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Understanding transcriptomes requires documenting the structures, modifications, and abundances of RNAs as well as their proximity to other molecules. The methods that make this possible depend critically on enzymes (including mutant derivatives) that act on nucleic acids for capturing and sequencing RNA. We tested two 3' nucleotidyl transferases, Saccharomyces cerevisiae poly(A) polymerase and Schizosaccharomyces pombe Cid1, for the ability to add base and sugar modified rNTPs to free RNA 3' ends, eventually focusing on Cid1. Although unable to polymerize ΨTP or 1meΨTP, Cid1 can use 5meUTP and 4thioUTP. Surprisingly, Cid1 can use inosine triphosphate to add poly(I) to the 3' ends of a wide variety of RNA molecules. Most poly(A) mRNAs efficiently acquire a uniform tract of about 50 inosine residues from Cid1, whereas non-poly(A) RNAs acquire longer, more heterogeneous tails. Here we test these activities for use in direct RNA sequencing on nanopores, and find that Cid1-mediated poly(I)-tailing permits detection and quantification of both mRNAs and non-poly(A) RNAs simultaneously, as well as enabling the analysis of nascent RNAs associated with RNA polymerase II. Poly(I) produces a different current trace than poly(A), enabling recognition of native RNA 3' end sequence lost by in vitro poly(A) addition. Addition of poly(I) by Cid1 offers a broadly useful alternative to poly(A) capture for direct RNA sequencing on nanopores.

In Trypanosoma brucei, mitochondrial pre-mRNAs undergo 3'-5' exonucleolytic processing, 3' adenylation and uridylation, 5' pyrophosphate removal, and, often, U-insertion/deletion editing. The 3' modifications are modulated by pentatricopeptide repeat (PPR) Kinetoplast Polyadenylation Factors (KPAFs). We have shown that KPAF3 binding to the 3' region stabilizes properly trimmed transcripts and stimulates their A-tailing by KPAP1 poly(A) polymerase. Conversely, poly(A) binding KPAF4 shields the nascent A-tail from uridylation and decay thereby protecting pre-mRNA upon KPAF3 displacement by editing. While editing concludes in the 5' region, KPAF1/2 dimer induces A/U-tailing to activate translation. Remarkably, 5' end recognition and pyrophosphate hydrolysis by the PPsome complex also contribute to mRNA stabilization. Here, we demonstrate that KPAF4 functions as a heterodimer with KPAF5, a protein lacking discernable motifs. We show that KPAF5 stabilizes KPAF4 to enable poly(A) tail recognition, which likely leads to mRNA stabilization during the editing process and impedes spontaneous translational activation of partially-edited transcripts. Thus, KPAF4/5 represents a poly(A) binding element of the mitochondrial polyadenylation complex. We present evidence that RNA editing substrate binding complex bridges the 5' end-bound PPsome and 3' end-bound polyadenylation complexes. This interaction may enable mRNA circularization, an apparently critical element of mitochondrial mRNA stability and quality control.

Obtaining neuron transcriptomes is challenging; their complex morphology and interconnected microenvironments make it difficult to isolate neurons without potentially altering gene expression. Multidendritic sensory neurons (md neurons) of Drosophila larvae are commonly used to study peripheral nervous system biology, particularly dendrite arborization. We sought to test if EC-tagging, a biosynthetic RNA tagging and purification method that avoids the caveats of physical isolation, would enable discovery of novel regulators of md neuron dendrite arborization. Our aims were twofold: discover novel md neuron transcripts and test the sensitivity of EC-tagging. RNAs were biosynthetically tagged by expressing CD:UPRT (a nucleobase-converting fusion enzyme) in md neurons and feeding 5-ethynylcytosine (EC) to larvae. Only CD:UPRT-expressing cells are competent to convert EC into 5-ethynyluridine-monophosphate which is subsequently incorporated into nascent RNA transcripts. Tagged RNAs were purified and used for RNA-sequencing. Reference RNA was prepared in a similar manner using 5-ethynyluridine (EUd) to tag RNA in all cells and negative control RNA-seq was performed on "mock tagged" samples to identify non-specifically purified transcripts. Differential expression analysis identified md neuron enriched and depleted transcripts. Three candidate genes encoding RNA-binding proteins (RBPs) were tested for a role in md neuron dendrite arborization. Loss-of-function for the m6A-binding factor Ythdc1 did not cause any dendrite arborization defects while RNAi of the other two candidates, the poly(A) polymerase Hiiragi and the translation regulator Hephaestus, caused significant defects in dendrite arborization. This work provides an expanded view of transcription in md neurons and a technical framework for combining EC-tagging with RNA-seq to profile transcription in cells that may not be amenable to physical isolation.

Objective To investigate the clinical symptoms and molecular biological characteristics in a family with oculopharyngeal muscular dystrophy (OPMD). Methods Clinical data of the family members were collected. Genomic DNA were isolated from peripheral blood of 6 family members including the probands for polymerase chain reaction (PCR). TA cloning followed by Sanger DNA sequencing was conducted for detecting PABPN1 gene mutations. Results The first symptom of the 4 family members in OPMD pedigree was ptosis at 50⁃years⁃old. The clinical symptoms of pharyngeal muscles involved in gradually dysphagia. Genetic testing found: the proband (Ⅱ3), the second sister of the proband (Ⅱ2), the fourth brother of the proband (Ⅱ4), and the fifth sister of the proband (Ⅱ5) had abnormal amplification of (GCN) repeat, (GCN)10/(GCN)13 in exon 1 of PABPN1 gene, which was identified in 4 members of the family who conducted the genetic testing. There was a dominant inheritance pattern in the lineage. The final diagnosis was OPMD, and the family was diagnosed as OPMD. Conclusions Clinical symptoms play a decisive role in the detection of the disease, thus genetic testing is the ctritical factor to the diagnosis and prevention of oculopharyngeal muscular dystrophy, which provides necessary information for choosing treatments and genetic counseling.

Gld2, a noncanonical cytoplasmic poly(A) polymerase, interacts with the RNA binding protein CPEB1 to mediate polyadenylation-induced translation in dendrites of cultured hippocampal neurons. Depletion of Gld2 from the hippocampus leads to a deficit in long-term potentiation evoked by theta burst stimulation. At least in mouse liver and human primary fibroblasts, Gld2 also 3' monoadenylates and thereby stabilizes specific miRNAs, which enhance mRNA translational silencing and eventual destruction. These results suggest that Gld2 would be likely to monoadenylate and stabilize miRNAs in the hippocampus, which would produce measurable changes in animal behavior. We now report that using Gld2 knockout mice, there are detectable alterations in specific miRNA monoadenylation in the hippocampus when compared to wild type, but that these modifications produce no detectable effect on miRNA stability. Moreover, we surprisingly find no overt change in animal behavior when comparing Gld2 knockout to wild-type mice. These data indicate that miRNA monoadenylation-mediated stability is cell type-specific and that monoadenylation has no measurable effect on higher cognitive function.

T cells maintain a quiescent state prior to activation. As inappropriate T cell activation can cause disease, T cell quiescence must be preserved. Despite its importance, the mechanisms underlying the “quiescent state” remain elusive. Here, we identify BTG1 and BTG2 (BTG1/2) as factors responsible for T cell quiescence. BTG1/2-deficient T cells show an increased proliferation and spontaneous activation due to a global increase in messenger RNA (mRNA) abundance, which reduces the threshold to activation. BTG1/2 deficiency leads to an increase in polyadenylate tail length, resulting in a greater mRNA half-life. Thus, BTG1/2 promote the deadenylation and degradation of mRNA to secure T cell quiescence. Our study reveals a key mechanism underlying T cell quiescence and suggests that low mRNA abundance is a crucial feature for maintaining quiescence. [ABSTRACT FROM AUTHOR]

microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants bear a methyl group on the ribose of the 3' terminal nucleotide. We showed previously that the methylation of miRNAs and siRNAs requires the protein HEN1 in vivo and that purified HEN1 protein methylates miRNA/miRNA* duplexes in vitro. In this study, we show that HEN1 methylates both miRNA/miRNA* and siRNA/siRNA* duplexes in vitro with a preference for 21-24 nt RNA duplexes with 2 nt overhangs. We also demonstrate that HEN1 deposits the methyl group on to the 2' OH of the 3' terminal nucleotide. Among various modifications that can occur on the ribose of the terminal nucleotide, such as 2'-deoxy, 3'-deoxy, 2'-O-methyl and 3'-O-methyl, only 2'-O-methyl on a small RNA inhibits the activity of yeast poly(A) polymerase (PAP). These findings indicate that HEN1 specifically methylates miRNAs and siRNAs and implicate the importance of the 2'-O-methyl group in the biology of RNA silencing.

Post-transcriptional addition of poly(A) tails to the 30 end of RNA is one of the fundamental events controlling the functionality and fate of RNA in all kingdoms of life. Although an enzyme with poly(A)-adding activity was discovered in Escherichia coli more than 50 years ago, its existence and role in prokaryotic RNA metabolism were neglected for many years. As a result, it was not until 1992 that E. coli poly(A) polymerase I was purified to homogeneity and its gene was finally identified. Further work revealed that, similar to its role in surveillance of aberrant nuclear RNAs of eukaryotes, the addition of poly(A) tails often destabilizes prokaryotic RNAs and their decay intermediates, thus facilitating RNA turnover. Moreover, numerous studies carried out over the last three decades have shown that polyadenylation greatly contributes to the control of prokaryotic gene expression by affecting the steady-state level of diverse protein-coding and non-coding transcripts including antisense RNAs involved in plasmid copy number control, expression of toxin-antitoxin systems and bacteriophage development. Here, we review the main findings related to the discovery of polyadenylation in prokaryotes, isolation, and characterization and regulation of bacterial poly(A)-adding activities, and discuss the impact of polyadenylation on prokaryotic mRNA metabolism and gene expression. [ABSTRACT FROM AUTHOR]

A polyA (pA) tail is an essential modification added to the 30 ends of a wide range of RNAs at different stages of their metabolism. Here, we describe the main sources of polyadenylation and outline their underlying biochemical interactions within the nuclei of budding yeast Saccharomyces cerevisiae, human cells and, when relevant, the fission yeast Schizosaccharomyces pombe. Polyadenylation mediated by the S. cerevisiae Trf4/5 enzymes, and their human homologues PAPD5/7, typically leads to the 30-end trimming or complete decay of non-coding RNAs. By contrast, the primary function of canonical pA polymerases (PAPs) is to produce stable and nuclear export-competent mRNAs. However, this dichotomy is becoming increasingly blurred, at least in S. pombe and human cells, where polyadenylation mediated by canonical PAPs may also result in transcript decay. [ABSTRACT FROM AUTHOR]

The role and function of long non-coding RNAs (lncRNAs) in modulating gene expression is becoming apparent. Vascular endothelial growth factor A (VEGF-A) is a key regulator of blood vessel formation and maintenance making it a promising therapeutic target for activation in ischemic diseases. In this study, we uncover a functional role for two antisense VEGF-A lncRNAs, RP1-261G23.7 and EST AV731492, in transcriptional regulation of VEGF-A during hypoxia. We find here that both lncRNAs are polyadenylated, concordantly upregulated with VEGF-A, localize to the VEGF-A promoter and upstream elements in a hypoxia dependent manner either as a single-stranded RNA or DNA bound RNA, and are associated with enhancer marks H3K27ac and H3K9ac. Collectively, these data suggest that VEGF-A antisense lncRNAs, RP1-261G23.7 and EST AV731492, function as VEGF-A promoter enhancer-like elements, possibly by acting as a local scaffolding for proteins and also small RNAs to tether. [ABSTRACT FROM AUTHOR]

Quantification of gene expression is a crucial research tool in the life sciences, which makes it important to identify any factors that could compromise its accuracy. One of these factors are non‐polyadenylated (poly(A)−) transcripts, including circular RNAs (circRNAs) that can skew quantification of gene expression as they resemble messenger RNAs (mRNAs). Here, we highlight the impact circRNAs and other poly(A)− transcripts have on gene expression profiling and the biological conclusions drawn from such experiments. We also propose easily adoptable strategies to increase the accuracy of gene expression quantification. [ABSTRACT FROM AUTHOR]

Star-PAP, a nuclear phosphatidylinositol (PI) signal-regulated poly(A) polymerase (PAP), couples with type I PI phosphate kinase α (PIPKIα) and controls gene expression. We show that Star-PAP and α together regulate 3'-end processing and expression of pre-mRNAs encoding key anti-invasive factors (KISS1R, CDH1, NME1, CDH13, FEZ1α and WIF1) in breast cancer. Consistently, the endogenous Star-PAP level is negatively correlated with the cellular invasiveness of breast cancer cells. While silencing Star-PAP or PIPKIα increases cellular invasiveness in low-invasiveness MCF7 cells, Star- PAP overexpression decreases invasiveness in highly invasive MDA-MB-231 cells in a cellular Star-PAP level-dependent manner. However, expression of the PIPKIα- noninteracting Star-PAP mutant or the phosphodeficient Star-PAP (S6A mutant) has no effect on cellular invasiveness. These results strongly indicate that α interaction and Star-PAP S6 phosphorylation are required for Star-PAP-mediated regulation of cancer cell invasion and give specificity to target anti-invasive gene expression. Our study establishes Star-PAP-α-mediated 3'-end processing as a key antiinvasive mechanism in breast cancer. [ABSTRACT FROM AUTHOR]

Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4-negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death-promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis [ABSTRACT FROM AUTHOR]

MiRNAs are endogenous noncoding RNA molecules. They play important gene-regulatory roles by binding to the mRNA of target genes thereby leading to either transcript degradation or translational repression. In virtually all diseases, distinct alterations of miRNA expression profiles have been found thus suggesting miRNAs as interesting biomarkers. Here, we present an SPR biosensor that utilizes disposable, injection-molded sensor chip/microfluidic hybrids combined with a lateral imaging optical system for parallel analysis of three one-dimensional spot arrays to detect miRNA-93. To increase the sensitivity of the biosensor we used two different amplification strategies. By adding an RNA-DNA-hybrid antibody for primary signal amplification, a limit of detection of 10 pmol/L was achieved. Based on that method we demonstrate the detection of miRNA-93 in total RNA lysate from HEK-293 cells. Utilizing an enzymatic signal amplification with Poly(A) polymerase, the sensitivity could be increased even further leading to a limit of detection of 1 fmol/L. [ABSTRACT FROM AUTHOR]

FAM46C is one of the most frequently mutated genes in multiple myeloma. Here, using a combination of in vitro and in vivo approaches, we demonstrate that FAM46C encodes an active non-canonical poly(A) polymerase which enhances mRNA stability and gene expression. Reintroduction of active FAM46C into multiple myeloma cell lines, but not its catalytically-inactive mutant, leads to broad polyadenylation and stabilization of mRNAs strongly enriched with those encoding endoplasmic reticulum-targeted proteins and induces cell death. Moreover, silencing of FAM46C in multiple myeloma cells expressing WT protein enhance cell proliferation. Finally, using a FAM46C-FLAG knock-in mouse strain, we show that the FAM46C protein is strongly induced during activation of primary splenocytes and that B lymphocytes isolated from newly generated FAM46C KO mice proliferate faster than those isolated from their WT littermates. Concluding, our data clearly indicate that FAM46C works as an onco-suppressor, with the specificity for B-lymphocyte lineage from which multiple myeloma originates. [ABSTRACT FROM AUTHOR]

Poly(A) tails are found at the 3′ end of almost every eukaryotic mRNA and are important for the stability of mRNAs and their translation into proteins. Thus, removal of the poly(A) tail, a process called deadenylation, is critical for regulation of gene expression. Most deadenylation enzymes are components of large multi-protein complexes. Here, we describe an in vitro deadenylation assay developed to study the exonucleolytic activities of the multi-protein Ccr4-Not and Pan2-Pan3 complexes. We discuss how this assay can be used with short synthetic RNAs, as well as longer RNA substrates generated using in vitro transcription. Importantly, quantitation of the reactions allows detailed analyses of deadenylation in the presence and absence of accessory factors, leading to new insights into targeted mRNA decay. [ABSTRACT FROM AUTHOR]

Genome-wide studies of aging have identified subsets of genes that show age-related changes in expression. Although the types of genes that are age regulated vary among different tissues and organisms, some patterns emerge from these large data sets. First, aging is associated with a broad induction of stress response pathways, although the specific genes and pathways involved differ depending on cell type and species. In contrast, a wide variety of functional classes of genes are downregulated with age, often including tissue-specific genes. Although the upregulation of age-regulated genes is likely to be governed by stress-responsive transcription factors, questions remain as to why particular genes are susceptible to age-related transcriptional decline. Here, we discuss recent findings showing that splicing is misregulated with age. While defects in splicing could lead to changes in protein isoform levels, they could also impact gene expression through nonsense-mediated decay of intron-retained transcripts. The discovery that splicing is misregulated with age suggests that other aspects of gene expression, such as transcription elongation, termination, and polyadenylation, must also be considered as potential mechanisms for age-related changes in transcript levels. Moreover, the considerable variation between genome-wide aging expression studies indicates that there is a critical need to analyze the transcriptional signatures of aging in single-cell types rather than whole tissues. Since age-associated decreases in gene expression could contribute to a progressive decline in cellular function, understanding the mechanisms that determine the aging transcriptome provides a potential target to extend healthy cellular lifespan. [ABSTRACT FROM AUTHOR]

Tobacco mosaic virus (TMV) possesses an upstream pseudoknotted domain (UPD), which is important for replication. After substituting the UPD with an internal poly(A) tract (43 nt), a mutant TMV-43A was constructed. TMV-43A replicated slower than TMV and induced a non-lethal mosaic symptom in Nicotiana benthamiana . In this study, deep sequencing was performed to detect the differences of small RNA profiles between TMV- and TMV-43A-infected N. benthamiana . The results showed that TMV-43A produced lesser amount of virus-derived interfering RNAs (vsiRNAs) than that of TMV. However, the distributions of vsiRNAs generation hotspots between TMV and TMV-43A were similar. Expression of genes related to small RNA biogenesis in TMV-43A-infected N. benthamiana was significantly lower than that of TMV, which leads to generation of lesser vsiRNAs. The expressions of host defense response genes were up-regulated after TMV infection, as compared to TMV-43A-infected plants. Host defense response to TMV-43A infection was lower than that to TMV. The absence of UPD might contribute to the reduced host response to TMV-43A. Our study provides valuable information in the role of the UPD in eliciting host response genes after TMV infection in N. benthamiana . (187 words) [ABSTRACT FROM AUTHOR]

We describe a tandem RNA isolation procedure (TRIP) that enables purification of in vivo formed messenger ribonucleoprotein (mRNP) complexes. The procedure relies on the purification of polyadenylated mRNAs with oligo(dT) beads from cellular extracts, followed by the capture of specific mRNAs with 3′-biotinylated 2′-O-methylated antisense RNA oligonucleotides, which are recovered with streptavidin beads. TRIP was applied to isolate in vivo crosslinked mRNP complexes from yeast, nematodes and human cells for subsequent analysis of RNAs and bound proteins. The method provides a basis for adaptation to other types of polyadenylated RNAs, enabling the comprehensive identification of bound proteins/RNAs, and the investigation of dynamic rearrangement of mRNPs imposed by cellular or environmental cues. [ABSTRACT FROM AUTHOR]

Poly ( ADP-ribose) polymerase ( PARP) plays an important role in endothelial dysfunction, leading to atherogenesis and vascular-related diseases. However, whether PARP regulates nitric oxide ( NO), a key regulator of endothelial function, is unclear so far. We investigated whether inhibition of PARP-1, the most abundant PARP isoform, prevents atherogenesis by regulating NO production and tried to elucidate the possible mechanisms involved in this phenomenon. In apolipoprotein E-deficient (apoE−/−) mice fed a high-cholesterol diet for 12 weeks, PARP-1 inhibition via treatment with 3,4-dihydro-54-(1-piperindinyl) butoxy-1(2H)-isoquinoline ( DPQ) or PARP-1 gene knockout reduced aortic atherosclerotic plaque areas (49% and 46%, respectively). Both the groups showed restored NO production in mouse aortas with reduced arginase II (Arg II) expression compared to that in the controls. In mouse peritoneal macrophages and aortic endothelial cells ( MAECs), PARP-1 knockout resulted in lowered Arg II expression. Moreover, phosphorylation of endothelial NO synthase ( eNOS) was preserved in the aortas and MAECs when PARP-1 was inhibited. Reduced NO production in vitro due to PARP-1 deficiency could be restored by treating the MAECs with oxidized low-density lipoprotein treatment, but this effect could not be achieved with peritoneal macrophages, which was likely due to a reduction in the expression of induced NOS expression. Our findings indicate that PARP-1 inhibition may attenuate atherogenesis by restoring NO production in endothelial cells and thus by reducing Arg II expression and consequently arginase the activity. [ABSTRACT FROM AUTHOR]

Alternative polyadenylation (APA), which is regulated by both cis-elements and trans-factors, plays an important role in posttranscriptional regulation of eukaryotic gene expression. However, comparing to the extensively studied transcription and alternative splicing, the extent of APA divergence during evolution and the relative cis- and trans-contribution remain largely unexplored. To directly address these questions for the first time in mammals, by using deep sequencing-based methods, we measured APA divergence between C57BL/6J and SPRET/EiJ mouse strains as well as allele-specific APA pattern in their F1 hybrids. Among the 24,721 polyadenylation sites (pAs) from 7,271 genes expressing multiple pAs, we identified 3,747 pAs showing significant divergence between the two strains. After integrating the allele-specific data from F1 hybrids, we demonstrated that these events could be predominately attributed to cis-regulatory effects. Further systematic sequence analysis of the regions in proximity to cis-divergent pAs revealed that the local RNA secondary structure and a poly(U) tract in the upstream region could negatively modulate the pAs usage. [ABSTRACT FROM AUTHOR]

Prevalent single cell RNA amplification and sequencing chemistries mainly focus on polyadenylated RNAs in eukaryotic cells by using oligo(dT) primers for reverse transcription. We develop a new RNA amplification method, "easier-sea", to reverse transcribe and amplify the total RNAs, both with and without polyadenylate tails, from a single cell for transcriptome sequencing with high efficiency, reproducibility, and accuracy. By distributing the reverse transcribed cDNA molecules into 1.5 X 10[sup 5] aqueous droplets in oil, the cDNAs are isothermally amplified using random primers in each of these 65-pL reactors separately. This new method greatly improves the ease of single-cell RNA sequencing by reducing the experimental steps. Meanwhile, with less chance to induce errors, this method can easily maintain the quality of single-cell sequencing. In addition, this polyadenylate-tail-independent method can be seamlessly applied to prokaryotic cell RNA sequencing. [ABSTRACT FROM AUTHOR]

Here we demonstrate that the RNase E-based degradosome is required for poly(A) polymerase I (PAP I)-dependent polyadenylation after Rho-independent transcription terminators for both mono- and polycistronic transcripts. Disruption of degradosome assembly in mutants lacking the polynucleotide phosphorylase (PNPase) binding domain led to a significant increase in the level of PNPase synthesized polynucleotide tails in the rpsJ and rpsM polycistronic transcripts and the lpp monocistronic transcript. The polynucleotide tails were mostly located within the coding sequences in the degradosome mutants compared to the wild type control where the majority of the PAP I synthesized poly(A) tails were after the Rho-independent transcription terminators. For the Rho terminated metNIQ operon, the tails for all three mRNAs were predominately polynucleotide and were located within the coding sequences in both wild type and degradosome mutant strains. Furthermore, by employing a pnp-R100D point mutant that encodes a catalytically inactive PNPase protein that still forms intact degradosomes, we show that a catalytically active PNPase is required for normal mRNA polyadenylation by PAP I. Our data suggest that polyadenylation requires a functional degradosome to maintain an equilibrium between free PNPase and the PAP I polyadenylation complex. [ABSTRACT FROM AUTHOR]

Alternative polyadenylation (APA) is an important layer of gene regulation that produces mRNAs that have different 3' ends and/or encode diverse protein isoforms. Up to 70% of annotated genes in plants undergo APA. Increasing numbers of poly(A) sites collected in various plant species demand new methods and tools to access and mine these data. We have created an open-access web service called PlantAPA (http://bmi.xmu.edu.cn/plantapa) to visualize and analyze genome-wide poly(A) sites in plants. PlantAPA provides various interactive and dynamic graphics and seamlessly integrates a genome browser that can profile heterogeneous cleavage sites and quantify expression patterns of poly(A) sites across different conditions. Particularly, through PlantAPA, users can analyze poly(A) sites in extended 3' UTR regions, intergenic regions, and ambiguous regions owing to alternative transcription or RNA processing. In addition, it also provides tools for analyzing poly(A) site selections, 3' UTR lengthening or shortening, non-canonical APA site switching, and differential gene expression between conditions, making it more powerful for the study of APA-mediated gene expression regulation. More importantly, PlantAPA offers a bioinformatics pipeline that allows users to upload their own short reads or ESTs for poly(A) site extraction, enabling users to further explore poly(A) site selection using stored PlantAPA poly(A) sites together with their own poly(A) site datasets. To date, PlantAPA hosts the largest database of APA sites in plants, including Oryza sativa, Arabidopsis thaliana, Medicago truncatula, and Chlamydomonas reinhardtii. As a user-friendly web service, PlantAPA will be a valuable addition to the community of biologists studying APA mechanisms and gene expression regulation in plants. [ABSTRACT FROM AUTHOR]

More than half of human genes use alternative cleavage and polyadenylation to generate alternative 3′ untranslated region (3′UTR) isoforms. Most efforts have focused on transcriptome-wide mapping of alternative 3′UTRs and on the question of how 3′UTR isoform ratios may be regulated. However, it remains less clear why alternative 3′UTRs have evolved and what biological roles they play. This review summarizes our current knowledge of the functional roles of alternative 3′UTRs, including mRNA localization, mRNA stability, and translational efficiency. Recent work suggests that alternative 3′UTRs may also enable the formation of protein–protein interactions to regulate protein localization or to diversify protein functions. These recent findings open an exciting research direction for the investigation of new biological roles of alternative 3′UTRs. [ABSTRACT FROM AUTHOR]

Summary: Alternative polyadenylation (APA) is now emerging as a widespread mechanism modulated tissue-specifically, which highlights the need to define tissue-specific poly(A) sites for profiling APA dynamics across tissues. We have developed an R package called TSAPA based on the machine learning model for identifying tissue-specific poly(A) sites in plants. A feature space including more than 200 features was assembled to specifically characterize poly(A) sites in plants. The classification model in TSAPA can be customized by selecting desirable features or classifiers. TSAPA is also capable of predicting tissue-specific poly(A) sites in unannotated intergenic regions. TSAPA will be a valuable addition to the community for studying dynamics of APA in plants. [ABSTRACT FROM AUTHOR]

Switching between different alternative polyadenylation (APA) sites plays an important role in the fine tuning of gene expression. New technologies for the execution of 3’-end enriched RNA-seq allow genome-wide detection of the genes that exhibit significant APA site switching between different samples. Here, we show that the independence test gives better results than the linear trend test in detecting APA site-switching events. Further examination suggests that the discrepancy between these two statistical methods arises from complex APA site-switching events that cannot be represented by a simple change of average 3’-UTR length. In theory, the linear trend test is only effective in detecting these simple changes. We classify the switching events into four switching patterns: two simple patterns (3’-UTR shortening and lengthening) and two complex patterns. By comparing the results of the two statistical methods, we show that complex patterns account for 1/4 of all observed switching events that happen between normal and cancerous human breast cell lines. Because simple and complex switching patterns may convey different biological meanings, they merit separate study. We therefore propose to combine both the independence test and the linear trend test in practice. First, the independence test should be used to detect APA site switching; second, the linear trend test should be invoked to identify simple switching events; and third, those complex switching events that pass independence testing but fail linear trend testing can be identified. [ABSTRACT FROM AUTHOR]

SG2NA belongs to a three member Striatin subfamily of WD-40 repeat superfamily. It has multiple protein–protein interaction domains that are involved in the assembly of supra-molecular signaling complexes. Earlier we had demonstrated that there are at least five variants of SG2NA, generated by alternative splicing. We now demonstrate that a 52 kDa novel variant is generated by the editing of the transcript for the 82 kDa isoform. The 52 kDa protein is abundant in mouse tissues but it is barely present in immortalized cells, suggesting its role in cell differentiation. Besides splicing and editing, expression of SG2NAs in tissues is also regulated by differential polyadenylation and mRNA/protein stability. Further, the longer UTR is seen only in the brain mRNA from 1 month old mouse and 8–10 day old chick embryo. Like alternative splicing, differential polyadenylation of Sg2na transcripts is also conserved in evolution. Taken together, these results suggest a highly versatile and dynamic mode of regulation of SG2NA with potential implications in tissue development. [ABSTRACT FROM AUTHOR]

Transcriptional cyclin-dependent kinases (CDKs) regulate RNA polymerase II initiation and elongation as well as cotranscriptional mRNA processing. In this report, we describe an important role for CDK12 in the epidermal growth factor (EGF)-induced c-FOS proto-oncogene expression in mammalian cells. This kinase was found in the exon junction complexes (EJC) together with SR proteins and was thus recruited to RNA polymerase II. In cells depleted of CDK12 or eukaryotic translation initiation factor 4A3 (eIF4A3) from the EJC, EGF induced fewer c-FOS transcripts. In these cells, phosphorylation of serines at position 2 in the C-terminal domain (CTD) of RNA polymerase II, as well as levels of cleavage-stimulating factor 64 (Cstf64) and 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF73), was reduced at the c-FOS gene. These effects impaired 3' end processing of c-FOS transcripts. Mutant CDK12 proteins lacking their Arg-Ser-rich (RS) domain or just the RS domain alone acted as dominant negative proteins. Thus, CDK12 plays an important role in cotranscriptional processing of c-FOS transcripts. [ABSTRACT FROM AUTHOR]