81 research outputs found
A Deadenylase Assay by Size-Exclusion Chromatography
The shortening of the 3′-end poly(A) tail, also called deadenylation, is crucial to the regulation of mRNA processing, transportation, translation and degradation. The deadenylation process is achieved by deadenylases, which specifically catalyze the removal of the poly(A) tail at the 3′-end of eukaryotic mRNAs and release 5′-AMP as the product. To achieve their physiological functions, all deadenylases have numerous binding partners that may regulate their catalytic properties or recruit them into various protein complexes. To study the effects of various partners, it is important to develop new deadenylase assay that can be applied either in vivo or in vitro. In this research, we developed the deadenylase assay by the size-exclusion chromatography (SEC) method. The SEC analysis indicated that the poly(A) or oligo(A) substrate and the product AMP could be successfully separated and quantified. The enzymatic parameters of deadenylase could be obtained by quantifying the AMP generation. When using the commercial poly(A) as the substrate, a biphasic catalytic process was observed, which might correlate to the two distinct states of poly(A) in the commercial samples. Different lots of commercial poly(A) had dissimilar size distributions and were dissimilar in response to the degradation of deadenylase. The deadenylation pattern, processive or distributive, could also be investigated using the SEC assay by monitoring the status of the substrate and the generation kinetics of AMP and A2. The SEC assay was applicable to both simple samples using the purified enzyme and complex enzyme reaction conditions such as using protein mixtures or crude cell extracts as samples. The influence of solutes with absorption at 254 nm could be successfully eliminated by constructing the different SEC profiles
Repression of Mitochondrial Translation, Respiration and a Metabolic Cycle-Regulated Gene, SLF1, by the Yeast Pumilio-Family Protein Puf3p
Synthesis and assembly of the mitochondrial oxidative phosphorylation (OXPHOS) system requires genes located both in the nuclear and mitochondrial genomes, but how gene expression is coordinated between these two compartments is not fully understood. One level of control is through regulated expression mitochondrial ribosomal proteins and other factors required for mitochondrial translation and OXPHOS assembly, which are all products of nuclear genes that are subsequently imported into mitochondria. Interestingly, this cadre of genes in budding yeast has in common a 3′-UTR element that is bound by the Pumilio family protein, Puf3p, and is coordinately regulated under many conditions, including during the yeast metabolic cycle. Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility. Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling. We also identified the cytoplasmic translation factor Slf1p as yeast metabolic cycle-regulated gene that is repressed by Puf3p at the post-transcriptional level and promotes respiration and extension of yeast chronological life span when over-expressed. Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity
A Transcription Elongation Factor That Links Signals from the Reproductive System to Lifespan Extension in Caenorhabditis elegans
In Caenorhabditis elegans and Drosophila melanogaster, the aging of the soma is influenced by the germline. When germline-stem cells are removed, aging slows and lifespan is increased. The mechanism by which somatic tissues respond to loss of the germline is not well-understood. Surprisingly, we have found that a predicted transcription elongation factor, TCER-1, plays a key role in this process. TCER-1 is required for loss of the germ cells to increase C. elegans' lifespan, and it acts as a regulatory switch in the pathway. When the germ cells are removed, the levels of TCER-1 rise in somatic tissues. This increase is sufficient to trigger key downstream events, as overexpression of tcer-1 extends the lifespan of normal animals that have an intact reproductive system. Our findings suggest that TCER-1 extends lifespan by promoting the expression of a set of genes regulated by the conserved, life-extending transcription factor DAF-16/FOXO. Interestingly, TCER-1 is not required for DAF-16/FOXO to extend lifespan in animals with reduced insulin/IGF-1 signaling. Thus, TCER-1 specifically links the activity of a broadly deployed transcription factor, DAF-16/FOXO, to longevity signals from reproductive tissues
Comparative Analysis of mRNA Targets for Human PUF-Family Proteins Suggests Extensive Interaction with the miRNA Regulatory System
Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 3′-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 3′-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs
Dynamics of Co-Transcriptional Pre-mRNA Folding Influences the Induction of Dystrophin Exon Skipping by Antisense Oligonucleotides
Antisense oligonucleotides (AONs) mediated exon skipping offers potential therapy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, a “window of analysis” that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structures were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered “engaged” if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localisation of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency) of 94% of 176 previously reported AONs. Four novel insights are inferred: (1) the lowest frequencies of engaged nucleotides are associated with the most efficient AONs; (2) engaged nucleotides at 3′ or 5′ ends of the target site attenuate AON performance more than at other sites; (3) the performance of longer AONs is less attenuated by engaged nucleotides at 3′ or 5′ ends of the target site compared to shorter AONs; (4) engaged nucleotides at 3′ end of a short target site attenuates AON efficiency more than at 5′ end
microRNA-Mediated Messenger RNA Deadenylation Contributes to Translational Repression in Mammalian Cells
Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3′ untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3′ poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3′ UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs
Alternative splicing of exon 10 in the tau gene as a target for treatment of tauopathies
Tau aggregation is one of the major features in Alzheimer's disease and in several other tauopathies, including frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), and progressive supranuclear palsy (PSP). More than 35 mutations in the tau gene have been identified from FTDP-17 patients. A group of these mutations alters splicing of exon 10, resulting in an increase in exon 10 inclusion into tau mRNA. Abnormal splicing with inclusion of exon 10 into tau mRNA has also been observed in PSP and AD patients. These results indicate that abnormal splicing of exon 10, leading to the production of tau with exon 10, is probably one of the mechanisms by which tau accumulates and aggregates in tauopathic brains. Therefore, modulation of exon 10 splicing in the tau gene could potentially be targeted to prevent tauopathies. To identify small molecules or compounds that could potentially be developed into drugs to treat tauopathies, we established a cell-based high-throughput screening assay. In this review, we will discuss how realistic, specific biological molecules can be found to regulate exon 10 splicing in the tau gene for potential treatment of tauopathies
Identification and analysis of the RNA degrading complexes and machinery of Giardia lamblia using an in silico approach
Role of Ccr4-Not complex in heterochromatin formation at meiotic genes and subtelomeres in fission yeast
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