a model, using natural 5´ UTRs from Kluyveromyces marxianus, a promising cell factory for producing heterologous proteins. Results We constructed a mini-library consisting of 207 5´ UTRs harboring poly(A) and 34 5´ UTRs without poly(A) from K. marxianus. The effects of each 5´ UTR on the production of a GFP reporter were evaluated individually in vivo, and the resulting protein abundance spanned an approximately 450-fold range throughout. The data were used to train a multi-layer perceptron neural network (MLP-NN) model that incorporated the length and position of poly(A) as features. The model exhibited good performance in predicting protein abundance (average R2 = 0.7290). The model suggests that the length of poly(A) is negatively correlated with protein production, whereas poly(A) located between 10 and 30 nt upstream of the start codon (AUG) exhibits a weak positive effect on protein abundance. Using the model as guidance, the deletion or reduction of poly(A) upstream of 30 nt preceding AUG tended to improve the production of GFP and a feruloyl esterase. Deletions of poly(A) showed inconsistent effects on mRNA levels, suggesting that poly(A) represses protein production either with or without reducing mRNA levels. Conclusion The effects of poly(A) on protein production depend on its length and position. Integrating poly(A) features into machine-learning models improves simulation accuracy. Deleting or reducing poly(A) upstream of 30 nt preceding AUG tends to enhance protein production. This optimization strategy can be applied to enhance the yield of K. marxianus and other microbial cell factories.

Background: The 5´ untranslated region (5´ UTR) plays a key role in regulating translation efficiency and mRNA stability, making it a favored target in genetic engineering and synthetic biology. A common feature found in the 5´ UTR is the poly-adenine (poly(A)) tract. However, the effect of 5´ UTR poly(A) on protein production remains controversial. Machine-learning models are powerful tools for explaining the complex contributions of features, but models incorporating features of 5´ UTR poly(A) are currently lacking. Thus, our goal is to construct such a model, using natural 5´ UTRs from Kluyveromyces marxianus, a promising cell factory for producing heterologous proteins. Results: We constructed a mini-library consisting of 207 5´ UTRs harboring poly(A) and 34 5´ UTRs without poly(A) from K. marxianus. The effects of each 5´ UTR on the production of a GFP reporter were evaluated individually in vivo, and the resulting protein abundance spanned an approximately 450-fold range throughout. The data were used to train a multi-layer perceptron neural network (MLP-NN) model that incorporated the length and position of poly(A) as features. The model exhibited good performance in predicting protein abundance (average R2 = 0.7290). The model suggests that the length of poly(A) is negatively correlated with protein production, whereas poly(A) located between 10 and 30 nt upstream of the start codon (AUG) exhibits a weak positive effect on protein abundance. Using the model as guidance, the deletion or reduction of poly(A) upstream of 30 nt preceding AUG tended to improve the production of GFP and a feruloyl esterase. Deletions of poly(A) showed inconsistent effects on mRNA levels, suggesting that poly(A) represses protein production either with or without reducing mRNA levels. Conclusion: The effects of poly(A) on protein production depend on its length and position. Integrating poly(A) features into machine-learning models improves simulation accuracy. Deleting or reducing poly(A) upstream of 30 nt preceding AUG tends to enhance protein production. This optimization strategy can be applied to enhance the yield of K. marxianus and other microbial cell factories. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, the synthesis, processing, and degradation of mRNA are important processes required for the accurate execution of gene expression programmes. Fully processed cytoplasmic mRNA is characterised by the presence of a 5'cap structure and 3'poly(A) tail. These elements promote translation and prevent non-specific degradation. Degradation via the deadenylation-dependent 5'-3' degradation pathway can be induced by transacting factors binding the mRNA, such as RNA-binding proteins recognising sequence elements and the miRNA-induced repression complex. These factors recruit the core mRNA degradation machinery that carries out the following steps: i) shortening of the poly(A) tail by the Ccr4-Not and Pan2-Pan3 poly (A)-specific nucleases (deadenylases); ii) removal of the 5'cap structure by the Dcp1-Dcp2 decapping complex that is recruited by the Lsm1-7-Pat1 complex; and iii) degradation of the mRNA body by the 5'-3' exoribonuclease Xrn1. In this review, the biochemical function of the nucleases and accessory proteins involved in deadenylation-dependent mRNA degradation will be reviewed with a particular focus on structural aspects of the proteins and enzymes involved. [ABSTRACT FROM AUTHOR]

Protein arginine methyltransferase 5 (Prmt5) is essential for normal B‐cell development; however, the roles of Prmt5 in tumor‐infiltrating B cells in tumor therapy have not been well elucidated. Here, we revealed that CD19‐cre‐Prmt5fl/fl (Prmt5cko) mice showed smaller tumor weights and volumes in the colorectal cancer mouse model; B cells expressed higher levels of Ccl22 and Il12a, which attracted T cells to the tumor site. Furthermore, we used direct RNA sequencing to comprehensively profile RNA processes in Prmt5 deletion B cells to explore underline mechanisms. We found significantly differentially expressed isoforms, mRNA splicing, poly(A) tail lengths, and m6A modification changes between the Prmt5cko and control groups. Cd74 isoform expressions might be regulated by mRNA splicing; the expression of two novel Cd74 isoforms was decreased, while one isoform was elevated in the Prmt5cko group, but the Cd74 gene expression showed no changes. We observed Ccl22, Ighg1, and Il12a expression was significantly increased in the Prmt5cko group, whereas Jak3 and Stat5b expression was decreased. Ccl22 and Ighg1 expression might be associated with poly(A) tail length, Jak3, Stat5b, and Il12a expression might be modulated by m6A modification. Our study demonstrated that Prmt5 regulates B‐cell function through different mechanisms and supported the development of Prmt5‐targeted antitumor treatments. [ABSTRACT FROM AUTHOR]

The mRNA polyadenylation plays essential function in regulation of mRNA metabolism. Mis-regulations of mRNA polyadenylation are frequently linked with aberrant gene expression and disease progression. Under the action of polyadenylate polymerase, poly(A) tail is synthesized after the polyadenylation signal (PAS) sites on the mRNAs. Alternative polyadenylation (APA) often occurs in mRNAs with multiple poly(A) sites, producing different 3′ ends for transcript variants, and therefore plays important functions in gene expression regulation. In this review, we first summarize the classical process of mRNA 3′-terminal formation and discuss the length control mechanisms of poly(A) in nucleus and cytoplasm. Then we review the research progress on alternative polyadenylation regulation and the APA site selection mechanism. Finally, we summarize the functional roles of APA in the regulation of gene expression and diseases including cancers.

To be properly expressed, genes need to be accompanied by a terminator, a region downstream of the coding sequence that contains the information necessary for the maturation of the mRNA 3ʹ end. The main event in this process is the addition of a poly(A) tail at the 3ʹ end of the new transcript, a critical step in mRNA biology that has important consequences for the expression of genes. Here, we review the mechanism leading to cleavage and polyadenylation of newly transcribed mRNAs and how this process can affect the final levels of gene expression. We give special attention to an aspect often overlooked, the effect that different terminators can have on the expression of genes. We also discuss some exciting findings connecting the choice of terminator to the biogenesis of small RNAs, which are a central part of one of the most important mechanisms of regulation of gene expression in plants. [ABSTRACT FROM AUTHOR]

Next, the gene number in each of the 32 chromosomes was then extracted using the genome annotation file, and PAS in different gene positions were also examined. We compared the CS position with the gene location on the chromosome, and the gene containing two or more cleavage sites means the gene was potentially regulated by alternative polyadenylation. (A) The gene number, PAS site number, and ratio of PAS site to the gene on a particular chromosome. We selected four genes (gene number: LOC118274515, LOC118275636, LOC118264954, LOC118275420) to explore the regulation of variable tailing on genes. [Extracted from the article]

Poly(A)‐binding protein (PABP) is an essential element of cellular translational machinery. Recent studies have revealed that poly(A) tail modifications can modulate mRNA stability and translational potential, and that oligoadenylate‐derived PABP ligands can act as effective translational inhibitors with potential applications in pain management. Although extensive research has focused on protein‐RNA and protein‐protein interactions involving PABPs, further studies are required to examine the ligand specificity of PABP. In this study, we developed a microscale thermophoresis‐based assay to probe the interactions between PABP and oligoadenylate analogs containing different chemical modifications. Using this method, we evaluated oligoadenylate analogs modified with nucleobase, ribose, and phosphate moieties to identify modification hotspots. In addition, we determined the susceptibility of the modified oligos to CNOT7 to identify those with the potential for increased cellular stability. Consequently, we selected two enzymatically stable oligoadenylate analogs that inhibit translation in rabbit reticulocyte lysates with a higher potency than a previously reported PABP ligand. We believe that the results presented in this study and the implemented methodology can be capitalized upon in the future development of RNA‐based biological tools. [ABSTRACT FROM AUTHOR]

Abstract Background The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3′ to 5′ direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3′-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. Results To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17–19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5′ parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3′-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5′-ends of RNAs was detected. Conclusions In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5′-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3′-5′ direction.

Abstract Background Cells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and proteins. A common source of aberrant mRNAs is premature polyadenylation, which can result in non-functional protein products. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide via ribosome-associated quality control. Results Here, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at poly(A) sequences during ribosome-associated quality control. We show that MKRN1 directly binds to the cytoplasmic poly(A)-binding protein (PABPC1) and associates with polysomes. MKRN1 is positioned upstream of poly(A) tails in mRNAs in a PABPC1-dependent manner. Ubiquitin remnant profiling and in vitro ubiquitylation assays uncover PABPC1 and ribosomal protein RPS10 as direct ubiquitylation substrates of MKRN1. Conclusions We propose that MKRN1 mediates the recognition of poly(A) tails to prevent the production of erroneous proteins from prematurely polyadenylated transcripts, thereby maintaining proteome integrity.

Cis-acting RNA elements are crucial for the regulation of polyadenylated RNA stability. The element for nuclear expression (ENE) contains a U-rich internal loop flanked by short helices. An ENE stabilizes RNA by sequestering the poly(A) tail via formation of a triplex structure that inhibits a rapid deadenylation-dependent decay pathway. Structure-based bioinformatic studies identified numerous ENE-like elements in evolutionarily diverse genomes, including a subclass containing two ENE motifs separated by a short double-helical region (double ENEs [dENEs]). Here, the structure of a dENE derived from a rice transposable element (TWIFB1) before and after poly(A) binding (~24 kDa and ~33 kDa, respectively) is investigated. We combine biochemical structure probing, small angle X-ray scattering (SAXS), and cryo-electron microscopy (cryo-EM) to investigate the dENE structure and its local and global structural changes upon poly(A) binding. Our data reveal 1) the directionality of poly(A) binding to the dENE, and 2) that the dENEpoly(A) interaction involves a motif that protects the 3'-most seven adenylates of the poly(A). Furthermore, we demonstrate that the dENE does not undergo a dramatic global conformational change upon poly(A) binding. These findings are consistent with the recently solved crystal structure of a dENE+poly(A) complex [S.-F. Torabi et al., Science 371, eabe6523 (2021)]. Identification of additional modes of poly(A)-RNA interaction opens new venues for better understanding of poly(A) tail biology. [ABSTRACT FROM AUTHOR]

Summary: Multiple factors influence translation termination efficiency, including nonsense codon identity and immediate context. To determine whether the relative position of a nonsense codon within an open reading frame (ORF) influences termination efficiency, we quantitate the production of prematurely terminated and/or readthrough polypeptides from 26 nonsense alleles of 3 genes expressed in yeast. The accumulation of premature termination products and the extent of readthrough for the respective premature termination codons (PTCs) manifest a marked dependence on PTC proximity to the mRNA 3′ end. Premature termination products increase in relative abundance, whereas readthrough efficiencies decrease progressively across different ORFs, and readthrough efficiencies for a PTC increase in response to 3′ UTR lengthening. These effects are eliminated and overall translation termination efficiency decreases considerably in cells harboring pab1 mutations. Our results support a critical role for poly(A)-binding protein in the regulation of translation termination and also suggest that inefficient termination is a trigger for nonsense-mediated mRNA decay (NMD).

La-related protein 4 (LARP4) directly binds both poly(A) and poly(A)-binding protein (PABP). LARP4 was shown to promote poly(A) tail (PAT) lengthening and stabilization of individual mRNAs presumably by protection from deadenylation (Mattijssen et al., 2017). We developed a nucleotide resolution transcriptome-wide, single molecule SM-PAT-seq method. This revealed LARP4 effects on a wide range of PAT lengths for human mRNAs and mouse mRNAs from LARP4 knockout (KO) and control cells. LARP4 effects are clear on long PAT mRNAs but become more prominent at 30–75 nucleotides. We also analyzed time courses of PAT decay transcriptome-wide and for ~200 immune response mRNAs. This demonstrated accelerated deadenylation in KO cells on PATs < 75 nucleotides and phasing consistent with greater PABP dissociation in the absence of LARP4. Thus, LARP4 shapes PAT profiles throughout mRNA lifespan with impact on mRNA decay at short lengths known to sensitize PABP dissociation in response to deadenylation machinery.

La-Related Protein 1 (LARP1) is an RNA-binding protein that regulates the stability and translation of mRNAs encoding the translation machinery, including ribosomal proteins and translation factors. These mRNAs are characterized by a 5ʹ-terminal oligopyrimidine (TOP) motif that coordinates their temporal and stoichiometric expression. While LARP1 represses TOP mRNA translation via the C-terminal DM15 region, the role of the N-terminal La-Module in the recognition and translational regulation of TOP mRNAs remains elusive. Herein we show that the LARP1 La-Module also binds TOP motifs, although in a cap-independent manner. We also demonstrate that it recognizes poly(A) RNA. Further, our data reveal that the LARP1 La-Module can simultaneously engage TOP motifs and poly(A) RNA. These results evoke an intriguing molecular mechanism whereby LARP1 could regulate translation and stabilization of TOP transcripts. [ABSTRACT FROM AUTHOR]

RNA polyadenylation is an important step in the messenger RNA (mRNA) maturation process, and the first step is recognizing the polyadenylation signal (PAS). The PAS type and distribution is a key determinant of post-transcriptional mRNA modification and gene expression. However, little is known about PAS usage and alternative polyadenylation (APA) regulation in livestock species. Recently, sequencing technology has enabled the generation of a large amount of sequencing data revealing variation in poly(A) signals and APA regulation in Sus scrofa. We identified 62,491 polyadenylation signals in Sus scrofa using expressed sequence tag (EST) sequences combined with RNA-seq analysis. The composition and usage frequency of polyadenylation signal in Sus scrofa is similar with that of human and mouse. The most highly conserved polyadenylation signals are AAUAAA and AUUAAA, used for over 63.35% of genes. In addition, we also analyzed the U/GU-rich downstream sequence (DSE) element, located downstream of the cleavage site. Our results indicate that APA regulation was widely occurred in Sus scrofa, as in other organisms. Our result was useful for the accurate annotation of RNA 3′ ends in Sus scrofa and the analysis of polyadenylation signal usage in Sus scrofa would give the new insights into the mechanisms of transcriptional regulation.

Generating mRNA in vitro to encode therapeutic or cell-modifying proteins is rapidly gaining favor. An important factor that determines efficiency of translation from in vitro transcribed mRNA is the length of the 3′ poly(A) sequence. However, reproducibly generating and maintaining templates from circular plasmids to have consistent lengths of the homo poly(A) sequences is challenging. The procedure reported here entails repeated restriction digestion with type IIS enzymes, ligation and circular plasmid propagation. The homopolymeric sequence of approximately 100 bp that is generated using the method is approximately equal to the number of 3′ A residues found in the mRNA of mammalian cells. Evaluating expression in vivo of a reporter transcript produced using this method showed efficient expression in vivo.

Short Interspersed Elements (SINEs) are mobile genetic elements of higher eukaryotes, which originated during evolution from various tRNAs and less often from 5S rRNA and 7SL RNA. Similar to the genes of these RNAs, SINEs are transcribed by RNA polymerase III. The transcripts of some mammalian SINEs have an ability to undergo AAUAAA-dependent polyadenylation, which is unique for the RNA polymerase III transcripts. It is well known that this polyadenylation of many RNA polymerase II transcripts (e.g., mRNAs) increases their lifetime in the cell. The aim of this work is to examine whether the stability of SINE transcripts increases as a result of AAUAAA-dependent polyadenylation. HeLa cells were transfected with SINE DNA, both containing and not containing the polyadenylation signal (AATAAA). One day later, the transcription was inhibited by actinomycin D, and the decrease in the level of the SINE transcripts was monitored by northern hybridization. For all the eight studied SINEs, the half-life of nonpolyadenylated transcripts was 20–30 minutes, and for polyadenylated transcripts, this parameter exceeded 3 hours. Interestingly, the insertion of an additional 80-bp DNA fragment into the middle region of B2 SINE did not significantly reduce the stability of the polyadenylated transcripts. It is most likely that the increase in the lifetime of the polyadenylated SINE transcripts is due to the fact that the poly(A) tail interacts with the poly(A)-binding proteins (PABPs), thus protecting the RNA from degradation by the exonucleases acting from the 3'-end. The results make it possible to design SINE-based vectors intended for the expression of short noncoding RNAs, which are stable in a cell due to polyadenylation. [ABSTRACT FROM AUTHOR]

The polyadenylic acid [poly(A)] tail of mRNA plays a noteworthy role in the initiation of the translation, maturation, and stability of mRNA. It also significantly contributes to the production of alternate proteins in eukaryotic cells. Hence, it has recently been recognized as a prospective drug target. Binding affinity of bis(N-p-tolylbenzohydroxamato)Cobalt(II), [N-p-TBHA-Co(II)] (1) and bis(N-p-naphthylbenzohydroxamato)Copper(II), [N-p-NBHA-Cu(II)] (2) complexes with poly(A) have been investigated by biophysical techniques namely, absorption spectroscopy, fluorescence spectroscopy, diffuse reflectance infrared Fourier transform spectroscopy, circular dichroism spectroscopy, viscometric measurements and through molecular docking studies. The intrinsic binding constants (Kb) of complexes were determined following the order of N-p-TBHA-Co(II)] > N-p-NBHA-Cu(II), along with hyperchromism and a bathochromic shift for both complexes. The fluorescence quenching method revealed an interaction between poly(A)-N-p-TBHA-Co(II)/poly(A)-N-p-NBHA-Cu(II). The mode of binding was also determined via the fluorescence ferrocyanide quenching method. The increase in the viscosity of poly(A) that occurred from increasing the concentration of the N-p-TBHA-Co(II)/N-p-NBHA-Cu(II) complex was scrutinized. The characteristics of the interaction site of poly(A) with N-p-TBHA-Co(II)/N-p-NBHA-Cu(II) were adenine and phosphate groups, as revealed by DRS-FTIR spectroscopy. Based on these observations, a partial intercalative mode of the binding of poly(A) has been proposed for both complexes. Circular dichroism confirmed the interaction of both the complexes with poly(A). The molecular docking results illustrated that complexes strongly interact with poly(A) via the relative binding energies of the docked structure as −259.39eV and −226.30eV for N-p-TBHA-Co(II) and N-p-NBHA-Cu(II) respectively. Moreover, the binding affinity of N-p-TBHA-Co(II) is higher in all aspects than N-p-NBHA-Cu(II) for poly(A). [ABSTRACT FROM AUTHOR]

Removal of the poly(A) tail, or deadenylation, is a crucial step in destabilizing mRNAs in eukaryotes. In this chapter, we describe a cell-free deadenylation assay that uses cytoplasmic cell extracts from human HEK293 cells transiently transfected with DNA encoding RNA-binding proteins (RBP), and in vitro-transcribed, radiolabeled, RNA probes. We include methods to evaluate the effects of RBPs or deadenylases on various in vitro-transcribed probes, with or without poly(A) tails. Finally, we also demonstrate the adaptability of these assays to test purified protein components in our cell-free deadenylation assay. In our experience, these methods are well suited for the initial assessment of the effects of RBPs on the deadenylation of mRNAs., (© 2024. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

The poly-adenosine, or poly(A) tail, plays key roles in controlling the stability and translation of messenger RNAs in all eukaryotes, and, as such, facile assays that can measure poly(A) length are needed. This chapter describes an approach that couples RNase H-mediated cleavage of an RNA of interest with high-resolution denaturing gel electrophoresis and northern blot-based detection. Major advantages of this method include the ability to directly measure the abundance of any RNA and the length of its poly(A) tail without amplification steps. The assay provides high specificity, sensitivity, and reproducibility for accurate quantitation using standard molecular biology equipment and reagents. Overall, the high-resolution northern blotting approach offers a cost-effective means of poly(A) RNA analysis that is especially useful for small numbers of transcripts and comparisons between experimental conditions or time points., (© 2024. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

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MicroRNAs are small noncoding RNAs that regulate translation and mRNA stability by binding target mRNAs in complex with Argonaute (AGO) proteins. AGO interacts with a member of the TNRC6 family proteins to form a microRNP complex, which recruits the CCR4‐NOT complex to accelerate deadenylation and inhibits translation. MicroRNAs primarily repress translation of target mRNAs but have been shown to enhance translation of a specific type of target reporter mRNAs in various experimental systems: G0 quiescent mammalian cells, Xenopus laevis oocytes, Drosophila embryo extracts, and HeLa cells. In all of the cases mentioned, a common feature of the activated target mRNAs is the lack of a poly(A) tail. Here, we show let‐7‐microRNP‐mediated translational activation of nonadenylated target mRNAs in a mammalian cell‐free system, which contains over‐expressed AGO2, TNRC6B, and PAPD7 (TUTase5, TRF4‐1). Importantly, translation of nonadenylated mRNAs was activated also by tethered TNRC6B silencing domain (SD), in the presence of PAPD7. Deletion of the poly(A)‐binding protein (PABP) interacting motif (PAM2) from the TNRC6B‐SD abolished the translational activation, suggesting the involvement of PABP in the process. Similar results were also obtained in cultured HEK293T cells. This work may provide novel insights into microRNP‐mediated mRNA regulation. [ABSTRACT FROM AUTHOR]

Cleavage of mRNA molecules causes their rapid degradation, thereby playing an important role in regulation of gene expression and host genome defense from viruses and transposons in bacterial and eukaryotic cells. Current negative-readout, and repressor-based positive-readout reporters of mRNA degradation have limitations. Here we report the development of a single transcript that acts as a positive reporter of mRNA cleavage. We show that placement of bacterial CopT and CopA hairpins into the 5′ UTR and 3′ UTR of an mRNA results in inhibition of translation of the intervening coding sequence in Drosophila. An internal poly(A) tract inserted downstream of the coding sequence stabilizes transcripts cut within the 3′ UTR. When these components are combined in a transcript in which targets sites for RNA cleavage are placed between the poly(A) tract and CopA, cleavage results in translational activation, providing a single transcript-based method of sensing mRNA cleavage with a positive readout.

Molecules capable of inducing self-structure in poly(A) may arrest protein synthesis by preventing poly(A) chain elongation thereby inhibiting the function of mRNA. We examined whether new methylene blue can induce such structural organization in single stranded poly(A) and compared the results with those of the phenothiazinium dyes thionine and toluidine blue O. Strong association between the dyes and poly(A) was revealed from the spectroscopy and cyclic voltammetry. Thionine has a higher affinity compared to toluidine blue O and new methylene blue at 50 mM and 100 mM, but at 200 mM [Na + ] new methylene blue exhibited the highest binding affinity. Negative enthalpy and positive entropy changes, and enthalpy-entropy compensation behaviour were characteristic features for the complexation. Involvement of hydrophobic forces in the binding process is correlated to the negative heat capacity changes. Differential scanning calorimetry and circular dichroism results revealed that the dyes bind poly(A) and induce a stable secondary structure with a melting temperature of 333.15 K. The binding constants increased in the range of 50–200 mM [Na + ]. Molecular docking results revealed that not only van der Waals forces of attraction but hydrogen bonding also plays a crucial role in dye-poly(A) binding. Recognition and binding of small molecules to single stranded poly(A) may present a new avenue for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Thermodynamics of the induction of self-structure in polyadenylic acid by the cytotoxic isoquinoline alkaloid coralyne was investigated using calorimetry tools. The binding was an exothermic process driven by large negative enthalpy change. The equilibrium constant at [K + ] = (130 ± 0.01) mM and T = (298.15 ± 0.01) K was calculated to be (1.34 ± 0.07)·10 6 M −1 . Temperature dependent calorimetric studies suggested weakening of the self-structure at higher temperatures. The self-structure induction process was characterized by complete enthalpy-entropy compensation. The temperature dependence of the enthalpy change yielded negative heat capacity value. The equilibrium constant decreased with increasing [K + ] apparently due to the weakening of the self-structure at higher salt concentrations. Differential scanning calorimetry studies testified for the formation of self-structure in (50 ± 0.01)–(130 ± 0.01) mM [K + ]. Parsing of the standard molar Gibbs energy change revealed that the non-polyelectrolytic forces dominated the self-structure induction process. [ABSTRACT FROM AUTHOR]

The energetics of self-structure induction in polyadenylic acid in the presence of proflavine has been studied using isothermal titration calorimetry. The self-structure induction process was exothermic and driven by large positive entropy change. The equilibrium constant at T = (298.15 ± 0.01) K and [Na + ] = (130 ± 0.01) mM was calculated to be (1.01 ± 0.08) · 10 6 M −1 . Salt dependent calorimetric studies revealed that the equilibrium constant increased with increasing [Na + ] in the 50–130 mM range suggesting enhanced binding preference at higher salt concentrations, apparently due to easier self-structure formation at higher [Na + ]. Dissection of the standard molar Gibbs energy change clearly established that the self-structure induction was driven by non-polyelectrolytic forces and the polyelectrolytic contribution was relatively small. The equilibrium constant decreased with increasing temperature indicating destabilization of the self-structure at higher temperatures. Negative standard molar heat capacity value obtained from the temperature dependence of the enthalpy change suggested that hydrophobic forces are important for the self-structure induction. Furthermore, a complete enthalpy–entropy compensation phenomenon was also observed. [ABSTRACT FROM AUTHOR]

Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation.

The CCR4-NOT complex is a highly conserved, multifunctional machinery with a general role in controlling mRNA metabolism. It has been implicated in a number of different aspects of mRNA and protein expression, including mRNA degradation, transcription initiation and elongation, ubiquitination, and protein modification. The core CCR4-NOT complex is evolutionarily conserved and consists of at least three NOT proteins and two catalytic subunits. The L-shaped complex is characterized by two functional modules bound to the CNOT1/Not1 scaffold protein: the deadenylase or nuclease module containing two enzymes required for deadenylation, and the NOT module. In this review, we will summarize the currently available information regarding the three-dimensional structure and assembly of the CCR4-NOT complex, in order to provide insight into its roles in mRNA degradation and other biological processes.

In eukaryotic cells, the shortening and removal of the poly(A) tail (deadenylation) of cytoplasmic mRNA is a key event in regulated mRNA degradation. A major enzyme involved in deadenylation is the Ccr4-Not deadenylase complex, which can be recruited to its target mRNA by RNA-binding proteins or the miRNA repression complex. In addition to six non-catalytic components, the complex contains two enzymatic subunits with ribonuclease activity: Ccr4 and Caf1 (Pop2). In vertebrates, each deadenylase subunit is encoded by two paralogues: Caf1, which can interact with the anti-proliferative protein BTG2, is encoded by CNOT7 and CNOT8, whereas Ccr4 is encoded by the highly similar genes CNOT6 and CNOT6L. Currently, it is unclear whether the catalytic subunits work co-operatively or whether the nuclease components have unique roles in deadenylation. We therefore developed a method to express and purify a minimal human BTG2-Caf1-Ccr4 nuclease sub-complex from bacterial cells. By using chemical inhibition and well-characterized inactivating amino acid substitutions, we demonstrate that the enzyme activities of Caf1 and Ccr4 are both required for deadenylation in vitro. These results indicate that Caf1 and Ccr4 cooperate in mRNA deadenylation and suggest that the enzyme activities of Caf1 and Ccr4 are regulated via allosteric interactions within the nuclease module. [ABSTRACT FROM AUTHOR]

The shortening of the poly(A) tail of cytoplasmic mRNA (deadenylation) is a pivotal step in the regulation of gene expression in eukaryotic cells. Deadenylation impacts on both regulated mRNA decay as well as the rate of mRNA translation. An important enzyme complex involved in poly(A) shortening is the Ccr4-Not deadenylase. In addition to at least six non-catalytic subunits, it contains two distinct subunits with ribonuclease activity: a Caf1 subunit, characterised by a DEDD (Asp-Glu-Asp-Asp) domain, and a Ccr4 component containing an Endonuclease-Exonuclease-Phosphatase (EEP) domain. In vertebrate cells, the complexity of the complex is further increased by the presence of paralogues of the Caf1 subunit (encoded by either CNOT7 or CNOT8) and the occurrence of two Ccr4 paralogues (encoded by CNOT6 or CNOT6L). In plants, there are also multiple Caf1 and Ccr4 paralogues. Thus, the composition of the Ccr4-Not complex is heterogeneous. The potential differences in the intrinsic enzymatic activities of the paralogues will be discussed. In addition, the potential redundancy, cooperation and/or the extent of unique roles for the deadenylase subunits of the Ccr4-Not complex will be reviewed. Finally, novel approaches to study the catalytic roles of the Caf1 and Ccr4 subunits will be discussed.

Tandem repeats of short DNA sequences are commonly found in human DNA. These simple sequence repeats or microsatellites are highly polymorphic in the human genome. Since the anti-tumour agent cisplatin preferentially forms DNA adducts at runs of consecutive guanine nucleotides (poly(G)), the position and frequency of occurrence of poly(G) sequences in the updated human genome was investigated. There are more runs of consecutive guanines than would be expected by random chance. This especially true for poly(G) sequences longer than approximately n = 9. A plot of poly(G) length against log(observed/expected) frequency produced a straight line for n > 9. A similar observation was also found for poly(A) DNA sequence repeats. This data implied that the increase in observed/expected frequency is directly related to length of DNA repeat. It was proposed that long runs of consecutive guanine nucleotides could be a sensitive sensor of cellular DNA damage since a number of DNA damaging agents cause lesions at poly(G) sequences. [ABSTRACT FROM AUTHOR]

Recently, in addition to poly(A)+ long non-coding RNAs (lnc RNAs), many lnc RNAs without poly(A) tails, have been characterized in mammals. However, the non-polyA lnc RNAs and their conserved motifs, especially those associated with environmental stresses, have not been fully investigated in plant genomes. We performed poly(A)− RNA-seq for seedlings of Arabidopsis thaliana under four stress conditions, and predicted lnc RNA transcripts. We classified the lnc RNAs into three confidence levels according to their expression patterns, epigenetic signatures and RNA secondary structures. Then, we further classified the lnc RNAs to poly(A)+ and poly(A)− transcripts. Compared with poly(A)+ lncRNAs and coding genes, we found that poly(A)− lncRNAs tend to have shorter transcripts and lower expression levels, and they show significant expression specificity in response to stresses. In addition, their differential expression is significantly enriched in drought condition and depleted in heat condition. Overall, we identified 245 poly(A)+ and 58 poly(A)− lnc RNAs that are differentially expressed under various stress stimuli. The differential expression was validated by q RT- PCR, and the signaling pathways involved were supported by specific binding of transcription factors ( TFs), phytochrome-interacting factor 4 ( PIF4) and PIF5. Moreover, we found many conserved sequence and structural motifs of lnc RNAs from different functional groups (e.g. a UUC motif responding to salt and a AU-rich stem-loop responding to cold), indicated that the conserved elements might be responsible for the stress-responsive functions of lnc RNAs. [ABSTRACT FROM AUTHOR]

Highlights: [•] JGB, IB, SO and PSF bind to double stranded poly(A). [•] The binding followed the order JGB>IB>SO>PSF. [•] Binding was entropy dominated with a small enthalpy contribution to the Gibbs energy. [•] Conformational perturbation of ds poly(A) occurred on dye binding. [Copyright &y& Elsevier]

LINE-1 (L1) elements are the only active and autonomous transposable elements in humans. The core retrotransposition machinery is a ribonucleoprotein particle (RNP) containing the L1 mRNA, with endonuclease and reverse transcriptase activities. It initiates reverse transcription directly at genomic target sites upon endonuclease cleavage. Recently, using a direct L1 extension assay (DLEA), we systematically tested the ability of native L1 RNPs to extend DNA substrates of various sequences and structures. We deduced from these experiments the general rules guiding the initiation of L1 reverse transcription, referred to as the snap-velcro model. In this model, L1 target choice is not only mediated by the sequence specificity of the endonuclease, but also through basepairing between the L1 mRNA and the target site, which permits the subsequent L1 reverse transcription step. In addition, L1 reverse transcriptase efficiently primes L1 DNA synthesis only when the 3' end of the DNA substrate is single- stranded, suggesting so-far unrecognized DNA processing steps at the integration site. [ABSTRACT FROM AUTHOR]

[Display omitted] • BTG2, which is frequently mutated in non-Hodgkin lymphoma, can stimulate deadenylation of poly(A)-PABPC1 by the Caf1/CNOT7 subunit of the Ccr4-Not complex. • To understand the interaction between BTG2 and PABPC1 in detail, interfacing residues of PABPC1 and BTG2 were mapped using a mutagenesis approach. • PABPC1 variant R49E has reduced BTG2 binding, but can interact with poly(A). • A structural model of PABPC1-BTG2 was generated using NMR-guided molecular docking followed by validation using mutagenesis data. • A model of the quaternary poly(A)-PABPC1-BTG2-Caf1/CNOT7 complex provides structural insight into enhanced deadenylation by Caf1/CNOT7 of poly(A)-PABPC1 in the presence of BTG2. Degradation of cytoplasmic mRNA in eukaryotes involves the shortening and removal of the mRNA poly(A) tail by poly(A)-selective ribonuclease (deadenylase) enzymes. In human cells, BTG2 can stimulate deadenylation of poly(A) bound by cytoplasmic poly(A)-binding protein PABPC1. This involves the concurrent binding by BTG2 of PABPC1 and the Caf1/CNOT7 nuclease subunit of the Ccr4-Not deadenylase complex. To understand in molecular detail how PABPC1 and BTG2 interact, we set out to identify amino acid residues of PABPC1 and BTG2 contributing to the interaction. To this end, we first used algorithms to predict PABPC1 interaction surfaces. Comparison of the predicted interaction surface with known residues involved in the binding to poly(A) resulted in the identification of a putative interaction surface for BTG2. Subsequently, we used pulldown assays to confirm the requirement of PABPC1 residues for the interaction with BTG2. Analysis of RNA-binding by PABPC1 variants indicated that PABPC1 residues required for interaction with BTG2 do not interfere with poly(A) binding. After further defining residues of BTG2 that are required for the interaction with PABPC1, we used information from published NMR chemical shift perturbation experiments to guide docking and generate a structural model of the BTG2-PABPC1 complex. A quaternary poly(A)-PABPC1-BTG2-Caf1/CNOT7 model showed that the 3′ end of poly(A) RNA is directed towards the catalytic centre of Caf1/CNOT7, thereby providing a rationale for enhanced deadenylation by Caf1/CNOT7 in the presence of BTG2 and PABPC1. [ABSTRACT FROM AUTHOR]

Highlights: [•] It was a dual-readout assay for PaH. [•] PaH caused the aggregation of poly(A)-stabilized AuNPs for the strong affinity between poly(A) and PaH. [•] Poly(A) and gold nanoparticles were exploited to detect PaH with great sensitivity. [•] This RLS method was simple and fast without complicated labeling and separation. [•] PaH was detected with naked eyes for the first time. [ABSTRACT FROM AUTHOR]

The human genome is laden with both non-LTR (longterminal repeat) retrotransposons and microsatellite repeats. Both types of sequences are able to, either actively or passively, mutagenize the genomes of human individuals and are therefore poised to dynamically alter the human genomic landscape across generations. Non-LTR retrotransposons, such as L1 and Alu, are a major source of new microsatellites, which are born both concurrently and subsequently to L1 and Alu integration into the genome. Likewise, the mutation dynamics of microsatellite repeats have a direct impact on the fitness of their non-LTR retrotransposon parent owing to microsatellite expansion and contraction. This review explores the interactions and dynamics between non-LTR retrotransposons and microsatellites in the context of genomic variation and evolution. [ABSTRACT FROM AUTHOR]

The poly(A) polymorphism (L/S) in the VDR gene has been implicated in susceptibility of cancer, but a number of studies have reported inconclusive results. The aim of this study is to investigate the relationship between the poly(A) polymorphism in the VDR gene and cancer risk by meta-analysis. We searched PubMed database, EMBASE database, CNKI database, and Wanfang database, covering all studies until January 22, 2013. Statistical analysis was performed by using the software Revman4.2 and STATA 10.0. A total 8,186 cancer cases and 8,685 controls in 19 case-control studies from 15 studies were identified for data analysis. The results suggested that the S allele carriers (SS + SL) did not have an increased or decreased risk of cancer when compared with the homozygote LL carriers (odds ratio (OR) = 0.96, 95 % CI = 0.87-1.06, P = 0.43 for SS + SL vs. LL). In addition, in the subgroup analysis by ethnicity and cancer type, no significant association was found among Caucasians, African-Americans, prostate cancer, or breast cancer. This current meta-analysis suggested that the poly(A) polymorphism in the VDR gene may not contribute to the risk of cancer. Future studies are needed to validate our findings. [ABSTRACT FROM AUTHOR]

Abstract: We studied the substrate specificity of the exosome of Sulfolobus solfataricus using the catalytically active Rrp41–Rrp42-hexamer and complexes containing the RNA-binding subunits Rrp4 or Csl4. The conservation of both Rrp4 and Csl4 in archaeal and eukaryotic exosomes suggests specific functions for each of them. We found that they confer different specificities to the exosome: RNA with an A-poor 3′-end is degraded with higher efficiency by the Csl4-exosome, while the Rrp4-exosome strongly prefers poly(A)-RNA. High C-content and polyuridylation negatively influence RNA processing by all complexes, and, in contrast to the hexamer, the Rrp4-exosome prefers longer substrates. [Copyright &y& Elsevier]